CN107207599A - Anti- TRKA antibody and its derivative with enhanced inhibition activity are ache related for treating bone - Google Patents

Anti- TRKA antibody and its derivative with enhanced inhibition activity are ache related for treating bone Download PDF

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Publication number
CN107207599A
CN107207599A CN201580075478.3A CN201580075478A CN107207599A CN 107207599 A CN107207599 A CN 107207599A CN 201580075478 A CN201580075478 A CN 201580075478A CN 107207599 A CN107207599 A CN 107207599A
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antibody
trka
pain
humanization
bone
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A·沃姆斯利
S·K·迪贝
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Ichnos Sciences SA
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Glenmark Pharmaceuticals SA
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Priority claimed from US14/562,297 external-priority patent/US20150183885A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The present invention relates to antibody for TrkA acceptors and application thereof, including the anti-TrkA antibody of humanization and the method with anti-TrkA Antybody therapies.

Description

Anti- TRKA antibody and its derivative with enhanced inhibition activity are used to treat bone photo Close pain
Invention field
The present invention relates generally to anti-TrkA receptor antibodies and application thereof, including the anti-TrkA antibody of humanization, and using anti- The method of TrkA Antybody therapies.On the one hand, it is used for the present invention relates to the anti-TrkA antibody of the humanization with enhanced inhibition activity Treat neuroma and/or the ache related method of bone.
Another further aspect, the present invention relates to the anti-TrkA antibody of the humanization with enhanced inhibition activity, it can comprising heavy chain Become area, light chain variable district, people's constant region of light chain and the light chain constant region variants of human IgG 4, the antibody shows the exchangeability changed Matter.
Background of invention
Neurotrophic factor (Neurotrophin) belongs to peptide growth factor family (Barde YA (1994) J.Neurobiol.25(11):1329-33), (Levi-Montalcini R related to the member of NGF families first in structure (1987)EMBO J.6(5):1145-54).Neurotrophic factor adjusts peripheral neurons and the neuron point of central nervous system Change and survival and cynapse transmission.In addition, NFG works on a variety of non-neuronal tissues and cell, such as immunocyte. NFG is worked by two membrane receptors being present in target cell, low-affinity p75 acceptors, and with tyrosine kinase activity 140kDa high-affinity transmembrane glycoproteins TrkA (Kaplan DR et al., (1991) Science 252 (5005):554- 8；Klein R et al.,(1991)Cell 65(1):189-97).TrkA is in the neuron of neural crest, in sympathetic neuron In and in the cholinergic neuron of basal forebrain and corpus straitum express, in these positions its be NGF activity critical mediator (Holtzman DM et al.,(1992)Neuron 9(3):465-78；Verge VM et al.,(1992) J.Neurosci.12(10):4011-22).TrkA is also expressed in some non-neuronal tissues and cell, including bone-marrow-derived lymphocyte (Torcia M et al.,(1996)Cell 85(3):345-56)。
Nerve growth factor (NGF) is initially as the survival factors quilt felt in the nervous system of development with sympathetic neuron Identify (Gorin PD＆Johnson EM (1979) PNAS USA, 76 (10):5382-6).In adult, NGF is not existence institute It is required, but there is key effect for pain and hyperalgesic generation in several acute and chronic pain statuses.NGF exists High expression in damage and inflammation tissue, and trkA activation can be triggered by number of mechanisms on nociceptive neuron With reinforcing pain signal conduction.Inflammation is ache related in animal model to be significantly reduced by neutralizing NGF bioactivity (Woolf CJ et al.,(1994)Neuroscience 62(2):327-31；McMahon SB et al.,(1995) Nat.Med.1(8):774-80；Koltzenburg M et al.,(1999)Eur.J.Neurosci.11(5):1698- 704) increase for, pointing out the neurotrophic factor level is to produce the complete hyperalgia to react necessary.It is worth noting , the suppression of other neurotrophic factors does not cause the hyperalgesic antagonism to the induction, points out the effect to be specific to NGF(McMahon SB et al.,(1995)Nat.Med.1(8):774-80)；In addition, NGF suppresses also in different neuropathy Cause analgesic activity (Koltzenburg M et al., (1999) Eur.J.Neurosci.11 during rationality is ache related (5):1698-704；Ro LS et al.,(1999)Pain 79(2-3):265-74；Theodosiou M et al., (1999)Pain,81(3):245-55；Christensen MD&Hulsebosch CE(1997)Exp.Neurol.147(2): 463-75)。
There is still unsatisfied huge medical need on pain therapy.Main antalgesic classification, nonsteroid anti-inflammatory Medicine (NSAID) and opioid drug, (Hefti FF et al., (2006) Trends are limited because of its effect and tolerance Pharmacol.Sci.27(2):85-91).It is sufficient that chronic pain patient less than 30% can use current treatment to obtain Alleviate, and there are many side effects, (Kalso E et al., (2004) Pain especially in the case of chronic administration 112(3):372-80).Recognize that NGF has central role in the Theory of Pain Mechanism of adult so that the exploitation that has an opportunity is a kind of brand-new Pain treatment.
It can be a kind of more preferable therapeutic choice to target TrkA rather than NGF, because this receptor does not disturb receptor-mediated by p75 NGF functions (p75 acceptors in neurodevelopment have extensive function).
The present inventor have rated in deep body pain, anti-using humanization especially in the ache related treatment method of bone Effect and security of TrkA antibody, wherein it can be that the bone injury for example caused by exterior trauma causes that the bone photo, which closes pain, Pain or caused by the lesion for involving bone pain (can for example cause the cancer of bone injury) or by cause bone photo close pain Pain caused by the inappropriate innervation of pain and/or other influences.The present inventor is also evaluated caused by treatment neuroma Effect and security of the anti-TrkA antibody of humanization are used in the method for pain, wherein the neuroma is because of wound or other lesions And improper formation.
Summary of the invention
Present disclose relates generally to the anti-TrkA antibody of humanization, its preparation and application.
On the one hand, the disclosure provides the anti-TrkA antibody of humanization or its fragment, and it is included：
A) include and be selected from SEQ ID NO:The heavy-chain variable domains of 1-5 sequence；With
B) include and be selected from SEQ ID NO:The light variable domains of 6-13 sequence,
The wherein CDR2 of heavy-chain variable domains includes at least one 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and/or wherein weight chain variable structure The non-CDR region in domain is including 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor selected from the amino acid position by 37,42 and 89 groups constituted, wherein each group membership Amino acid position provided using the numbering system described in Kabat.
In another further aspect, the disclosure provides the seperated nuclear acid of encoding humanized anti-TrkA antibody or its fragment, includes this point The carrier of freestone acid and nucleic acid or the host cell of carrier comprising the separation.The disclosure also provides generation humanization anti-TrkA The method of antibody or its fragment.
In other respects, the disclosure provide the composition comprising the anti-TrkA antibody of humanization or its fragment and comprising with treatment The anti-TrkA antibody of humanization of agent connection or the immunoconjugates of its fragment.
Especially, it is used to treat bone photo pass the present invention relates to the anti-TrkA antibody of the humanization combined with people TrkA or its fragment Pain, wherein the anti-TrkA antibody or its fragment include heavy-chain variable domains 1,2 and 3 and light variable domains 1,2 With 3, and heavy-chain variable domains include and are selected from SEQ ID NO:1-5 sequence, light variable domains, which are included, is selected from SEQ ID NO:The non-CDR region of 6-13 sequence, wherein heavy-chain variable domains is selected from the amino acid position by 37,42 and 89 groups constituted Put comprising 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, wherein the amino acid position of each group membership is provided using the numbering system described in Kabat.
In other respects, the disclosure provides the anti-TrkA antibody of humanization or its fragment, composition or immunoconjugates, is used for Medicine, for treat pain, for treat chronic ache, for treat Acute Pain, for treat with following one or more Related pain：Pancreatitis, kidney stone, mullerianosis, IBD, Crohn's disease, post-operation adhesion, cholecystolithiasis, headache, Dysmenorrhoea, musculoskeletal pain, sprain, splanchnodynia, ovarian cyst, prostatitis, cystitis, interstitial cystitis, postoperative pain, Migraines, trigeminal neuralgia, from burn and/or the pain of wound, the pain related to wound, neurogenic pain and The related pain of musculoskeletal disease, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, periarticular pathologies, tumour Pain, the pain from Bone tumour, HIV, for treating cancer, neural disorder, Alzheimer disease, diabetes, sugar Characteristic of disease nephrosis, viral disorder, illness, leprosy or the inflammation of HIV mediations are urinated, and for diagnosis or prognosis.
In another further aspect, this disclosure relates to for treating deep body pain (deep somatic pain), especially bone Ache related method, it at least includes step：The anti-TrkA of humanization of the present invention for applying effective dose to individual in need resists Body or its fragment.
According to the present invention, bone is ache related to be intended to refer to, and individual is because of damage, the degeneration or any closed with one or more bone photos Lesion and any sensation of pain undergone.
Pain can show as feeling of numbness, tingle, compacted pain, thermal sensation or creeping chill or girdle sensation, dull pain are for example oppressed or Pain is for example stabbed, hits, tears or punctured to girdle sensation, vexed pain or tingle, sharp pain, pain can be it is lasting, bounce, Or lag rule or irregular intermittent, or only occur when the region is touched.
More particularly, it can be fracture (bone frature), bone split (bone chip), the disconnected (bone of bone that bone is ache related Break) or any other loss or inflammation consequence.This can cause because of accident, or because excessively using or repeating to move Caused by injury region, or caused by bone is weak, the bone it is weak can because anhormonia, infection, osteocarcinoma, metastatic malignant tumour, Leukaemia, myeloma or other involve the cancer of blood or bone relevant cell or interrupted because of bone blood supply and cause.
According to the present invention, it can be the consequence for the lesion for involving bone that bone is ache related.The lesion for involving bone refers to, directly or One or more bones of individual are affected indirectly to cause any lesion of sensation of pain.The example of lesion includes cancer, sense Dye and immune system dysfunction.
Especially, bone is ache related can be related with osteocarcinoma, especially secondary bone cancer and/or metastatic carcinoma cell, or Result from.
Alternatively, bone is ache related to be caused by one or more neuromas, especially by osteocarcinoma or any other disease Neuroma caused by becoming.
Especially, the inventive method includes, using the anti-TrkA antibody of the humanization of the present invention of effective dose or its fragment, with most Smallization reduces abnormal neuron rudiment and/or the formation of neuroma or size.
According to the present invention, the anti-TrkA antibody of humanization of effective dose or its fragment are intended to refer to, when by clinician or other When titular medical practitioner is administered to individual, it is enough to cause one or more sensation of pain in the individual in need It is able to the amount alleviated or stopped.
Another further aspect, this disclosure relates to which the anti-TrkA antibody of humanization of the present invention or its fragment are used to treat deep body pain Bitterly, especially bone is ache related or neuroma is ache related.More particularly, it can be fracture, bone split, fracture that bone is ache related Disconnected or any other loss or inflammation consequence.This can cause because of accident, or because excessively using or repeating mobile damage Place cause, or by anhormonia, infection, osteocarcinoma, metastatic malignant tumour, leukaemia, myeloma or other involve blood or bone Bone is weak caused by the cancer of relevant cell or bone blood supply are interrupted causes.
According to the present invention, bone is ache related to be caused by the lesion for involving bone.The lesion for involving bone refers to, directly or One or more bones of ground connection influence individual are so as to cause any lesion of sensation of pain.The example of lesion include cancer, infection, And immune system dysfunction.
Especially, bone is ache related can be related with osteocarcinoma, especially secondary bone cancer and/or metastatic carcinoma cell, or Result from.
Alternatively, bone is ache related to be caused by one or more neuromas, especially by osteocarcinoma or any other disease Neuroma caused by becoming.
Especially, the present invention relates in the treatment using the anti-TrkA antibody of humanization of the present invention or its fragment, to minimize Or reduction abnormal neuron rudiment and/or the formation of neuroma or size.
Another further aspect, the disclosure provides treatment inflammatory pain (inflammatory pain), osteo-arthritic pain (osteoarthritic pain) and neurogenic pain (neuropathic pain) method.On the one hand, it is anxious in vivo In property Inflammatory hyperalgesia model, acute inflammation pain sensation mistake result in using the anti-TrkA antibody of humanization with 0.01mg/kg dosage Quick notable reverse.On the other hand, humanization is applied with 0.01mg/kg dosage in chronic inflammatory hyperalgesia model in vivo Anti- TrkA antibody result in the hyperalgesic notable and lasting reverse of chronic inflammatory.On the other hand, arthroxerosis in vivo In hyperalgesia model, arthroxerosis hyperalgia result in using the anti-TrkA antibody of humanization with 0.01mg/kg dosage It is notable and lasting reverse.Another further aspect, in vivo in the chronic constriction injury model of neurogenic pain, with 1.0mg/ Kg dosage applies the notable reverse that the anti-TrkA antibody of humanization result in neurogenic pain.
Another further aspect, the disclosure provides the product comprising the anti-TrkA antibody of humanization or its fragment, composition or conjugate.
Another further aspect, the disclosure provides the medicine box comprising the anti-TrkA antibody of humanization or its fragment, composition or conjugate.
Because protein is more greater and more complicated than traditional organic and inorganic drug, this has also triggered stablizes effective with production The specific question related containing antibody pharmaceutical formulation.In order that albumen retains biological activity, preparation must fully preserve egg The Conformational Integrity of at least core sequence of casamino acid, and multiple functional groups of protected protein are not degraded simultaneously.Albumen It is (that is, any to be related to modifying protein, caused newly by key formation or fracture that matter degradation pathway can be related to chemical instability The process of chemical entities) or physical instability (that is, the change of protein higher structure).Chemical instability can be because of deacylation Amine, racemic, hydrolysis, oxidation, β eliminations or disulfide bond are exchanged and caused.Physical instability can because denaturation, aggregation, precipitation or Absorption causes.
According to a further aspect of the present invention there is provided stable aqueous pharmaceutical preparations, its anti-TrkA comprising therapeutically effective amount resists Body, buffer, surfactant and tension regulator (tonicifying agent), wherein preparation pH is about 5 to about 7.
According to the present invention in this respect, anti-TrkA antibody is included：
A) include and be selected from SEQ ID NO:The heavy-chain variable domains of 1-5 sequence；With
B) include and be selected from SEQ ID NO:The light variable domains of 6-13 sequence,
The wherein CDR2 of heavy-chain variable domains includes at least one 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor and/or wherein heavy-chain variable domains Non- CDR region selected from 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is included by the amino acid position of 37,42 and 89 groups constituted, wherein each group membership Amino acid position provides (Kabat EA et al., (1991) Sequences of using numbering system shown in Kabat proteins of immunological interest.5th Edition-US Department of Health and Human Services,NIH publication n°91-3242)。
According to the present invention in this respect, anti-TrkA antibody exists with least 0.1mg/ml concentration.
According to the present invention in this respect, anti-TrkA antibody exists with least 0.2mg/ml to 175mg/ml concentration.
According to the present invention in this respect, anti-TrkA antibody exists with least 1mg/ml concentration.
According to the present invention in this respect, anti-TrkA antibody exists with least 10mg/ml concentration.
According to the present invention in this respect, anti-TrkA antibody exists with least 100mg/ml concentration.
According to the present invention in this respect, anti-TrkA antibody exists with least 150mg/ml concentration.
According to the present invention in this respect, buffer is selected from：Citrate, acetate, histidine, phosphate, three (three (hydroxyl first Base) aminomethane).
According to the present invention in this respect, stabilizer/tension regulator is selected from：Sodium acetate, sodium acid carbonate, sodium carbonate, sodium chloride, Potassium acetate, saleratus, potassium carbonate, potassium chloride, sucrose, polyalcohol, carbohydrate, amino acid such as histidine, arginine and sweet ammonia Acid, methionine, proline, lysine, glutamic acid, amine and trehalose.
According to the present invention in this respect, surfactant is selected from：Tween 20/40/80, TWEEN-20/80, Bo Luosha Nurse, lauryl sodium sulfate.
According to the present invention in this respect, the pH of preparation is 5.75.
According to the present invention in this respect, the pH of preparation is 6.0.
According to the present invention in this respect, preparation can also include one or more excipient and filler.
There is provided the low concentration aqueous of the anti-TrkA antibody of regulation to pH6.0 according to a preferred embodiment of the invention Preparation, comprising：
The anti-TrkA antibody of 10mg/ml；
25mM citrates；
150mM NaCl；
0.05%Tween 80.
There is provided the low concentration aqueous of the anti-TrkA antibody of regulation to pH6.0 according to a preferred embodiment of the invention Preparation, comprising：
The anti-TrkA antibody of at least 10mg/ml；
50mM histidines；
150mM NaCl；
0.05%Tween 80.
There is provided the highly concentrated of the anti-TrkA antibody of regulation to pH5.75 or 6.0 according to a preferred embodiment of the invention Aqueous formulation is spent, comprising：
The anti-TrkA antibody of 100mg/ml；
50mM histidines；
150mM NaCl；
0.05%Tween 80.
There is provided the highly concentrated of the anti-TrkA antibody of regulation to pH5.75 or 6.0 according to a preferred embodiment of the invention Aqueous formulation is spent, comprising：
The anti-TrkA antibody of 150mg/ml；
50mM histidines；
150mM NaCl；
0.05%Tween 80.
The present inventor overcomes the aqueous liquid preparation with typically containing albumen (especially antibody) when developing these preparations Related the problem of.Several alternative preparations attempted during the research project lack steady in a long-term at 5 DEG C, 25 DEG C or 40 DEG C Property, antibody occurs in that degraded and obvious unstable especially in several alternative preparations combined without above-listed specific components Property.
In addition, it is possible to for two aspects of anti-TrkA amount of antibody and dosage number of every dose of administration, the subcutaneous administration system The exploitation of agent also increases possible dosage regimen.In addition, preliminary data be also shown that according to the present invention subcutaneous administration preparation be It is a kind of more efficiently using by way of anti-TrkA antibody the method and formulation previously tested.
In accordance with a further aspect of the present invention, with the dosage of at least 0.1mg/kg body weight, especially at least 0.3mg/kg, 1mg/ Kg, 3mg/kg, 10mg/kg or 30mg/kg dosage, individual in need is administered to by preparation.
According to a further aspect of the present invention, preparation is applied once to patient in need.
According to a further aspect of the present invention, preparation is applied to patient in need, wherein it is small to be spaced at least 1 between being administered twice When, it is spaced at least 4 hours/6 hours/12 hours/24 hours between being preferably administered twice.
According to a further aspect of the present invention, preparation is applied to patient in need on demand.
According to a further aspect of the present invention, preparation was in 5 DEG C of stabilizations at least 1 year, preferably at least 2 years.
According to a further aspect of the present invention, preparation is 25 DEG C of stable at least three month, most preferably preferably 6 months, 1 year.
According to a further aspect of the present invention, preparation is in 40 DEG C of stable at least one moons, preferably 3 months.
Wherein stability can be used selected from following one or more methods measurement：Determine clarity, the coloring of preparation The change of degree, opacity and granulometric impurity (visible particle)；280nm wavelength optical absorption measurement is to determine egg present in preparation The concentration of white matter；PAGE gel develops to determine that the weight of antibody changes and/or degraded；Antibody knot is determined by ELISA Close any change of property；The change that positive/negative antibody materials are constituted in preparation is determined by HPLC-CEX；By HPLC-IEF, The change of the isoelectric focusing spectrum of antibody present in preparation is determined using Capillary Electrophoresis；Analyzed by HPLC-SEC, it is determined that system The change of antibody in agent.
According to a further aspect of the present invention, preparation includes a certain amount of anti-TrkA antibody, and thus preparation can be with least The dosage of 0.1mg/kg body weight, especially at least 0.3mg/kg, 1mg/kg, 3mg/kg, 10mg/kg or 30mg/kg dosage, are applied With to individual in need.
According to a further aspect of the invention, the method for also providing the individual pain for the treatment of, including administration include therapeutically effective amount The invention formulation of anti-TrkA antibody.
Brief description
Fig. 1：The surface plasma body resonant vibration measurement of anti-TrkA antibody.Data are expressed as stoichiometric number and (are abbreviated as RU；Y-axis) Vs. time (X-axis) (Figure 1A) MNAC13 antibody.(Figure 1B) BXhVH5VL1 antibody.(Fig. 1 C) GBR VH5 (V37A) VL1 antibody. (Fig. 1 D) GBR VH5 (K3Q, V37A) VL1 antibody.
Fig. 2：The anti-TrkA antibody heat endurance measured using differential scanning calorimetry.Data are expressed as excess molar heat capacity (excess molar heat capacity, are abbreviated as Cp [kcal/mol/ DEG C]；Y-axis) vs. temperature (X-axis).(Fig. 2A) MNAC13 antibody (FAB fragments Tm is Tm1, at 74 DEG C).(FAB fragments Tm is Tm3 to (Fig. 2 B) BXhVH5VL1 antibody, 76.5 ℃).(Fig. 2 C) GBR VH5 (V37A) VL1 antibody (FAB fragments Tm is Tm1, at 73.6 DEG C).(Fig. 2 D) GBR VH5 (K3Q, V37A) VL1 antibody (FAB fragments Tm is Tm1, at 73 DEG C).(Fig. 2 E) Fig. 2 C and Fig. 2 D superposition.(Fig. 2 F) Fig. 2 B and Fig. 2 D's Superposition.(Fig. 2 G) Fig. 2A and Fig. 2 D superposition.
Fig. 3：The functional biological activity of anti-TrkA antibody.The TF-1 cells propagation that the anti-TrkA antibody of humanization is induced NGF Influence；Data are expressed as % breeder reactions (Y-axis) vs. antibody concentrations (μ g/ml；X-axis).(Fig. 3 A) GBR VH5 (V37A) VL1,GBR VH5(K3Q,V37A)VL1vs.BXhVH5VL1.(Fig. 3 B) GBR VH5 (V37A) VL1, GBR VH5 (K3Q, V37A) VL1vs.MNAC13.(Fig. 3 C) GBR VH5 (K3Q, V37A) VL1vs.GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P. (Fig. 3 D) BXhVH5VL1, BXhVH5VL3, GBR VH5 (V37A) VL1vs.GBR VH5 (V37A) VL3.(Fig. 3 E) BXhVH5VL1,BXhVH3VL1,GBR VH5(V37A)VL1vs.GBR VH3(V37A)VL1。
Fig. 4：The anti-TrkA antibody of humanization reverses acute inflammation pawl pain.To a rear solid end subplantar injection CFA for AMB1 mouse, Inducing acute Inflammatory hyperalgesia, and it is measured as % homonymies (injection) and offside (does not inject) the weight ratio born on pawl (%ipsi/contra, average value ± s.e.m.).In CFA injections (0hr), 23hr takes load-bearing reading afterwards, afterwards the 24hr after CFA Start to process, single i.p. injects 0.0001 (white post), 0.001 (horizontal hachure post), 0.01 (check post) and 0.1mg/kg (rhombus hachure post) anti-TrkA antibody or 0.1mg/kg Isotype control antibodies (black post), afterwards upon administration 4,8,24, 48,72,96 and 120hr measures load-bearing.As positive control, with 10mg/kg Indomethacins (indomethacin) p.o. processing Mouse (vertical hatching post).
Fig. 5：The anti-TrkA antibody of humanization reverses chronic inflammatory arthritis.Pass through a hind leg knee joint to AMB1 mouse Middle intra-articular injection CFA, induces the chronic inflammatory hyperalgia in joint, and is measured as % homonymies (injection) and offside (is not noted Penetrate) the weight ratio (%ipsi/contra, average value ± s.e.m.) that bears on limb.It is (first to be used in fact before CFA injections are faced Test (naive)) and the 3rd after CFA, take load-bearing reading within 7 and 10 days, the 13rd day start to process after CFA afterwards, single i.p. notes Penetrate 0.01 (hollow square, dotted line), 1 (triangles) and the anti-TrkA antibody of 10mg/ml (open circles) or 10mg/kg isotypes Control antibodies (filled circles), afterwards 4,8,24,48,72,96hr measurement load-bearing upon administration (in bracket).As positive control, 60mg/kg celecoxibs (celecoxib) were reinstated from the 13rd day and twice a day handle mouse (open diamonds), the 13rd after CFA Its 1 and 8hr measurements load-bearing upon administration, then the 1hr measurements load-bearing upon administration in the 14-17 days after CFA.
Fig. 6：The anti-TrkA antibody of humanization reverses arthroxerosis pain.Into a hind leg knee joint for AMB1 mouse Intra-articular injection MIA, inducing chronic osteoarthritis hyperalgia, and it is measured as % homonymies (injection) and offside (not injecting) The weight ratio (%ipsi/contra, average value ± s.e.m.) born on limb.Before MIA injections are faced and the 3rd, 7 after MIA injections Baseline (BL) load-bearing reading was taken with 10 days, afterwards the 14th day start to process after MIA, single i.p. injects 1 (open triangles), 10 (open diamonds) and 100 μ g/kg (filled circles, dotted line) anti-TrkA antibody or 10mg/kg Isotype control antibodies (solid squares) (Fig. 6 A).Compareed as comparison material, after MIA the 14th day with C16H25NO2 (tramadol) 10mg/kg or Pregabalin (pregabalin) 30mg/kg p.o. handle animal, every other day use C16H25NO2 30mg/kg within the 16-22 days after MIA afterwards Or Pregabalin 100mg/kg processing animals (Fig. 6 B).After MIA the 14th day upon administration 4,8 and 24 hours, it is and right afterwards In antibody treatment group at every 24 hours, for C16H25NO2 and Pregabalin treatment group 1 and 24 hour upon administration, commented using load-bearing All animals are estimated.
Fig. 7：The anti-TrkA antibody of humanization reverses neurogenic pain.Pass through chronic constriction injury AMB1 mouse ischiums Nerve, inducing chronic neurogenic pain sensation allergy and the abnormality pain sensation, and it is measured respectively as the threshold force (g) needed for pawl retraction With the time delay (sec) from coldplate retraction pawl.Before surgery and after surgery 7 days on the day of before processing starts (0hr), Read off.Then single i.p. injects 1000 μ g/kg Isotype control antibodies (triangles) or 10 (filled circles), and 100 is (solid It is square) and the 1000 anti-TrkA antibody of μ g/kg (solid diamond), handle animal.It is interim when after this 7 days administration, once a day Using Pregabalin (filled inverted triangles) 30mg/kg/10ml p.o. or salt solution (open circles).In 4h, 24h and afterwards every one It is until the 7th day after administration, record reading after administration.On all dates, after Pregabalin or saline administration 1h record to Reading after medicine.
Fig. 8：Right SCG (4 mouse/processing=4 neuromeres/places of newborn AMB1 mouse of the morphometric analysis through processing Reason), the mouse reinstates GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P (mark GBR) (triangle) on the 1st from after being born, Tanezumab (pros) or PBS (circle) is handled 4 weeks.With one-way analysis of variance (one-way ANOVA) and Dunnett is afterwards Examine, compared with PBS processing, carry out statistical analysis：*p<0.05,**p<0.01,***p<0.001.
Fig. 9：Left and right SCG (8-9 mouse/processing=15-18 of adult AMB1 mouse of the morphometric analysis through processing Neuromere/processing), wherein the mouse is with GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P (mark GBR) (triangle), Tanezumab (pros) or PBS (circle) is handled 4 weeks.With one-way analysis of variance (one-way ANOVA) and Dunnett is afterwards Examine, compared with PBS processing, carry out statistical analysis：*p<0.05,**p<0.01,***p<0.001.
Figure 10：The frequency curve of the diameter of all SCG neuron cell bodies of adult AMB1 mouse through processing, wherein institute State mouse GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P (mark GBR) (1904 neurons, circle), PBS (1547 Neuron, square) or tanezumab (1420 neurons, rhombus) processing.Entered using Graphpad Prism and canonical parameter Row branch mailbox (Bining).
Figure 11：From PBS processing adults SCG, from tanezumab processing animal SCG (minimum), From the SCG (maximum) of the animal of GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P (mark GBR) processing representative H＆E dyes Color is cut into slices.Black arrow labeled neurons cell body.
Figure 12：Pass through the sciatic nerve inducing neural pathologic pain of chronic constriction injury AMB1 mouse.Mechanical hyperalgesia Allergy (A) is measured as the threshold force needed for pawl retraction, and the cold abnormality pain sensation (B) is measured as the time delay from cold drawing retraction claw (sec).On the day of processing starts (0h), (- 7d) and reading is taken on the day of before processing starts (0h) within 7 days after surgery before surgery Number.Then, single i.p. pump pickles control (circle) or 0.3 (triangle) and 1mg/kg (inverted triangle) GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P (mark GBR) or 0.3 (pros) and 1mg/kg (rhombus) tanezumab, handle animal.In 4h, the 1st It and afterwards every other day until administration after the 9th day record administration after reading, and upon administration the 14th day record final reading.
Figure 13：Multiple intra-articular injection CFA, inducing chronic Inflammatory joint pain in AMB1 mouse knee joints.D0:First Secondary CFA injects day；D3,D10,D17,D24:The the 3,10,17th and 24 day after first time CFA injections.Arrow is indicated the 0,7,14th With intra-articular injection in 21 days (PBS or CFA).4h, 8h, 24h, 48h, 76h, 96h and 120h:4,8,24,48 after D24 administrations, 76,96 and 120 hours.*:P<0.05, compared to CFA+ excipient groups, one-way analysis of variance.False mould (sham)+excipient group (rhombus), n=6；CFA+ excipient group (pros), n=8；CFA+GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P (three Angle) and CFA+Tanezumab (intersection) group, n=10.
Figure 14：The 2nd, 5,7,14 and 20 day after the fracture, longitudinal in-vivo monitoring mouse.Post-operative values are related to preoperative value.A) Activity Assessment；B) the analysis of the ground reaction force (GRF) of operation limb.As a result it is expressed as average value ± SEM；N=5-7；Asterisk Represent p<0.05, anti-TrkA vs.PBS.
Figure 15：The femur of fracture is constituted with the representative Histological section and poroma (callus) of the fast green dyeing of sarranine-O/ Evaluation.A-C:PBS- processing, D-F:Anti-NGF antibodies, G-I:Anti- TrkA antibody；Engineer's scale=500 μm.J-L:The 7th (J), 14 (K) and 25 (L) day tissue morphology metric evaluation poroma compositions.As a result it is expressed as average value ± SEM；N=5-8；White Post：PBS processing, light grey post：Anti-ngf antibodies；Black gray expandable post：Anti- TrkA antibody.
Figure 16：In the mouse handled with PBS, anti-ngf antibodies or anti-TrkA antibody after the healing stage of 25 days fractured bones The bending rigidity of scab.As a result it is expressed as average value ± SEM；N=6-8.
Figure 17：Stability data of low concentration (10mg/ml) preparation at 5 DEG C.
Figure 18：Stability data of low concentration (10mg/ml) preparation at 25 DEG C.
Figure 19：Stability data of low concentration (10mg/ml) preparation at 40 DEG C.
Figure 20：Stability datas of high concentration (100mg/ml) the preparation A (pH 5.75) at 5 DEG C.
Figure 21：Stability datas of high concentration (100mg/ml) the preparation A (pH 5.75) at 25 DEG C.
Figure 22：Stability datas of high concentration (100mg/ml) the preparation A (pH 5.75) at 40 DEG C.
Figure 23：Stability datas of high concentration (100mg/ml) the preparation B (pH 6) at 5 DEG C.
Figure 24：Stability datas of high concentration (100mg/ml) the preparation B (pH 6) at 25 DEG C.
Figure 25：Stability datas of high concentration (100mg/ml) the preparation B (pH 6) at 40 DEG C.
Figure 26：Stability datas of high concentration (150mg/ml) the preparation A (pH 5.75) at 5 DEG C.
Figure 27：Stability datas of high concentration (150mg/ml) the preparation A (pH 5.75) at 25 DEG C.
Figure 28：Stability datas of high concentration (150mg/ml) the preparation A (pH 5.75) at 40 DEG C.
Figure 29：Stability datas of high concentration (150mg/ml) the preparation B (pH 6) at 5 DEG C.
Figure 30：Stability datas of high concentration (150mg/ml) the preparation B (pH 6) at 25 DEG C.
Figure 31：Stability datas of high concentration (150mg/ml) the preparation B (pH 6) at 40 DEG C.
Detailed description of the invention
This disclosure relates to the anti-TrkA antibody of humanization or its fragment, its preparation and application.
Term " TrkA ", " people TrkA ", " TrkA acceptors " or " people TrkA acceptors " is equally used herein, such as without other Special instruction, it is intended that " people TrkA ".People TrkA includes people TrkA variant, isotype and species homologue herein.Cause This, the antibody of the disclosure can in some cases with the TrkA cross reactions from inhuman other species.In some implementations In scheme, antibody can be specific to one or more people TrkA albumen completely, it is possible to do not show species or other types of Inhuman cross reactivity.
TrkA be also referred to as high-affinity IgE receptors or the type of neurotrophic tyrosine kinase receptor 1 or TRK1- convertibilities protein tyrosine kinase or tropomyosin associated kinase A or tyrosine kinase receptor or EGFR-TK by Body A or Trk-A or gp140trk or p140-TrkA or MTC or TRK.TrkA is receptor tyrosine kinase, by adjusting sympathetic god Propagation, differentiation and survival through member and neural neuron, participate in development and the maturation of maincenter and peripheral neverous system.TrkA is NGF (TrkA major ligand) high-affinity receptor；It can also combine NTF3/ neurotrophic factor -3s and be activated.
The complete amino acid sequence of people TrkA isotypes known to four kinds can see UniProt/Swiss-Prot logins Number P04629 (Consortium TU, (2012) Nucleic Acids Res.40 (D1):D71-D5).These four isotypes lead to Cross variable sheer generation：Isotype TrkA-I is present in most of non-neuronal tissues (UniProt/Swiss-Prot logins Number P04629-2), and isotype TrkA-II is main that (UniProt/Swiss-Prot accession number is expressed in neuronal cell P04629-1), isotype TrkA-III specifically expresses (UniProt/ by multipotent neural stem cell and neural crest ancester cell Swiss-Prot accession number P04629-4).4th isotype is from isotype TrkA-II in 1-71 different and deleting residues of residue 393-398, referred to as isotype 3 (UniProt/Swiss-Prot accession number P04629-3).4th isotype is in residue 1-71 Different from isotype TrkA-II and lack residue 393 to 398, being referred to as isotype 3, (UniProt/Swiss-Prot is logged in Number P04629-3).TrkA-II isotypes are TrkA main known isotypes.Isotype TrkA-I to NTF3 neurotrophies because Son has enhanced reactivity, and isotype TrkA-III has constitutive activity and does not combine NGF.It is preferable to carry out at one In scheme, TrkA isotypes used herein are with SEQ ID NO:72 TrkA-II isotypes and include SEQ ID NO: Its extracellular region of 25 sequences.
Term " anti-TrkA antibody or its fragment " or " the anti-TrkA antibody of humanization or its fragment " include combining herein People TrkA, such as unpack format people TrkA antibody or its fragment, especially with reference to TrkA-II isotypes (SEQ ID NO: 72) antibody or its fragment, more particularly people TrkA extracellular regions (the SEQ ID NO with reference to TrkA-II isotypes:25) unit price The antibody of form or its fragment, and affinity (KD) value be 500nM or smaller, preferably 350nM or smaller, more preferably 150nM or smaller, even more preferably still 100nM or smaller, most preferably 50nM or smaller, especially 30nM or smaller.Generally, The anti-TrkA antibody of humanization or its fragment can suppress TrkA functional activation and/or can block or reduce one or more By the bioactivity of NGF and TrkA zygotic induction.
Herein, " anti-ngf antibodies " are the antibody for referring to combine NGF, preferably people NGF.Generally, anti-ngf antibodies energy Enough suppress TrkA functional activation and/or can block or reduce TrkA one or more bioactivity.Anti-ngf antibodies with NGF (such as hNGF) binding affinity can be 500nM or smaller, preferably 100nM or smaller.Generally, anti-ngf antibodies should Show any one or more in following characteristics：(a) with reference to NGF and NGF bioactivity is suppressed and/or by NGF signal transduction work( The downstream pathway that can be mediated；(b) block or reduce NGF receptor activations (including TrkA Receptor dimerizations and/or autophosphorylation)； (c) increase NGF is removed；(d) suppress (reduction) NGF synthesis, produce or discharge.Anti-ngf antibodies are known in the art, referring to example Such as PCT Publication WO 01/78698, WO 01/64247, the US patent No.s 5,844,092,5,877,016 and 6,153,189； Hongo etc., (2000) Hybridoma, 19:215-227；GenBank accession number U39608, U39609, L17078 or L17077.
Term " the anti-TrkA antibody of humanization or its fragment that can suppress TrkA functional activation " refers to herein, shows The anti-TrkA antibody of any one or more humanization of following characteristics is shown：(a) with reference to TrkA and suppress TrkA bioactivity and/or By the downstream pathway of NGF combinations or the mediation of NTF3/ NT3 signal transductions function；(b) prevent, alleviate or treat and appoint In terms of what pain；(c) block or reduce TrkA activation or Dimerized and/or autophosphorylation；(d) increase TrkA is removed； (e) suppress or reduce TrkA synthesis and/or cell surface expression.
Term " can block or reduce the anti-TrkA antibody of humanization or its piece of TrkA one or more bioactivity Section " herein refer to, can directly or indirectly reduce, suppress, in and/or destroy TrkA bioactivity the anti-TrkA of humanization Antibody.
Term " TrkA bioactivity " or " TrkA bioactivity " are related to herein, but are not limited to, any below Or it is multinomial：NGF or other neurotrophic factors can be combined；Being capable of homodimerization or heterodimer and/or from phosphoric acid Change；The signal transduction path of NGF inductions can be activated；Cell differentiation, propagation, survival, growth, migration can be promoted and other are thin Born of the same parents' physiology changes, including (in the case of neuron, including periphery and axoneuron) neuron morphology changes, cynapse shape Into, synaptic function, neurotransmitter and/or neuropeptide release and damage after regenerate；And can mediated pain and with Bone tumour phase The cancer pain of pass.
Term " IC50 " is being used to describe half maximum suppression concentration (IC50) herein, and it is that compound suppresses biological function The validity of (the anti-TrkA antibody of such as humanization suppresses the TF-1 cells propagation of NGF inductions) is measured.
Term " antibody " used herein includes full length antibody and its any antigen-binding fragment or single-stranded.Antibody, it is special It is not naturally occurring antibody, is that the unit is by four polypeptide chains group with the glycoprotein of the Y shape unit presence of one or more copies Into.Each Y-shaped contains 2 identical heavy chains (H) copies and 2 identical light chains (L) copies, and heavy chain and light chain are relative because of its Molecular weight and gain the name.Every light chain is matched with heavy chain, and every heavy chain is matched with another heavy chain.Covalent interchain disulfide bond and Noncovalent interaction links together these chains.Antibody, particularly naturally occurring antibody, containing variable region, it is two The antigen binding site of individual copy.Y-shaped is cut into 3 independent molecules, 2 by papain (a kind of proteolytic enzyme) It is referred to as " Fab " fragment (Fab=antigen-binding fragments), one is referred to as " Fc " fragment or " Fc areas " (crystallizable of Fc= Section).Fab fragments are made up of complete light chain and Partial heavy.Heavy chain contain 1 variable domains (weight chain variable district or VH) and 3 or 4 constant domains (CH1, CH2, CH3 and CH4, depending on Antibody types or isotype).CH1 and CH2 domains it Between region be referred to as hinge area, it causes there is flexibility between 2 Fab arms of Y shape antibody molecule, it is allowed to which it is opened and closed To be adapted to reference to 2 antigenic determinants for being separated by fixed range.IgA, IgD and IgG heavy chain all have 4 domains, i.e. 1 variable domains (VH) and 3 constant domains (CH1-3).IgE and IgM heavy chain has 1 variable domains and 4 Constant domain (CH1-4).The constant region of antibody can mediate and host tissue or the factor combination, the host tissue or because Attached bag includes the first composition (C1q) of the classical path of various cells (for example, effector cell) and complement system of immune system.Every Light chain is generally connected by 1 covalent disulfide bonds with heavy chain.Every light chain contains 1 variable domains (light variable domains Or VL) and 1 light chain constant domain.Light chain constant domain can be κ light chain constant domains, be referred to herein as IGKC, or can be lambda light chain constant domain, it is referred to herein as IGLC.IGKC is used with C κ or CK equivalences herein, With identical implication.IGLC is used with C λ or CL equivalences herein, with identical implication.Term used herein " IGLC domains " refers to all lambda light chain constant domains, for example, selected from IGLC1, IGLC2, IGLC3, IGLC6 and IGLC7 All lambda light chain constant domains.VH and VL areas can be further subdivided into the hypervariable region for being named as complementary determining region (CDR), its Between spread region that is more conservative, being named as framework region (FR or FW or " non-CDR region ").Each VH and VL includes 3 CDR With 4 FR, arranged in the following order from aminoterminal to c-terminus：FRl、CDRl、FR2、CDR2、FR3、CDR3、FR4.Heavy chain and Contain the binding structural domain with antigen interactions in the variable region of light chain.
Determined by the gene of constant region, antibody is divided into different classes of, also referred to as isotype.People's constant light is divided into κ And λ (C λ) light chain (CK).Heavy chain is divided into mu (μ), delta (δ), gamma (γ), alpha (α) or epsilon (ε), fixed respectively Justice isotype IgM, IgD, IgG, IgA and IgE of antibody.Therefore, " isotype " used herein is it is meant that by constant region Any immunoglobulin class and/or subclass that chemistry and antigen property are defined.Known human immunoglobulin(HIg) isotype is IgGl(IGHG1),IgG2(IGHG2),IgG3(IGHG3),IgG4(IGHG4),IgAl(IGHA1),IgA2(IGHA2),IgM (IGHM), IgD (IGHD), and IgE (IGHE).So-called human immunoglobulin(HIg) vacation-γ IGHGP genes represent other people and exempted from Epidemic disease immunoglobulin constant heavy gene, the gene has been sequenced, but because the transition zone (switch region) of change is without compiling Code protein (Bensmana M et al., (1988) Nucleic Acids Res.16 (7):3108).Although turning with change Area is changed, human immunoglobulin(HIg) vacation-γ IGHGP genes still have the open read frame of all heavy chain constant region (CH1-CH3) and hinge. The protein domain of all open read frames coding of its heavy chain constant domain, can be all with the architectural feature with prediction Human immunoglobulin(HIg) constant domain, aligns well.Other vacation-γ the isotypes be referred to herein as IgGP or IGHGP.It is also reported that other vacation immunoglobulin genes, such as human immunoglobulin heavy chain's constant domainThe false bases of P1 and P2 Because of (IGHEP1 and IGHEP2).IgG classes are most commonly used for therapeutic purposes.In people, the category include subclass IgGl, IgG2, IgG3 and IgG4.In mouse, the category includes subclass IgGl, IgG2a, IgG2b, IgG2c and IgG3.
Term " chimeric antibody " or " inosculating antibody TrkA antibody " include variable region sequences from a kind of species herein Constant-region sequences are derived from the antibody of another species, for example, variable region sequences are from mouse antibodies, constant-region sequences are derived from people The antibody of antibody.
Term " humanized antibody " or " the anti-TrkA antibody of humanization " include, the grafting on people's Frame sequence herein It is derived from the antibody of another mammalian species such as CDR sequences of mouse germline.Can be in people's Frame sequence and in source Extra framework region modification is carried out from the CDR sequence of another mammalian species germline.
Terms used herein " Fab " or " Fab areas " are covered comprising many of VH, CH1, VL and CL immunoglobulin domains Peptide.Fab can refer to this region of separation, or this region in full length antibody or antibody fragment.
Terms used herein " Fc " or " Fc areas " include such polypeptide, and the polypeptide is included except the first constant region is immunized Antibody constant region beyond imrnuglobulin domain.Therefore, Fc refers to IgA, IgD and IgG last 2 constant regions immunoglobulin knot Structure domain and IgE and IgM last 3 constant region immunoglobulin domains and the flexible hinge of these domain N-terminals.It is right In IgA and IgM, Fc can include J chains.Include immunoglobulin domains C γ 2 and C γ 3 and C γ l and C γ for IgG, Fc Hinge between 2.Although the border in Fc areas can change, human IgG heavy chain Fc areas be normally defined including residue C226 or P230 is to its c-terminus, wherein according to EU numbering systems (Edelman GM et al., (1969) PNAS USA 63 (1):78- 85) number.For human IgG1, Fc areas are defined herein as including residue P232 to its c-terminus, wherein numbering system according to EU Unified editing number.Fc can refer to this region of separation, or this region in Fc polypeptides (such as antibody).
The terms " hinge " or " hinge area " or " antibody hinge region " are covered comprising the constant knot of antibody first and second The flexible polypeptide of amino acid between structure domain." hinge area " is herein defined as sequence area of the length for 6-62 amino acid, only exists In IgA, IgD and IgG, the cysteine residues of 2 heavy chains of bridge joint are contained.In structure, IgG CH1 domains terminate at EU Position 220, IgG CH2 domains start from residue EU positions 237.Therefore, for IgG, antibody hinge is defined herein as Including position 221 (D221 in IgG1) to 231 (A231 in IgG1), wherein being numbered according to EU numbering systems.
Term " parental antibody ", " parental immunoglobulin ", " parental generation antibody " or " parental immunoglobulin ", herein It is used interchangeably, including can be then modified to produce the unmodified antibody of variant.The parental antibody can be natural anti- Body or the variant or engineered forms of natural antibody.Parental antibody can refer to antibody composition, comprising parental antibody in itself, Or encode its amino acid sequence." parent murine antibody " or " corresponding parent murine antibody " is here and hereinafter meant that, is modified to produce Change body, antibody or immunoglobulin with reference to people TrkA, mouse antibody MNAC13 disclosed in especially WO00/73344.
Term " variant antibodies " or " antibody variants " include compared with parent because at least one amino acid is repaiied herein Adorn and be different from the antibody sequence of parent antibody sequence.Variant antibodies sequence preferably has extremely with parent antibody sequence herein Few about 80%, most preferably at least about 90%, more preferably at least about 95% amino acid sequence identity.Antibody variants can refer to antibody Itself, the composition comprising the antibody variants or encode its amino acid sequence.
Term " amino acid modified " includes 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, insertion and/or missing in peptide sequence herein." ammonia Base acid substitution " or " substitution " are in this article referred to, and the amino acid of specific location is put by another amino acid in parental polypeptide sequence Change.For example, substitution R94K refers to variant polypeptide, the arginine quilt of weight chain variable framework region variants, wherein position 94 in the present case Lysine is replaced.For precedent, 94K refers to the substitution that position 94 is carried out with lysine.For the purposes herein, multiple substitution allusion quotation Separated by oblique line type.For example, R94K/L78V refers to, substitution R94K and L78V dual variant is included." amino acid insertion " or " insertion " is in this article referred to, and specific location adds amino acid in parental polypeptide sequence.For example, insertion -94 refers in place Put 94 insertions." amino acid deletions " or " missing " are in this article referred to, and remove the amino of specific location in parental polypeptide sequence Acid.For example, R94- refers to the arginine of deletion sites 94.
Herein, term " conservative modification " or " conservative sequence modification " are intended to finger, and this is amino acid modified not significantly Influence or the binding characteristic for changing the antibody comprising the amino acid sequence.Conservative sex modification includes 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, insertion and lacked Lose.Modification can introduce antibody of the present invention by standard technique known in the art, and what such as direct mutagenesis and PCR were mediated lures Become.Conserved amino acid substitution refers to that amino acid residue is by another radical amino acid replacement with similar side chain.With similar side The amino acid residue families of chain have been defined in the art.These families include the amino acid with basic side chain (for example, relying Propylhomoserin, arginine, histidine), the amino acid (for example, aspartic acid, glutamic acid) with acid side-chain, with uncharged Polar side chain amino acid (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, Tryptophan), the amino acid with non-polar sidechain is (for example, alanine, valine, leucine, isoleucine, proline, phenylpropyl alcohol Propylhomoserin, methionine), the amino acid (for example, threonine, valine, isoleucine) with β branched building blocks and with fragrance The amino acid (for example, tyrosine, phenylalanine, tryptophan, histidine) of race's side chain.Therefore, in the CDR region of antibody of the present invention or One or more of framework region amino acid residue could alternatively be other amino acid residues from identical side chain family, and can To test the function that the antibody (variant antibodies) of the change retains.
For all human immunoglobulin heavy chain's constant domains, according to " EU numbering systems " (Edelman GM etc., ibid Quotation) numbering.For people's κ immunoglobulin light chain constants domain (IGKC), according to " EU numbering systems " (Edelman GM Deng ibid quotation) numbering.For people's lambda immunoglobulin light chain constant domain (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7), (Kabat EA etc., (1991) Sequences of proteins of is numbered according to " Kabat numbering systems " immunological interest 5th Edition-US Department of Health and Human Services, NIH publication n ° 91-3242), Dariavach P etc. are described in, (1987) PNAS USA 84 (24):9074-8 and Frangione B etc., (1985) PNAS USA82 (10):3415-9.
Term " variable domains " refers to that mediate antigen combines and limits specific knot of the specific antibodies to specific antigen Structure domain.In natural antibody, antigen binding site is constituted by defining specific two variable domains：One is located at heavy chain In, herein referred to as heavy-chain variable domains (VH) are another to be located in light chain, herein referred to as light variable domains (VL).Under certain situation, specificity can be only present in the only one of two domains, such as from cameloid (camelids) in the single domain antibody of heavy chain antibody.Generally about 110, V areas amino acid length, by be referred to as framework region (FR or " non-CDR region ") 15-30 amino acid relatively constant amino acid sequence chain and be referred to as " hypervariable region " 7-17 amino acid long The shorter region that the extreme of degree is variable is constituted, and wherein framework region is separated by hypervariable region.The varistructure of native heavy and light chain Domain includes 4 FR, and these FR largely take β sheet configurations, is connected by three hypervariable regions for forming ring.Height in every chain Become area to be closely packed together by FR, facilitate the formation of the antigen binding site of antibody together with the hypervariable region from another chain (referring to Kabat EA etc., ibid quotation).The terms " hypervariable region " refer to that the amino acid for being responsible for the antibody of antigen binding is residual Base.Hypervariable region generally comprises the amino acid residue from " complementary determining region " or " CDR ", and the latter has maximum sequence variability And/or participate in antigen recognizing.For all variable domains, it is numbered according to Kabat (Kabat EA etc., ibid quotation).
There are a variety of CDR to be defined on to use, and be included herein.Kabat defines the variability based on sequence, is most often to make CDR definition (Kabat EA etc., ibid quotation).Chothia then refers to position (the Chothia＆Lesk J of structure ring (1987)Mol Biol 196:901-917).AbM define be Kabat and Chothia define it is compromise, for Oxford Molecular AbM antibody moulds build software (Martin ACR etc., (1989) PNAS USA 86:9268–9272；Martin ACR etc., (1991) Methods Enzymol.203:121–153；Pedersen JT etc., (1992) Immunomethods 1: 126–136；Rees AR etc., (1996) In Sternberg M.J.E. (ed.), Protein Structure Prediction.Oxford University Press,Oxford,141–172).Recently contact definition is introduced (MacCallum RM etc., (1996) J Mol Biol 262:732-745), it is based on to available compound in albumen database The analysis of thing structure.(international ImMunoGeneTics information systems) CDR define (http:// Www.imgt.org) based on all immunoglobulins to all species and φt cell receptor V-REGIONs IMGT numbering (International ImMunoGeneTics information systems；Lefranc MP etc., (1999) Nucleic Acids Res.27(1):209-12；Ruiz M etc., (2000) Nucleic Acids Res.28 (1):219-21；Lefranc MP (2001)Nucleic Acids Res.29(1):207-9；Lefranc MP(2003)Nucleic Acids Res.31(1): 307-10；Lefranc MP etc., (2005) Dev.Comp.Immunol.29 (3):185-203；Kaas Q etc., (2007) Briefings in Functional Genomics&Proteomics,6(4):253-64)。
Preferably definition is performed as follows (according to Kabat EA etc. in all complementary determining regions (CDR) referred in the present invention People is numbered, ibid quotation)：LCDR1:24-34；LCDR2:50-56；LCDR3:89-98；HCDR1:26-35；HCDR2: 50-65；HCDR3:95-102." the non-CDR region " of variable domains is referred to as framework region (FR)." the non-CDR in VL areas herein Area " includes amino acid sequence：1-23 (FR l), 35-49 (FR2), 57-88 (FR3), and 99-107 (FR4).VH areas herein " non-CDR region " include amino acid sequence：1-25 (FR l), 36-49 (FR2), 66-94 (FR3), and 103-113 (FR4).
Term " full length antibody " used herein includes, and constitutes the structure of the native biological form of antibody, including can Become area and constant region.For example, in most of mammals (including people and mouse), the full length antibody of IgG classes is the tetramer, by 2 pairs of identical composition, two immunoglobulin chains of each pair have 1 light chain and 1 heavy chain for every a pair, and every light chain is comprising exempting from Epidemic disease imrnuglobulin domain VL and CL, every heavy chain include immunoglobulin domains VH, CH1 (C γ 1), CH2 (C γ 2) and CH3 (Cγ3).In some mammals, such as camel and alpaca, IgG antibody only can be made up of 2 heavy chains, every heavy chain bag The variable domains of Han Yu Fc areas connection.
Antibody fragment in this article refers to antigen-binding fragment, includes, but not limited to (i) by VL, VH, CL and CH1 structure The Fab fragments of domain composition, including Fab ' and Fab '-SH；(ii) the Fd fragments being made up of VH and CH1 domains；(iii) by single The Fv fragments of the VL and VH domains composition of antibody；(iv) dAb fragments (the Ward et al being made up of single variable domains (1989)Nature 341:544-546)；(v) fragments of F (ab') 2 --- include the bivalent fragment of 2 connected Fab fragments； (vi) Single Chain Fv Molecule A (scFv), wherein VH domains are connected with VL domains by peptide linker, and the joint allows 2 structures Associate to form antigen binding site (Bird et al (1988) Science 242 in domain:423-426；Huston et al(1988) PNAS USA 85:5879-5883)；(vii) Bispecific single chain Fv dimers (PCT/US92/09965)；(viii) " disome Antibody (diabody) " or " three body antibody (triabodies) " --- the multivalence or polyspecific piece built by Gene Fusion Section (Tomlinson I et al., (2000) Methods Enzymol 326:461-479；WO94/13804；Holliger et al.,(1993)PNAS USA 90:6444-6448)；(ix) and identical or different antibody genetic fusion scFv (Coloma&Morrison(1997)Nature Biotech 15:159-163)。
Term " effector function " includes raw caused by antibody Fc district and the interaction of Fc acceptors or part herein Thing chemical event.Effector function includes the effector function that Fc γ R are mediated, and such as ADCC be (antibody dependent cellular mediation Cytotoxicity) and ADCP (phagocytosis of antibody dependent cellular mediation), and complement-mediated effector function such as CDC (cytotoxicity of complement-mediated).The effector function of antibody can change in the following way：Change, that is, strengthen or reduce, It is preferred that the affinity of enhancing antibody pairing effect molecule such as Fc acceptors or complement component.Binding affinity can generally pass through modification Effector molecule binding site changes, and desirably positioning purpose site and modifies this in an appropriate manner in this case At least a portion in site.Also, it is contemplated that the effector molecule binding site changed on antibody need not significantly change overall knot Affinity is closed, but the geometry of interaction can be changed, so that effect mechanism is invalid in non-effective combination.May be used also To consider, it can also be combined by modifying indirect participation effector molecule but participate in the site that effector function is exercised, to change Change effect subfunction.By changing the effector function of antibody, many aspects of immune response can be controlled, for example, enhancing or Suppress the various reactions of immune system, obtain possible diagnosis and treat effective effect.
Herein, term " subject " includes anyone or non-human animal.Term " non-human animal " includes all vertebras Animal, such as mammal and nonmammalian, such as primate, sheep, dog, cat, horse, cow, chicken, amphibian, Reptile etc..It is preferred that, subject is people.
Antibody of the present invention
First aspect present invention is provided, the anti-TrkA antibody of humanization or its fragment, and it is included：
A) include and be selected from SEQ ID NOs:The heavy-chain variable domains of 1-5 sequence；With
B) include and be selected from SEQ ID NOs:The light variable domains of 6-13 sequence；
The wherein CDR2 of heavy-chain variable domains includes at least one 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and/or wherein weight chain variable structure The non-CDR region in domain is including 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor selected from the amino acid position by 37,42 and 89 groups constituted, wherein each group membership Amino acid position provided using numbering system shown in Kabat.
In some embodiments, the CDR2 of heavy-chain variable domains replaces comprising at least one conserved amino acid.
In some embodiments, the non-CDR region of heavy-chain variable domains is selected from the ammonia by 37,42 and 89 groups constituted Base acid position replaces comprising conserved amino acid.
In some embodiments, the CDR2 of heavy-chain variable domains includes sequence SEQ ID NO:15.
In some embodiments, heavy-chain variable domains, which are included, is selected from SEQ ID NOs:1,3 and 5 sequence, light chain can Structure changes domain, which is included, is selected from SEQ ID NOs:6 and 8 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment include heavy-chain variable domains and light chain variable The combination of domain, the combination, which is included, is selected from SEQ ID NO:1 and SEQ ID NO:6、SEQ ID NO:3 and SEQ ID NO: 6、SEQ ID NO:3 and SEQ ID NO:8、SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:5 and SEQ ID NO:8 sequence；It is preferred that SEQ ID NO:5 and SEQ ID NO:6 sequence or SEQ ID NO:5 and SEQ ID NO:8 sequence Row；More preferably SEQ ID NO:5 and SEQ ID NO:6 sequence.
In some embodiments, the disclosure is provided the anti-TrkA antibody of humanization or the heavy-chain variable domains of its fragment Not comprising SEQ ID NO:71 sequence.
In some embodiments, the disclosure is provided the anti-TrkA antibody of humanization or the heavy-chain variable domains of its fragment Lack the serine of position 87, wherein the amino acid position of each group membership is provided using numbering system shown in Kabat.
In some embodiments, the disclosure is provided the anti-TrkA antibody of humanization or the heavy-chain variable domains of its fragment Threonine is included in position 87, wherein the amino acid position of each group membership is provided using numbering system shown in Kabat.
In some embodiments, the CDR2 of heavy-chain variable domains 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, which is included in, is selected from by 50,60 Hes The groups of 62 compositions, the group for being preferably selected from being made up of 60 and 62, amino acid position 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, wherein each group membership Amino acid position is provided using numbering system shown in Kabat.
In some embodiments, the CDR2 of heavy-chain variable domains 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor do not include be selected from by Y50A, The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the group of P60A and T62S compositions, wherein the amino acid position of each group membership is numbered using shown in Kabat System is provided.
In some embodiments, if the anti-TrkA antibody of humanization or fragment are in the non-CDR region of heavy-chain variable domains 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor be A49S, then the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the anti-TrkA antibody of humanization or fragment in heavy-chain variable domains CDR2 It is not Y50A.
In some embodiments, the ammonia of the anti-TrkA antibody of humanization or fragment in the non-CDR region of heavy-chain variable domains Base acid substitution is not A49S, and/or the amino acid of the anti-TrkA antibody of humanization or fragment in heavy-chain variable domains CDR2 takes Generation is not Y50A.
In some embodiments, the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of antibody or the non-CDR region of the heavy-chain variable domains of its fragment is included Selected from the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor by V37A, G42E and V89L group constituted, preferably V37A, wherein the amino acid position of each group membership Provided using numbering system shown in Kabat.
In some embodiments, antibody, which is included, contains SEQ ID NO:The heavy-chain variable domains of 3 sequences, wherein heavy chain The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the non-CDR region of variable domains is included selected from the group being made up of V37A, T40A, G42E, R44G, A49S and V89L 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, V37A, T40A, G42E, R44G and V89L are preferably selected from, wherein the amino acid position of each group membership is used Numbering system is provided shown in Kabat.
In some embodiments, combination of the antibody comprising heavy-chain variable domains and light variable domains, described group Close and include SEQ ID NO:3 and SEQ ID NO:6 sequences include SEQ ID NO:3 and SEQ ID NO:8 sequences, wherein heavy chain The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the non-CDR region of variable domains is included selected from the group being made up of V37A, T40A, G42E, R44G, A49S and V89L 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, V37A, T40A, G42E, R44G and V89L are preferably selected from, wherein the amino acid position of each group membership is used Numbering system is provided shown in Kabat.
In some embodiments, the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the non-CDR region of heavy-chain variable domains include be selected from by K3Q, V37A, The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the group of G42E, A49S, V89L and R94K composition, is preferably selected from K3Q, V37A, G42E, V89L and R94K, more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor K3Q and V37A are preferably comprised, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor V37A is most preferably comprised, wherein the amino acid position of each group membership Put and provided using numbering system shown in Kabat.Equivalent most preferably such embodiment, wherein heavy-chain variable domains The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of non-CDR region, which is included, to be selected from by V37A and K3Q, the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the group of V37A compositions, wherein each composition The amino acid position of member is provided using numbering system shown in Kabat.
In some embodiments, antibody, which is included, contains SEQ ID NO:The heavy-chain variable domains of 5 sequences, wherein heavy chain The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the non-CDR region of variable domains is included selected from the group being made up of K3Q, V37A, G42E, A49S, V89L and R94K 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, be preferably selected from K3Q, V37A, G42E, V89L and R94K, more preferably comprising 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor K3Q and V37A, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor V37A is most preferably comprised, wherein the amino acid position of each group membership is given using numbering system shown in Kabat Go out.Equivalent the most preferred embodiment is that wherein antibody, which is included, contains SEQ ID NO:The heavy-chain variable domains of 5 sequences, its The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the middle non-CDR region of heavy-chain variable domains, which is included, to be selected from by V37A and K3Q, and the amino acid of the group of V37A compositions takes Generation, wherein the amino acid position of each group membership is provided using numbering system shown in Kabat.
Another further aspect, the present invention provides the anti-TrkA antibody of humanization or its fragment, and it is included：
A) include and be selected from SEQ ID NOs:The heavy-chain variable domains of 31-49 sequence, and
B) include and be selected from SEQ ID NOs:The light variable domains of 6-13 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) include and be selected from SEQ ID NOs:The heavy-chain variable domains of 32,36,39,43,48 and 49 sequence, and
B) include and be selected from SEQ ID NOs:6-13 or selected from SEQ ID NOs:The light variable domains of 6 and 8 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) include and be selected from SEQ ID NOs:The heavy-chain variable domains of 32,36,48 and 49 sequence, and
B) sequence SEQ ID NO are included:6 or SEQ ID NO:8 light variable domains.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) include and be selected from SEQ ID NOs:The heavy-chain variable domains of 32 and 36 sequence, and
B) SEQ ID NO are included:The light variable domains of 6 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) SEQ ID NO are included:The heavy-chain variable domains of 36 sequence, and
B) SEQ ID NO are included:The light variable domains of 6 sequence.
In some embodiments, group of the anti-TrkA antibody of humanization comprising heavy-chain variable domains and light variable domains Close, the combination is selected from combined sequence SEQ ID NO:32 and SEQ ID NO:6、SEQ ID NO:32 and SEQ ID NO:8、 SEQ ID NO:36 and SEQ ID NO:6、SEQ ID NO:48 and SEQ ID NO:6、SEQ ID NO:49 and SEQ ID NO: 6 and SEQ ID NO:49 and SEQ ID NO:8, it is preferably selected from combined sequence SEQ ID NO:32 and SEQ ID NO:6、SEQ ID NO:32 and SEQ ID NO:8、SEQ ID NO:36 and SEQ ID NO:6、SEQ ID NO:49 and SEQ ID NO:6th, with And SEQ ID NO:49 and SEQ ID NO:8；It is most preferably selected from combined sequence SEQ ID NO:36 and SEQ ID NO：6.
In some embodiments, the anti-TrkA antibody of humanization or its fragment also include heavy chain and/or constant region of light chain, preferably Heavy chain and/or constant region of light chain and hinge area.It is preferred that, heavy chain constant region is people source, it is possible to be such as human IgG1 (IGHG1),IgG2(IGHG2),IgG3(IGHG3),IgG4(IGHG4),IgA1(IGHA1),IgA2(IGHA2),IgM (IGHM), IgD (IGHD), or IgE (IGHE) isotype.It is further preferred that heavy chain constant region is people IGHG1 isotypes or people IGHG4 Isotype.It is preferred that, constant region of light chain is people source, it is possible to be people κ (CK) or people λ (C λ) light chain constant domain, preferably Human kappa light chain constant domain.
In some embodiments, the anti-TrkA antibody of humanization or its fragment also include heavy chain and/or constant region of light chain and hinge Sequence, wherein heavy chain constant region and hinge area are people IGHG1 isotypes or people's IGHG4 isotypes.
In some embodiments, the anti-TrkA antibody of humanization or its fragment also include heavy chain and/or constant region of light chain and hinge Sequence, wherein heavy chain constant region and hinge area are people's IGHG4 isotypes, and wherein hinge area includes 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor S228P, Wherein amino acid position is provided using EU numbering systems.
Another further aspect, the present invention provides the anti-TrkA antibody of humanization or its fragment, and it is included：
A) include and be selected from SEQ ID NOs:The heavy chain of 50-70 sequence, and
B) include and be selected from SEQ ID NOs:The light chain of 29 and 30 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) include and be selected from SEQ ID NOs:The heavy chain of 51,52,56,57,60,64,69 and 70 sequence, and
B) include and be selected from SEQ ID NOs:29 and 30, preferably SEQ ID NO:The light chain of 29 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) include and be selected from SEQ ID NOs:The heavy chain of 51,52,56,57,69 and 70 sequence, and
B) include and be selected from SEQ ID NOs:29 and 30, preferably SEQ ID NO:The light chain of 29 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) include and be selected from SEQ ID NOs:The heavy chain of 51,52,56,57 and 70 sequence, and
B) include and be selected from SEQ ID NOs:29 and 30, preferably SEQ ID NO:The light chain of 29 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are included：
A) include and be selected from SEQ ID NOs:The heavy chain of 51,52,56 and 57 sequence, and
B) include and be selected from SEQ ID NOs:29 and 30, preferably SEQ ID NO:The light chain of 29 sequence.
In a preferred embodiment, the anti-TrkA antibody of humanization or its fragment are included：
A) SEQ ID NO are included:The heavy chain of 57 sequence, and
B) SEQ ID NO are included:The light chain of 29 sequence.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are full length antibodies.
In some embodiments, the anti-TrkA antibody of humanization or its fragment are antibody fragments, selected from Fab, Fab ', Fab '- SH, Fd, Fv, dAb, F (ab') 2, scFv, Bispecific single chain Fv dimers, diabody, three body antibody and with it is identical or not The scFv of synantibody genetic fusion；It is preferred that scFv or Fab；More preferably scFv dimers or diabody or F (ab') 2.
In some embodiments, humanization TrkA antibody or its fragment include variant Fc areas, and it is relative to parental antibody Fc Area is amino acid modified comprising at least one, wherein the antibody containing variant Fc areas shows the effector work(different from parental antibody Energy.
The amino acid modified one or more functional characters for typically changing antibody in Fc areas, such as serum half-life, The cytotoxicity of complement fixation, the effector function related to Fc acceptors or ligand binding, and/or antigen dependent cell.Under The Fc areas modification that face is listed is provided according to the EU numberings of Fc areas residue.In one embodiment, modify CH1 hinge area to change Become the quantity for for example increasing or decreasing cysteine residues in hinge area.This method is further described in the US patent No.s 5,677, 425 (Bodmer etc.).Change the quantity of cysteine residues in CH1 hinge areas can for example promote light chain and heavy chain assembling or Increase or decrease the stability of antibody.In another embodiment, mutant antibodies Fc hinge areas are partly declined with the biology for reducing antibody Phase.More particularly, one or more amino acid mutations are introduced into the CH2-CH3 domain interface regions of Fc- hinge fragments, so that SpA relative to natural Fc- hinge domains is combined, and there is the antibody staphylococcal protein A (SpA) weakened to combine.The party Method is described in greater detail in U.S. Patent number 6,165,745 (Ward etc.).In another embodiment, modified antibodies are given birth to increasing its Thing half-life period.Various schemes are all possible.For example, one or more of following mutation can be introduced：T252L、T254S And T256F, such as U.S. Patent number 6, described in 277,375 (Ward).Or, can be in CH1 in order to increase biological half life Or change antibody in CL areas, to include the redemption receptor binding domain for two rings for being derived from IgG Fc areas CH2 domains, referring to U.S. State's patent No. 5,869,046 and 6,121,022 (Presta etc.).In still another embodiment, by the way that at least one amino acid is residual Base is substituted by different amino acid residues, changes Fc areas, so as to change (one or more) effector function of antibody.For example, Can be by one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors selected from amino acid residue 234,235,236,237,297,318,320 and 322 Different amino acid residue, so that antibody has the effector ligand affinity changed but retains the antigen binding energy of parental antibody Power.The effector part of affinity to be changed can be, for example, the C1 compositions of Fc acceptors or complement.This method is described in Winter et al. U.S. Patent number 5,624,821 and 5,648,260.In another example, amino acid residue will can be selected from 329,331 and 332 one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors are different aminoacids residue, so that there is antibody the C1q changed to combine And/or reduction or elimination complement-dependent cytotoxicity (CDC).This method is described in Idusogie et al. United States Patent (USP) Numbers 6,194,551.In another example, thus it is possible to vary one in CH2 domain N-terminals region in amino acid position 231 to 238 Or more amino acid, thus change the ability of antibody complement-fixing.This method is described in Bodmer et al. PCT Publication WO 94/29351.In another example, it can modify Fc areas to increase by modifying one or more amino acid positioned at following position Affinity of the ability and/or increase antibody of antibody mediates antibody dependent cellular cytotoxicity (ADCC) to Fc γ acceptors：238, 239,248,249,252,254,255,256,258,265,267,268,269,270,272,276,278,280,283,285, 286,289,290,292,293,294,295,296,298,301,303,305,307,309,312,315,320,322,324, 326,327,329,330,331,333,334,335,337,338,340,360,373,376,378,382,388,389,398, 414,416,419,430,434,435,437,438 or 439.This method is described in Presta PCT Publication WO 00/42072. Furthermore, it is possible to chemical modification antibody of the present invention (for example, one or more chemical moieties can be attached on antibody), or Antibody of the present invention can be modified to change its glycosylation.
The property of antibody of the present invention
In some embodiments, the anti-TrkA antibody of humanization or its fragment can suppress TrkA functional activation.
In some embodiments, one or more biology that the anti-TrkA antibody of humanization can block or reduce TrkA are lived Property.
In some embodiments, the anti-TrkA antibody of humanization or its fragment combination people TrkA, and affinity (KD) value is 500nM or smaller, preferably 350nM or smaller, more preferably 150nM or smaller, even more preferably 100nM or smaller, most preferably 50nM or smaller, especially 30nM or smaller, the affine force value can be for example using the instrument (GE of BIAcore 2000 Healthcare Europe GmbH, Glattbrugg, Switzerland) or equivalent instrument known in the art, by that will resist Body is captured on Instrument sensor chip, uses restructuring unit price people TrkA extracellular domains (SEQ ID NO:25) as analyte, lead to Cross surface plasma body resonant vibration (SPR) measurement.Herein, the statement " unit price " related to the affinity measurement using TrkA acceptors Refer to such people TrkA receptor domains (such as people TrkA extracellular domains), (be different from for example by the domain amino acid end melting It is bonded to the artificial Dimerized or polymer occurred during Fc portion of immunoglobulin) artificial dimer does not occur for the domain Change or polymer, or (being different from the native dimeric body occurred when such as domain is combined with its native ligand NGF) is described Native dimeric body does not occur for domain.
It is known in the art to the standard assays of such as people TrkA binding affinity to assess antibody, including for example ELISAs, BIAcore, western blot, RIA, flow cytometry.Suitable determination test is retouched in detail in embodiment State.Binding kineticses (such as binding affinity such as K of antibodyD) standard assays known in the art, example can also be passed through Such as by Scatchard orNetwork analysis is assessed.RA KiCan be by known in the art Standard Competition determination test assess.Can be in TF-1 cell proliferation tests, the anti-TrkA antibody of evaluation engineeringization suppresses The ability of TrkA functional activations.Half maximum suppression concentration (IC50), suppresses the validity of biological function as compound Measure, can be used for selecting preferred antibody.
In some embodiments, the anti-TrkA antibody of humanization or its fragment in TF-1 cell proliferation tests with corresponding parent This mouse antibody, which is compared, has at least suitable or lower IC50, wherein the IC50 can such as usage factor dependence people it is red white Blood disease cell line TF-1, is measured by the ability of the cell propagation of antibody blocking cell surface TrkA/ β-NGF mediations (Kitamura T et al.,(1989)J Cellular Physiology 140(2):323-34).Preferably, humanization resists TrkA antibody or its fragment have 1 μ g/ml or lower, more preferably 0.75 μ g/ml in TF-1 cell proliferation tests, even more excellent Select 0.5 μ g/ml or lower, especially most preferably 0.3 μ g/ml or lower, 0.1 μ g/ml or lower IC50.
In some embodiments, the anti-TrkA antibody of humanization or its fragment have more than 65 DEG C, preferably greater than 70 DEG C of FAB Fragment thermostability temperature.For the analysis of FAB fragment heat endurances, differential scanning calorimetry can be used, wherein identifying The midpoint melting temperature of FAB fragments in total length IgG.Such calorimetric measurement is well known by persons skilled in the art, Ke Yigen According to such as Garber＆Demarest (2007) BBRC 355:751-7 is implemented.Surprisingly, it has been found that, the present inventor source The FAB fragments thermostability temperature for changing antibody is suitable with the FAB fragment thermostability temperatures of parent murine antibody, while having quite Affinity (being measured by SPR) and improve efficiency (being measured by TF-1 cell proliferation tests).Therefore, the disclosure is also provided The anti-TrkA antibody of humanization, it has the FAB fragment thermostability temperature suitable with parent murine antibody and suitable people TrkA Affinity and improved inhibition activity.
" having suitable FAB fragments thermostability temperature with parent murine antibody ", refers to, people when using in this context The FAB fragment thermostability temperatures of the anti-TrkA antibody of sourceization or the FAB fragments thermostability temperature of its fragment in parent murine antibody ± 20% in the range of, preferably in the range of ± 15%, more preferably in the range of ± 10%, even more preferably in ± 5% scope It is interior.Preferably, FAB fragment heat endurance of the FAB fragments thermostability temperature of humanized antibody of the present invention than parent murine antibody Temperature is low to be no more than 15%.
" suitable people TrkA affinity ", refers to when using in this context, the anti-TrkA antibody of humanization or its fragment Affinity in the range of ± the 20% of parent murine antibody, preferably in the range of ± 15%, more preferably in the range of ± 10%.It is excellent Selection of land, the K of humanized antibody of the present inventionDThan the K of parent murine antibodyDIt is low by least 5%, preferably at least 10%.
The disclosure, which is also provided, can be used for the anti-TrkA antibody of humanization or its fragment for the treatment of pain.
The effect (see embodiment 2) of the anti-TrkA antibody of humanization is tested in acute inflammation hyperalgia mouse, wherein By inducing the acute inflammation hyperalgia to rear solid end subplantar injection complete Freund's adjuvant (CFA).With 0.01mg/kg or more Dosage causes significant hyperalgia using the anti-TrkA antibody of humanization and reversed, and is observed when this is with using NSAID Indomethacins That arrives is similar.
The effect (see embodiment 3) of the anti-TrkA antibody of humanization is tested in chronic inflammatory hyperalgia mouse, wherein Pass through the intra-articular injection CFA inductions chronic inflammatory hyperalgia into knee joint.With 0.01mg/kg or more single dose Hyperalgesic notable reverse is generated using the anti-TrkA antibody of humanization, this comes with multiple dosing COX-2 selective Ns SAID plugs That is observed during former times cloth is suitable.
The effect of the anti-TrkA antibody of humanization is tested in chronic osteoarthritic hyperalgia mouse (referring to embodiment 4), wherein inducing the chronic osteoarthritic hyperalgia into knee joint by intra-articular injection sodium iodoacetate (MIA).With 0.01mg/kg or more single dose apply the anti-TrkA antibody of humanization produce it is hyperalgesic it is notable reverse, this with by many It is suitable observed by secondary administration opioid drug C16H25NO2 and Pregabalin.
The anti-TrkA antibody of humanization is tested in the neurogenic pain mouse that sciatic nerve spinal cord in chronic compression is induced Effect (CCI models；Referring to embodiment 5).The anti-TrkA antibody of humanization is applied with 0.01mg/kg or more single dose and produces machine Tool hyperalgia and the notable reverse of cold allodynia, the reverse and multiple dosing in the maximum dose level 1mg/kg of test It is suitable observed by Pregabalin.
Therefore, a preferred embodiment of the invention provides the anti-TrkA antibody of humanization and is used to treat with acute inflammation The patient of property pain, chronic inflam-matory pain, osteo-arthritic pain and/or neurogenic pain.
Nucleic acid, carrier and host cell
The disclosure also provides the seperated nuclear acid for encoding anti-TrkA antibody and its fragment, carrier and comprising the nucleic acid or carrier Host cell.Nucleic acid may reside in full cell, cell lysate or can be partially purified or substantially pure form. When passing through standard technique (including alkali/SDS processing, CsCl bands, column chromatography, agarose gel electrophoresis and other are known in the art Technology) nucleic acid is purified from other cell components or other pollutants (such as other nucleus or protein) Afterwards, nucleic acid is " separation " or " turning into substantially pure ", and the technology is compiled (1987) see, for example, Ausubel F et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.The nucleic acid of the present invention can be such as DNA or RNA, and can contain or can not contain Intron sequences.In a preferred embodiment, nucleic acid is cDNA molecules.
The nucleic acid of the present invention can use standard molecular biological technique to obtain, for example can by standard PCR amplification or CDNA clone technology obtains encoding antibody light and heavy chain or encodes the cDNA of VH and VL fragments.For from immunoglobulin gene The antibody that library (for example, using display technique of bacteriophage) is obtained, can reclaim one or more cores of encoding antibody from library Acid.It is well known in the art by the method that exogenous nucleic acid introduces host cell, and will changes with host cell used.Skill Art includes but is not limited to the transfection of glucan mediation, and calcium phosphate precipitation, chlorination Calcium treatment, the transfection of polyethyleneimine gathers The transfection of solidifying amine (polybrene) mediation, protoplast fusion, electroporation, virus or phage-infect, liposomal encapsulated multinuclear Thuja acid, and DNA microinjections are directly entered into nucleus.In the case of mammalian cell, transfection can be it is instantaneous or Stable.
In some embodiments, the seperated nuclear acid for encoding anti-TrkA antibody and its fragment is included selected from SEQ ID NO： 73-116 nucleotide sequence, is typically selected from SEQ ID NO：113,114,115 and 116 coding light chain variable region or light chain Nucleic acid molecules and/or selected from SEQ ID NO：73-112 encoding heavy chain variable region or the nucleic acid molecules of heavy chain.
Currently preferred nucleic acid molecules are to be selected from SEQ ID NO：The nucleic acid of 113 and 114 coding light chain variable region point Son and/or selected from SEQ ID NOs:The nucleic acid molecules of 74,78,81,85,90 and 91 encoding heavy chain variable region.More preferably Selected from SEQ ID NO：Nucleic acid molecules of 74 and 78 encoding heavy chain variable region and/or selected from SEQ ID NO：113 and 114 volume The nucleic acid molecules of code light chain variable district.Most preferably include SEQ ID NO：The encoding heavy chain variable region of 74 or 78 nucleotide sequences Nucleic acid molecules and/or include SEQ ID NO：The nucleic acid molecules of the coding light chain variable region of 113 nucleotide sequences.
Other preferred nucleic acid molecules of the present invention are to be selected from SEQ ID NO：The nucleic acid point of 115 and 116 coding light chain Son and/or selected from SEQ ID NO：The nucleic acid molecules of 93,94,98,99,102,106,111 and 112 encoding heavy chain.More preferably , include SEQ ID NO：Nucleic acid molecules of the encoding heavy chain of 93,94,98 and 99 nucleotide sequences and/or selected from SEQ ID NO：The nucleic acid molecules of 115 and 116 coding light chain.Most preferably, comprising SEQ ID NO：93,94,98 and 99 nucleotide sequences Encoding heavy chain nucleic acid molecules and/or include SEQ ID NO：The nucleic acid molecules of the coding light chain of 115 nucleotide sequences.
Once obtaining the DNA fragmentation of coding VH and VL sections, then can further it be manipulated by standard recombinant dna technology These DNA fragmentations, for example, change into full length antibody chain gene, or change into the piece corresponding with above-mentioned fragment by variable region gene Section gene, as such as Fab fragment genes, or changes into scFv genes.In these operations, coding VL or VH DNA fragmentation can It is operatively coupled on another DNA fragmentation for encoding another protein (such as antibody constant region or flexible joint).Herein The term " being operably connected " used is intended to indicate that, connects two DNA fragmentations so that by the amino of two DNA fragmentation codings Acid sequence keeps meeting frame.Pass through the another of separation DNA and the encoding heavy chain constant (CH1, CH2 and CH3) by VH is encoded DNA molecular is operably connected, and the separation DNA for encoding VH areas can be converted into total length heavy chain gene.People's heavy chain constant region base The sequence of cause is (see, for example, Kabat, EA et al., ibid quotation) known in the art, and can be with by standard PCR amplification Obtain the DNA fragmentation for including these regions.Heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant regions, but most preferably IgG4 constant regions, preferably people IGHG4 constant regions, wherein hinge area include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor S228P.For Fab fragment heavy chain genes, coding VH DNA can be operatively attached to only encoding heavy chain CH1 constant regions Another DNA molecular., can be with by the way that the DNA for encoding VL is operably connected with coding constant region of light chain CL another DNA molecular The separation DNA for encoding VL areas is converted into full-length light chains gene (and Fab light chain genes).The sequence of people's light chain constant region gene It is (see, for example, Kabat EA et al., ibid quotation) known in the art, and can be included by standard PCR amplification The DNA fragmentation in these regions.In preferred embodiments, constant region of light chain can be κ or λ constant regions, preferably κ constant regions. In order to produce scFv genes, coding VH- and VL DNA fragmentation is operably connected to coding flexible joint, for example, encodes amino Another fragment of acid sequence (Gly4-Ser) 3, so that VH and VL sequences can be expressed as continuous single chain protein, wherein VL and VH Area is connected by flexible joint (see, for example, Bird et al., ibid；Huston et al., ibid；McCafferty etc., (1990) Nature 348：552-554).The various technologies of the antibody fragment for producing antibody are developed.Traditionally, these pieces Section be by derived from the proteolytic digestion of complete antibody (see, for example, Morimoto etc., (1992) J.Biochem.Biophysical Methods, 24：107-117 and Brennan etc., (1985) Science, 229：81).So And, these fragments can directly be produced by recombinant host cell now.For example, can be separated from above-mentioned antibody phage libraries Antibody fragment.Or, Fab'-SH fragments and chemical coupling can be directly reclaimed from Escherichia coli to form the fragments of F (ab') 2 (Carter etc., (1992) Bio/Technology, 10：163-167)., can be from recombinant host cell according to another method The fragments of F (ab') 2 are directly separated in culture.Produce the other technologies of antibody fragment for those skilled in the art will be it is aobvious and It is clear to.In other embodiments, selected antibody is Single-Chain Fv Fragment of Murine (scFv), see, for example, WO93/16185；It is beautiful State's patent No. 5,571,894 and 5,587,458.Antibody fragment can also be for example " described in U.S. Patent number 5,641,870 " " linear antibodies ".
The nucleic acid of coding antibody of the present invention can be incorporated in carrier, preferred expression carrier with marking protein.A variety of expression Carrier can be used for protein expression.Expression vector can include the outer carrier of self-replacation sex chromosome or be incorporated into host genome In carrier.Construction of expression vector is with compatible with host cell species.Therefore, the carrier used in the present invention, preferred expression Carrier includes but is not limited to can be in mammalian cell, bacterium, insect cell, marking protein in yeast and vitro system Carrier.As known in the art, various expression vectors can be obtained by commercial sources or other modes, and available for the present invention Middle expression antibody.
Expression vector is generally comprised and controlled or regulatory sequence, selected marker, any fusion partner and/or other yuan The protein that part is operably connected." it is operably connected " and refers to herein, nucleic acid is placed in the work(with another nucleotide sequence In energy relation.Term " regulatory sequence " is intended to include, and can control promoter, the enhancer of transcription or the translation of antibody chain gene With other expression control elements (such as polyadenylation signal).Such regulatory sequence is described in such as Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990) in).Generally, these expression vectors include the transcription and translation regulation core being operably connected with the nucleic acid of encoding antibody Acid, and it is generally applicable to the host cell of marking protein.Generally, transcription and translation regulatory sequence can include starting Subsequence, ribosome bind site, transcription initiation and terminator sequence, translation initiation and terminator sequence, and enhancer or activation Agent sequence.As in this area it is also known that, expression vector usually contains Select gene or mark, with allow selection containing expression carry The host cell of the conversion of body.Select gene is it is known in the art that and will change with host cell used.For example, Generally selecting property marker gene can assign medicine to the host cell for having imported carrier, and (such as G418, hygromycin or ammonia first are talked endlessly Purine) resistance.It is preferred that selectable marker gene include dihyrofolate reductase (DHFR) gene (with methotrexate select/expand one Rising is used for dhfr- host cells) and neo genes (being used for G418 selections).
Suitable host cells for cloning or expressing the DNA in this paper carriers are prokaryotic, yeast or Higher eukaryotic Cell.Suitable prokaryotes for this purpose include eubacteria, including Gram-negative or gram-positive organism, for example Enterobacteriaceae (Enterobacteriaceae) such as Escherichia (Escherichia), such as Escherichia coli (E.coli), Enterobacter (Enterobacter), Klebsiella (Klebsiella), Proteus (Proteus), salmonella Belong to (Salmonella), such as salmonella typhimurium (Salmonella typhimurium), Serratia (Serratia), such as serratia marcescens (Serratia marcescans) and Shigella (Shigella), Yi Jiya Spore Bacillus (Bacilli) such as bacillus subtilis (B.subtilis) and bacillus licheniformis (B.licheniformis), it is false Zygosaccharomyces (Pseudomonas) such as pseudomonas aeruginosa (P.aeruginosa) and streptomyces (Streptomyces).Close Suitable escherichia coli cloning host includes Escherichia coli 294 (ATCC31,446), Escherichia coli B, Escherichia coli X1776 (ATCC 31,537) with Escherichia coli W3110 (ATCC 27,325).
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable clone or expressive host. Saccharomyces cerevisiae or common Saccharomyces cerevisiae are the most frequently used low eucaryon host microorganisms.Place for expressing recombinant antibodies of the present invention Chief cell is preferably mammalian host cell, including Chinese hamster ovary cell (Chinese hamster ovary celI) (is included in Urlaub＆ Chasin(1980)PNAS USA 77：Dhfr-CHO cells described in 4216-4220, it is together with DHFR selectable markers Use, see, for example, Kaufman＆Sharp (1982) J.Mol.Biol.159：Described in 601-621), NSO myeloma cell, COS cells, SP2 cells and HEK293-EBNA1 cells (Catalog number (Cat.No.)：CRL-10852).Particularly, in order to NSO Myeloma cell is used together, and another preferred expression system is public affairs in WO87/04462, WO89/01036 and EP0338841 The GS gene expression systems opened.
The structure of antibody and preparation
The antibody that anti-TrkA polypeptides are produced can be obtained by immune animal, i.e., by using well-known routine side Case, to animal, preferably non-human animal applies the polypeptide, see, for example, Handbook of Experimental Immunology, Weir DM (editor), Vol 4, Blackwell Scientific Publishers, Oxford, England (1986).Can be with Many warm-blooded animals, such as rabbit, mouse, rat, sheep, ox, camel or pig is immunized.However, mouse, rabbit, pig and rat, especially It is mouse, it is typically most suitable.Antibody can also be produced by recombinant DNA technology well known by persons skilled in the art.Separately Outside, antibody can also be produced by zymetology or chemical cleavage natural antibody.The humanized antibody of the present invention can be by the future One or more CDR from non-human animal (such as mouse) VH and/or VL areas or part thereof are transferred to from people VH and/or VL areas One or more framework regions on build.Preferably, the humanized antibody of the present invention can be built in the following way：Will One or more CDR in VH and/or VL areas of mouse MNAC13 antibody disclosed in WO00/73344 or part thereof, which are transferred to, to be come from On one or more framework regions in people VH and/or VL areas.Optionally, when need or expect reduction antibody immunogenicity and/or When keeping binding affinity, the people's Framework residues that can be will be present in VH and/or VL areas are replaced into corresponding inhuman (such as small Mouse) residue.Optionally, being present in the non-human amino acid residues in CDR can be replaced as people's residue.Can be based on as described above The sequence of the non-human monoclonal antibodies of preparation, prepares the chimeric or humanized antibody of the present invention.Can be from interested inhuman miscellaneous Hand over knurl to obtain the DNA of encoding heavy chain and light chain immunoglobulins, and using standard molecular biological technique it is engineered into Contain non-mouse (such as people) immunoglobulin sequences.For example, in order to produce chimeric antibody, methods known in the art can be used Mouse variable region is connected on human constant region (see, for example, Cabilly et al. U.S. Patent number 4,816,567).In order to produce Humanized antibody, can use methods known in the art to insert mouse CDR region in people's framework (see, for example, Winter U.S. State patent No.5,225,539 and Queen et al. United States Patent (USP) No.5,530,101；5,585,089；5,693,762 and 6, 180,370)。
The humanized antibody of the present invention can be built, wherein the heavy chain based on potential receptor molecule variable region and mouse antibody can The homology become between area considers, selects the people's acceptor molecule of the weight chain variable district.It is preferred that germline candidate's acceptor molecule, to drop Low potential immunogenicity.Germline database is by reading over to heavy chain FW3 areas end and partially into the antibody sequence of CDR3 sequences Composition.In order to select FW4 regions, the database of ripe antibody sequence generated from selected germline molecule, Huo Zheke may search for To use the antibody sequence generated from selected germline molecule from people's donor.People's acceptor molecule is preferably selected from and mouse donor molecule Identical heavy chain classification, and with variable region identical specification (canonical) structure type with mouse donor molecule.For choosing The people's acceptor molecule of weight chain variable district is selected, the second Consideration is included between mouse donor molecule and people's acceptor molecule in CDR length On homology.It is preferred that selecting people's acceptor antibody molecule by carrying out homologue retrieval to V-BASE databases, but it is also possible to Use other databases of such as Kabat databases and public ncbi database.
The humanized antibody of the present invention can be built, wherein the light chain based on potential receptor molecule variable region and mouse antibody can The homology become between area considers, selects the people's acceptor molecule of the light chain variable district.It is preferred that germline candidate's acceptor molecule, to drop Low potential immunogenicity.Germline database is by reading over to heavy chain FW3 areas end and partially into the antibody sequence of CDR3 sequences Composition.In order to select FW4 regions, the database of ripe antibody sequence generated from selected germline molecule, Huo Zheke may search for To use the antibody sequence generated from selected germline molecule from people's donor.People's acceptor molecule is preferably selected from and mouse donor molecule Identical light chain classification, and with the variable region identical canonical structure type with mouse donor molecule.For selection light chain variable The people's acceptor molecule in area, the second Consideration includes the homology between mouse donor molecule and people's acceptor molecule in CDR length. It is preferred that selecting people's acceptor antibody molecule by carrying out homologue retrieval to V-BASE databases, but it is also possible to using such as Other databases of Kabat databases and public ncbi database.
, can be by when for example, by by antibody is produced in channel genes mammalian host cell in recombinant antibodies form The enough long-times of host cell culture, to allow antibody to be expressed in host cell or more preferably allow antibody-secreting to life In the culture medium of long host cell, antibody is thus produced.Host cell for producing the antibody for combining people TrkA can be each Plant in culture medium and cultivate.Commercially available culture medium such as Ham's F10 (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), pole Limit basal medium (MEM；Sigma-Aldrich Chemie GmbH), RPMI-1640 (Sigma-Aldrich Chemie GmbH, Basel, Switzerland), EX-CELL 293, HEK293- serum free mediums (Sigma, Buchs, Switzerland) and Dulbecco Eagle the culture mediums ((DMEM of improvement；Sigma-Aldrich Chemie GmbH)) it is applied to culture host cell.It can make Antibody is reclaimed from culture medium with standard protein purification method.
Antibody can be operatively attached to fusion partner to allow to such as targeting, purifying, screening, presenting and expressing Protein etc..Fusion partner can be connected by joint sequence with antibody sequence.Joint sequence generally comprises a small amount of amino Acid, typically less than 10 amino acid, but it is also possible to use longer joint.Typically, flexible and degradation-resistant joint is selected Sequence.As it will appreciated by a person of ordinary skill, any one of various sequences can be used as joint.Example Such as, common joint sequence includes amino acid sequence GGGGS.Fusion partner can be targeting sequence or signal sequence, and it will Antibody and any associated fusion partner are directed to required cell position or extracellular medium.As known in the art, Protein targeting can be secreted into growth medium by some signal sequences, or targeted to the inner membrance and outer membrane positioned at cell Between periplasmic space.Fusion partner can also be the sequence of encoded peptide or protein, wherein the peptide or protein matter causes Purifying and/or screening can be carried out.Such fusion partner includes but is not limited to, and polyhistidyl tags (His labels) are (for example H6 and H10 or for immobilized metal affinity chromatography (IMAC) system (such as Ni2+ affinity columns)) other labels), GST fusion Body, MBP fusions, Strep- labels, bacterial enzyme BirA BSP biotinylation target sequences, and by the epitope tag of antibody target (such as c-myc labels, FLAG labels etc.).As it will appreciated by a person of ordinary skill, these labels can be used for purify, screening or Both.
The sign of anti-TrkA antibody and purifying
Determination test measurement and people TrkA combination and/or measurement can be used to block what TrkA was combined with its part NGF Ability, carrys out screening antibodies.One example of combination mensuration experiment is ELISA.In addition, surface plasma body resonant vibration (SPR) is analyzed (as used in embodiment) can be used for the measurement dynamic (dynamical) association and dissociation speed constant of antibody binding.The one of blocking test Individual example is the determination test based on flow cytometry, and it measures the blocking combined to NGF with TrkA.Resist for assessing anti-TrkA The determination test of body function activity, for example, can use TF-1 cell proliferation assays, wherein usage factor dependence people is red Leukemia Cell Lines TF-1 determines ability (the Kitamura T of the cell propagation of antibody blocking cell surface TrkA/ β-NGF mediations et al.,(1989)J.Cellular Physiology 140(2):323-34)。
The antibody of the present invention can isolated or purified in various ways known to those skilled in the art.Standard purification methods Including, the chromatographic technique carried out at atmospheric pressure, or at elevated pressure using such as FPLC and HPLC system, including ion exchange, hydrophobic phase Interaction, affine, size exclusion or gel filtration and reverse-phase chromatography.Purification process also includes electrophoresis, and immunology is precipitated, dialysis With chromatofocusing technology.Ultrafiltration and diafiltration techniques, are combined with protein compression, are also useful.In order to purify TrkA antibody, Can be for example, growing selected host cell in rolling bottle for monoclonal antibody-purified.It can filter and concentrated supernatant, Afterwards affinity chromatography is carried out with Protein A sepharose (Pharmacia, Piscataway, NJ).The antibody of elution can lead to Gel electrophoresis and high performance liquid chromatography inspection are crossed, to ensure purity.Therefore, currently preferred antibody is combined with people TrkA Separation and/or the antibody of purifying.
Immunoconjugates
On the other hand, the present invention is provided and therapeutic agent such as cytotoxin, medicine (such as immunodepressant) or radiation toxin Connect, the anti-TrkA antibody of people's TrkA associativities or its fragment.This conjugate is referred to herein as " immunoconjugates ".Comprising One or more cytotoxic immunoconjugates are referred to as " immunotoxin ".Cytotoxin or cytotoxic agent are included to cell Any medicament of harmful (for example killing cell).Example includes taxol (taxol), cytochalasin B (cytochalasin B), Gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), emetine (emetine), mitogen is mould Plain (mitomycin), Etoposide (etoposide), Teniposide (tenoposide), vincristine (vincristine), Vinblastine (vinblastine), colchicine (colchicin), Doxorubicin (doxorubicin), daunorubicin (daunorubicin), dihydroxy anthracene nucleus diketone (dihydroxy anthracin dione), mitoxantrone (mitoxantrone), mithramycin (mithramycin), actinomycin D (actinomycin D), 1- dehydrogenation testosterones (1- Dehydrotestosterone), glucocorticoid (glucocorticoids), procaine (procaine), totokaine (tetracaine), lidocaine (lidocaine), Propranolol (propranolol) and puromycin (puromycin) and Its analog or homologue.Therapeutic agent also includes such as antimetabolite (for example, methotrexate (methotrexate), 6- sulfydryls Purine (6-mercaptopurine), 6- thioguanines (6-thioguanine), cytarabine (cytarabine), 5- fluorine urine Pyrimidine (5-fluorouracil), Dacarbazine (decarbazine)), alkylating agent is (for example, mustargen (mechlorethamine), thiotepa (thioepa), Chlorambucil (chlorambucil), melphalan (melphalan), BCNU (carmustine (BSNU)) and lomustine (lomustine (CCNU)), endoxan (cyclothosphamide), busulfan (busulfan), dibromo mannitol (dibromomannitol), streptozotocin (streptozotocin), mitomycin C (mitomycin C) and cis- dichlorodiamine platinum (II) (cis- Dichlorodiamine platinum (II) (DDP)) cis-platinum (cisplatin)), anthracycline (anthracyclines) (example As daunorubicin (daunorubicin) (was originally：Daunomycin (daunomycin)) and Doxorubicin (doxorubicin))), Antibiotic was (for example, more raw rhzomorph (dactinomycin) (was originally：D actinomycin D (actinomycin)), bleomycin (bleomycin), mithramycin (mithramycin) and anthramycin (anthramycin (AMC))) and antimitotic agent (such as vincristine and vinblastine).The other examples for the therapeutic cells toxin that can be connected with the antibody of the present invention include Times carcinomycin (duocarmycins), Calicheamicin (calicheamicins), maytansine (maytansines) and Ao Lisi Statin (auristatins) and its derivative.One example of Calicheamicin antibody conjugates be it is commercially available (American Home Products).Joint technique obtained by this area can be used by cytotoxin It is connected with the antibody of the present invention.The example for having been used to the joint categories by cytotoxin and antibody conjugate includes but is not limited to Hydrazone, thioether, ester, disulphide and containing peptide linker.Joint can be selected, for example, is easy in lysosomal compartment be broken by low pH Joint, or the joint easily cut by protease, the protease can be the albumen that precedence table reaches for example in tumor tissues Enzyme such as cathepsin (such as cathepsin B, C, D).On cytotoxin type, joint and for by therapeutic agent with it is anti- The conjugated method of body it is discussed further, referring also to Saito G etc., (2003) Adv.Drug Deliv.Rev.55:199-215； Trail PA etc., (2003) Cancer Immunol.Immunother.52:328-337；Payne G(2003)Cancer Cell 3:207-212；Allen TM(2002)Nat.Rev.Cancer 2:750-763；Pastan I&Kreitman RJ (2002)Curr.Opin.Investig.Drugs 3:1089-1091；Senter PD&Springer CJ(2001) Adv.Drug Deliv.Rev.53:247-264.The antibody of the present invention can also be connected to produce cell with radio isotope Toxicity radiopharmaceutical, also referred to as radioimmunoconjugate.The radioactivity of diagnosis or therapeutical uses can be used for antibody conjugate Isotope example includes but is not limited to iodine -131, indium -111, Yttrium-90 and lutetium -177.The method for preparing radioimmunoconjugate exists Had built up in this area.The example of radioimmunoconjugate is commercially available, including(EDEC Pharmaceuticals) and(Corixa Pharmaceuticals), and similar method can be used for from this Invention Antibody preparation radioimmunoconjugate.The antibody mediated immunity conjugate of the present invention can be used for the given biological respinse of modification, and And drug moiety should not be construed as limited to the chemotherapeutant of classics.For example, drug moiety can have required biology living The protein or polypeptide of property.This proteinoid may include such as enzyme activity toxin or its active fragment, such as abrin, castor Ricin A, Pseudomonas exotoxin or diphtheria toxin；Protein, such as TNF or interferon-γ；Or it is biological anti- Answer conditioning agent, such as lymphokine, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin-6 (IL- 6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth because Son.The technology that these therapeutic agents are connected with antibody is it is well known that see, for example, Arnon etc., " being used for medicine in treatment of cancer The immune targeting of thing monoclonal antibody ", Monoclonal Antibodies and Cancer Therapy, Reisfeld Deng (eds.), pp.243-56 (Alan R.Liss, Inc.1985)；Hellstrom etc., " antibody is used for medicine delivery ", The (eds.) such as Controlled Drug Delivery (second edition), Robinson, pp.623-53 (Marcel Dekker, Inc.1987)；Thorpe, " the antibody carrier of cytotoxic agent in treatment of cancer:Summary ", Monoclonal Antibodies'84:The (eds.), pp.475-506 such as Biological And Clinical Applications, Pinchera (1985)；" about the analysis of the therapeutic application of radiolabelled antibody in treatment of cancer, result and future prospect ", The (eds.), pp.303- such as Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin 16 (Academic Press1985) and Thorpe etc., Immunol.Rev.62:119-58(1982).
On the other hand, the present invention is provided and therapeutic agent such as cytotoxin, medicine (such as immunodepressant) or radiation toxin Apply together, anti-TrkA antibody being combined with people TrkA or its fragment.
Pharmaceutical composition
On the other hand, the present invention provides the combination of the antibody comprising the present invention or its fragment and pharmaceutically acceptable carrier Thing, such as pharmaceutical composition.Such composition can include a kind of or combination (for example two or more are different) originally Invention antibody and/or immunoconjugates, and/or therapeutic agent as described above such as cytotoxin, medicine (such as immunodepressant) Or radiation toxin.For example, the pharmaceutical composition of the present invention can include the different epitopes or living with complementation combined on target antigen The combination of the antibody (or immunoconjugates) of property.The pharmaceutical composition of the present invention can also be in combined therapy (that is, with other medicaments Combination) middle administration, see below further general introduction.
As used herein, " pharmaceutically acceptable carrier " includes any and all solvent of physical compatibility, disperses to be situated between Matter, is coated, antibacterium and antifungal agent, isotonic agent and absorption delaying agent etc..Preferably, carrier is suitable for intravenous, intramuscular, skin Under, parenteral, spinal cord or Epidermal administration (such as by injecting or being transfused).Depending on method of administration, reactive compound, i.e. antibody Or immunoconjugates can be coated with the material to protect compound from that may make the sour and other natural bar of its inactivation The effect of part.Pharmaceutically acceptable carrier include aseptic aqueous solution or dispersion and for extemporaneous preparation of sterile inject solution or The aseptic powdery of dispersion.The purposes that these media and reagent are used for pharmaceutically active substance is known in the art.Any routine Medium or reagent, unless it is incompatible with reactive compound, otherwise it is contemplated that in pharmaceutical composition of the invention.Combination The reactive compound of supplement can also be incorporated in thing.
On the other hand, the present invention provides the anti-TrkA antibody of humanization or its fragment and other medicine for including the present invention The composition of activating agent.Preferably, pharmaceutically active agents be it is following in one or more：
A) analgestic, b) another anti-TrkA antibody, c) NGF, d) anticancer, e) anti-ngf antibodies, following article is further general State.
On the other hand, the present invention provides the composition comprising immunoconjugates and pharmaceutically acceptable carrier, wherein described Immunoconjugates are included and therapeutic agent connection and the people TrkA antibody combined or its fragment.The immunoconjugates that can use and Therapeutic agent is as described above.
The pharmaceutical composition of the present invention can also include pharmaceutically acceptable antioxidant.Pharmaceutically acceptable antioxygen The example of agent includes：(1) water soluble antioxidant, such as ascorbic acid, cysteine hydrochloride, niter cake, burnt sulfurous Sour sodium, sodium sulfite etc.；(2) oil-soluble inhibitor, such as ascorbyl palmitate, butylated hydroxyanisol (BHA), fourth Base hydroxy-methylbenzene (BHT), lecithin, propylgallate, alpha-tocopherol etc.；(3) metal-chelator, such as citric acid, second Ethylenediamine tetraacetic acid (EDTA) (EDTA), D-sorbite, tartaric acid, phosphoric acid etc..Available for the suitable aqueous in pharmaceutical composition of the present invention and The example of non-aqueous carrier includes water, ethanol, polyalcohol (such as glycerine, propane diols, polyethylene glycol etc.) and its suitable mixing Thing, such as organic ester of vegetable oil, such as olive oil and injectable, ethyl oleate.Appropriate mobility can be for example by making With coating material, such as lecithin, in the case of a dispersion by the particle diameter needed for maintenance, and by using surface-active Agent, to keep.These compositions can also contain auxiliary agent, such as preservative, wetting agent, emulsifying agent and dispersant.To ensure to prevent Microorganism is present, and can use above-mentioned sterilizing methods and by comprising various antibacteriums and antifungal agent, such as para hydroxybenzene Formic acid esters, methaform, phenol sorbic acid etc. is realized.Furthermore, it is also possible to expect to include isotonic agent such as sugar, chlorination in the composition Sodium etc..In addition, injectable drug form extension absorb can by comprising delay absorb reagent (for example aluminum monostearate and Gelatin) realize.
Therapeutical uses and other purposes
The antibody of the present invention can be used in medicine treating various disease/illnesss (various classifications such as set forth below).
Therefore, the method that the present invention provides the following illnesss for the treatment of, it is included to subject in need, and suitably lactation is moved Thing subject, particularly human experimenter, using the antibody described herein or derivative of therapeutically effective amount, thus treat the disease Disease.
The present invention also provides antibody as described herein or derivative and is preparing the use in being used to treat the medicine of following illnesss On the way.
Here term " treatment " includes treating existing illness/patient's condition.It also includes prophylactic treatment.It also includes changing Kind one or more ill symptomses, even if given illness/illness of patient there is no healing.For example, can alleviate or mitigate Pain.
One preferred medical usage is treatment pain.According to International Association for Pain Research (" IASP "), pain generally quilt It is defined as " relevant or being described according to the damage with actual or potential tissue damage sensation beastly and emotion Experience, or both have both at the same time ".In the pain of form of ownership, basic key element is that the high threshold acceptor and nerve specially changed are fine The activation of dimension, to alert the potential tissue damage of organism.The participation of inflammatory cell and process is the common of many pain statuses Factor.Term " Acute Pain " refer to immediately, the pain of usual high threshold, by for example hurting, weigh wounded, burn equivalent damage causes Or caused by chemical stimulation.Term " chronic ache " used herein refers to, in addition to Acute Pain, inflammatory and neuropathy Other pain in rationality source.Chronic ache is often considered to have relatively long duration, such as several months or several years, and It can be continuous or interruption.The antibody of the present invention can be used for treatment chronic ache or Acute Pain.The chronic pain of preferred therapeutic Bitterly.
Pain may, for example, be following any one or can be with associated：Inflammatory pain (inflammatory pain), Post-surgical pain (post-surgical pain), postoperative pain (post-operative pain) (including toothache (dental pain)), neurogenic pain (neuropathic pain), peripheral neuropathy (peripheral Neuropathy), diabetic neuropathy (diabetic neuropathy), nephrosis (diabetic Nephropathy), it is neural after fracture pain (fracture pain), gout arthralgia (gout joint pain), bleb Bitterly (post-herpetic neuralgia), cancer pain (cancer pain), osteoarthritis or rheumatoid arthritis pain Bitterly, sciatica (sciatica), pain (the pains associated with sickle related to sickle cell crisis Cell crises), have a headache (such as antimigraine (migraines), tension headache (tension headache), cluster headache (cluster headache)), dysmenorrhoea (dysmenorrhea), endometriosis (endometriosis), fibroid (uterine fibroids), musculoskeletal pain (musculoskeletal pain), chronic back pain (chronic low Back pain), fibromyalgia (fibromyalgia) is sprained (sprains), splanchnodynia (visceral pain), ovarian bursa Swollen (ovarian cysts), prostatitis (prostatitis), chronic Pelvic Pain Syndrome (chronic pelvic Pain syndrome), cystitis (cystitis), interstitial cystitis (interstitial cystitis), painful wing Guang syndrome (painful bladder syndrome) and/or painful bladder syndrome (bladder pain syndrome), Pain (the pain associated with chronic abacterial related to chronic non-bacterial prostatitis Prostatitis), incision pain (incisional pain), antimigraine (migraine), trigeminal neuralgia (trigeminal Neuralgia), the pain from burn and/or wound, the pain related to wound, the pain related to musculoskeletal disease, Ankylosing spondylitis (ankylosing spondilitis), periarticular pathologies (periarticular pathologies), Bone tumour pain (pain from bone metastases), HIV pain (pain from HIV), acromelalgia (erythromelalgia) or the pain as caused by pancreatitis or kidney stone, chromoma (malignant melanoma), Sjogren syndrome (Sjogren's syndrome), asthma (asthma) is (for example, uncontrolled asthma is high anti-with serious air flue Answering property (uncontrolled asthma with severe airway hyper-responsiveness)), chronic cough (intractable cough), demyelinating disease (demyelinating diseases), chronic alcoholism (chronic Alcoholism), apoplexy (stroke), thalamic pain syndrome (thalamic pain syndrome), as caused by toxin Pain, chemotherapy pain, inflammatory bowel disease (inflammatory bowel disorders), IBS (irritable Bowel syndrome), Inflammatory eye conditions (inflammatory eye disorders), inflammatory or unstable bladder illness (inflammatory or unstable bladder disorders), psoriasis (psoriasis), with inflammatory components Skin discomfort (skin complaints), tans severely (sunburn), carditis (carditis), dermatitis (dermatitis), flesh Scorching (myositis), neuritis (neuritis), collagen vascular diseases (collagen vascular diseases), chronic inflammation Venereal disease disease, inflammatory pain and related hyperalgia and allodynia, neurogenic pain and associated hyperalgesia or different Perseverance pain, diabetic neuropathic pain (diabetic neuropathy pain), cusalgia (causalgia), by handing over The pain (sympathetically maintained pain) that sense nerve is maintained, deafferentation syndromes (deafferentation syndromes), epithelial tissue damage or dysfunction, breathing, intestines and stomach, apparatus urogenitalis or blood The Visceromotor sexual disorder (disturbances of visceral motility) in area under control domain, allergic skin reaction (allergic skin reactions), pruritus (pruritis), leucoderma (vitiligo), general enterogastric diseases, knot Enteritis (colitis), gastric ulcer (gastric ulceration), duodenal ulcer (duodenal ulcers), blood vessel relaxes Contracting (vasomotor) or allergic rhinitis, bronchial disease, indigestion (dyspepsia), gastroesophageal reflux (gastroesophageal reflux), pancreatitis (pancreatitis), splanchnodynia (visceralgia) and bone fibres knot Structure depauperation (fibrous dysplasia of bone) (FD).
Pain may, for example, be following any or associated：Pancreatitis, kidney stone, endometriosis, IBD, Crohn disease, postoperative intestinal adhesion, cholecystolithiasis, headache, dysmenorrhoea, musculoskeletal pain is sprained, splanchnodynia, ovarian cyst, Prostatitis, cystitis, interstitial cystitis, postoperative pain, antimigraine, trigeminal neuralgia, burn and/or wound pain, with The related pain of wound, neurogenic pain, the pain related to musculoskeletal disease, rheumatoid arthritis, Bones and joints Inflammation, ankylosing spondylitis, periarticular pathologies, relaxing tumor pain, Bone tumour pain, HIV.
It is known to there are various models to be used to assess pain and available for screening antibodies/its derivative.It is, for example, possible to use wound Evil experiences hot plate test, such as disclosed in WO 00/73344.The experiment can be according to McMahon et al., 1995 (Nature Med.1：774-780), carried out using antibody/derivative as immunoadhesin.Antibody/derivative is subcutaneously defeated Inject the rear solid end three weeks by a definite date of adult rat or be transfused by osmotic mini-pump.Hot plate test (Eddy＆Leimbach can be used (1953)J.Pharm.Exp.Ther.107：385-393) --- the pain sensation mistake after the test simulation inflammation or neural partial injury Assess nociception sensitivity in quick situation, compartment of terrain.In this case, nociception Induced by Stimulation reaction (licks pawl and/or jump Jump), the reaction is considered as having higher comprehensive coordination than simple reflex.According to the experiment, by animal be put into plus The plate of heat temperature (being usually 56 DEG C) needed for is as in the column of substrate.In control-animal (being handled with irrelevant antibodies) and use Period of delay any in two kinds of reactions (licking pawl and jump) of measurement in the animal of anti-TrkA antibody/derivative processing.
As hot plate test an alternative it is possible to assess the nociception reaction to formalin.The experiment by Porro＆Cavazzuti, 1993 (Prog.Neurobiol, 41：It is 565-607) open, and for WO06/137106.It is related to, Apply given candidate before test, by analyze it is any it is subsequent lick pawl and reduce assess the reduction of pain reaction.Salt solution It is typically used as negative control.
Hyperalgesic assessment can use load-bearing method to determine.The mouse generally equably dispensing body between two limbs Weight.After limbs are carried out with pain stimulation, for example, pass through local injection complete Freund's adjuvant (CFA) or iodo acetic acid sodium (MIA) rear solid end (in vola, intraplantar) or knee joint (intra-articular) are arrived, weight can be reallocated to be placed on to reduce Weight and increase on injection limbs are placed on the weight on the limbs do not injected.Pain threshold detector is balanced using biped (incapacitance tester) measures load-bearing, wherein rear solid end is placed on separated sensor, and records after two The mean force that limb applies.Load-bearing can be used for assessing the acute inflammation hyperalgia as caused by intraplantar injection CFA, by intra-articular note Chronic inflammatory hyperalgia caused by CFA or the chronic osteoarthritic hyperalgia as caused by intra-articular injection MIA are penetrated, such as Respectively described in embodiment 2,3 and 4.
The assessment of mechanical hyperalgesia can be carried out by a variety of methods, for example, Von Frey or Randall- Selitto algometers (analgesiometer).It is applied in response to the wedge-shaped probe by Randall-Selitto algometers Rear solid end instep surface or the incremental pressure that rear solid end plantar surface is applied to by Von Frey, measuring claw withdrawal threshold.Right The period of delay that rear solid end bounces back is measured in animal according to animal and with anti-TrkA antibody/derivative processing.These methods can be used for commenting Mechanical hyperalgesia caused by estimating spinal cord in chronic compression damage (CCI) animal model, is shown in embodiment 5 and is described in detail.
CCI models are also well-known animal model.It is related to the spinal cord in chronic compression of sciatic nerve, and in grinding tooth The chronic ache of inducing neural pathologic property in animal such as mouse or rat.The model is by Bennett＆Xie in 1998 In Pain 33：Described in 87-107.It is used in such as WO 06/131592.Many neuropathic pain states One be mainly characterized by, generally harmless cold stimulation starts to cause pain.The reaction increase of non-pain stimulation is referred to as different Perseverance pain.It therefore, it can be tested the degree of the neuropathic pain state to measure induction with the cold drawing of allodynia, See in embodiment 5 and be described in detail.In the column that animal is put into the plate with temperature needed for being cooled to, the temperature can be at -5 DEG C extremely In the range of 15 DEG C.The delay that measurement rear solid end bounces back in control-animal and animal with the processing of anti-TrkA antibody/derivative Phase.Generally, in 5 DEG C and following surface temperature, the period of delay of retraction becomes different from baseline.
Antibody of the present invention can be used for treating cancer, neural disorder, Alzheimer's, diabetes, diabetic keratopathy kidney Disease, viral disease, the illness of HIV mediations, leprosy or inflammatory disease.In addition, antibody can be additionally used in treatment may with it is increased The related Other diseases of NGF levels, including such as lupus erythematosus, herpes zoster (shingles), PHN (postherpetic neuralgia) and hyperalgia.
Various cancers express TrkA.TrkA and NGF interaction may participate in tumor development (such as prostate cancer and pancreas The development of cancer).Actually in some form of cancer, excessive NGF can promote the growth and infiltration of nerve fibre.Pass through NGF effect is blocked, the formation of neuroma can be substantially reduced.In addition, as the simple alternative for providing blocking effect, can So that antibody and cytotoxic agent to be coupled, and available for targeted expression TrkA cancer cell.However, by antibody with it is toxin conjugated simultaneously Nonessential.ADCC (cytotoxicity of antibody dependent cellular mediation) can be caused by immune response, and wherein antibody passes through bag It can be made easily by immune system attack (such as by T cell, by complement activation etc.) by target cell.The cancer of preferred therapeutic Prostate cancer, thyroid cancer, lung cancer, prolactinoma (prolactinoma), melanoma or Bone cancer pain, including with Bone tumour Related cancer pain.Preferred cancer to be treated is Bone cancer pain, including the cancer pain related to Bone tumour.
Antibody, which can also be used for treatment, includes the various neural disorders of neurodegenerative disease, for example, antibody can be used for reducing The formation of neuroma.They can also be used for treatment Alzheimer disease or for treatment of nerve regeneration.The antibody of the present invention can use In these treatments, to reduce undesirable NGF rule-activator effects (" combination treatment " part see also hereafter).In addition, as above Described, antibody can be used for treatment neurogenic pain.This can be relevant with the lesion or dysfunction of nervous system.NGF is in sugar There is potential use in urine disease and treatment of leprosy, but NGF has undesirable agonist characteristics, including increase pain sensitivity Property, and this can be avoided by using the antibody of the present invention.
Another application is treatment inflammation.The peripheral location that NGF occurs in inflammatory process is by mast cell, fibroblast With the release of other cell types.Particularly, mast cell seems to have played important function.They produce NGF, while in its table Face expressive function TrkA acceptors.Seem, NGF/TrkA systems are lived by autocrine positive feedback mechanism mediating mast cell Change, so as to allow pain generation property inflammatory signals to be enlarged.The example of treatable inflammation includes urethra and pelvic cavity area The inflammatory forms in domain, osteoarthritis, multiple sclerosis, colitis, inflammatory bowel disease, cystitis, eczema, contact dermatitis, joint Inflammation, including chornic arthritis and rheumatoid arthritis, Crohn disease, psoriasis and asthma.
The antibody of the present invention can be used in treatment as described above reducing NGF undesirable rule-activator effect.
Antibody of the present invention or derivatives thereof can be controlled with one or more other activating agents, such as pharmaceutically active agents in combination It is used together in treatment.They can be used in medicine simultaneously, and order or coordination are applied.For example, antibody or derivative can be with analgesias Medicine, such as analgesia opiates or non-opioid analgesic agents combination.Disclosed in WO06/137106, a small amount of can block TrkA The molecule of bioactivity can strengthen the analgesic activity of opioid.This kind of analgesia opioid includes one or more select From following compound：Morphine (morphine), codeine (codeine), paracodin diacetylmorphine (dihydrocodeine diacetylmorphine), hydrocodone (hydrocodone), Hydromorphone (hydromorphone), levorphanol (levorphanol), Oxymorphone (oxymorphone), alfentanil (alfentanil), buprenorphine (buprenorphine), cloth support coffee sweet smell (butorphanol), fentanyl (fentanyl), Sufentanil (sufentanyl), pethidine (meperidine), methadone (methadone), Nabumetone (nabumetone), propoxyhene (propoxyphene), Pentazocine (pentazocine)；And its pharmaceutically acceptable derivative Thing (such as its pharmaceutically acceptable salt).Suitable non-opioid analgesic agents include on-steroidal anti-inflammatory drugs (NSAID) with And other antalgesics such as paracetamol (acetaminophen).The NSAID of conventional treatment acute inflammatory pain includes Ah Si Woods (asprin), brufen (ibuprofen), Indomethacin (indomethacin), naproxen (naproxen) and ketone Lip river Fragrant (ketoprofen), treat chronic inflam-matory pain includes celecoxib (celecoxib) and Meloxicam (meloxicam)。
Another combination is combination of one or more antibody with one or more other antibody of the present invention.It is preferred that combination It is the combination with one or more other anti-TrkA and/or anti-ngf antibodies.Relative to the treatment with monospecific antibody, such group Conjunction can provide increased effect in the treatment of one or more illnesss described herein.It is, for example, possible to use used herein Test procedure in find the combinations of two or more maximally efficient antibody.
Another combination is the combination of the antibody and NGF of the present invention.As described above, it has been already proposed to use NGF treats various diseases Disease, including Alzheimer's, diabetes, leprosy etc., but have been noted that because the activator for surrounding target is special Property causes the increase of pain sensitivity.Again, by using the antibody or derivative of the present invention, pain sensitivity can be reduced Property, so that the therapy more attractive based on NGF.
Another combination is the antibody and anticancer such as alkylating agent of the present invention, and antimetabolite, Topoisomerase II inhibitors are opened up Flutter isomerase I inhibitor, the combination of anti-mitosis medicine or platinum derivatives.
The antibody of the present invention can be applied by any suitable approach.This includes but is not limited to intraperitoneal, intramuscular, quiet In arteries and veins, subcutaneously, tracheal strips, orally, enteral are parenteral, intranasal or transdermal administration.
Injection (in intraperitoneal or encephalic --- generally in the ventricles of the brain --- or pericardium or intracapsular) liquid can typically be passed through Body preparation or by swallowing solid pharmaceutical preparation (with pill, tablet, the form of capsule) or liquid preparation (with emulsion and the shape of solution Formula), administration of antibodies is with topical application.
It is molten that the composition of parenteral generally comprises the immunoglobulin being dissolved in compatible, preferably aqueous solution Liquid.The concentration of antibody/derivative can change from less than 0.005% to 15-20%w/v in these preparations.This is main according to liquid Volume, viscosity of body etc., and selected according to required specific administration mode.Or, antibody can be prepared in solid form Using.Antibody can from different inertia or figuration combinations of substances, its can include part such as microcrystalline cellulose, gelatin or I Primary glue；Acceptor (recipient) such as lactose or starch；Reagent such as alginic acid, Primogel or cornstarch；Lubricant such as tristearin Sour magnesium, colloidal silica；Sweetener such as sucrose or saccharin；Or flavor enhancement, such as peppermint and gaultherolin.Other drugs are applied System includes hydrogel, hydroxymethyl cellulose, liposome, micro-capsule, microemulsion, microballoon etc..
If illness is localization, directly illness involve position/against the position carry out local injection be preferred Method of application.The anti-suitable systemic administration of TrkA antibody.Systemic administration can be carried out by injecting, such as continuous venoclysis, The dense note of rapid intravenous, is subcutaneously or intramuscularly injected.
Alternatively, other administration forms (such as oral, transmucosal, sublingual etc. by sucking) can also be used.However, It is possible if desired to which by locally administration (such as the intra-articular injection or subcutaneous, intramuscular injection) near affected tissue, delivering is anti- Body/derivative.
Anti- TrkA antibody/derivative can suitably be configured to be suitable for the pharmaceutical composition of predetermined method of administration.Injection With solution suitably containing the antibody/derivative being dissolved or dispersed in aqueous medium (such as water for injection), the medium is taken the circumstances into consideration Suitable buffer solution and molar concentration conditioning agent (such as phosphate, salt and/or glucose) can be contained.
Therapeutic scheme (i.e. dosage, time and repetition) can be expressed as, selected method of administration, the single to product Or repetitive administration (for example injecting).The interval that dosage is applied can be according to the degree of clinical response and duration and specific Individual and individual clinical history are modified.
Suitably, anti-TrkA antibody/derivative has long acting duration.Especially, such as determined from animal , the clinical effectiveness of antibody can extend up to 21 days after application.In addition, after, anti-TrkA antibody shows clinic The time of benefit can be so that the time than that can detect its presence in associated biomolecule matrix such as serum or blood plasma is long.
In view of expected long action time, (that is, effect suitably continued at least one week, or preferably at least two weeks, such as extremely Few three weeks or at least surrounding), suitably can be with no more than weekly frequency, such as no more than once every two weeks or every three Antibody/derivative once, is applied to subject by Zhou Yici or every four weeks.
The suitable unit dose of anti-TrkA antibody/derivative is usually 0.1mg/kg to 10mg/kg body weight.It is acute for treating Anti- TrkA antibody suitable dose with chronic inflam-matory pain is at least 0.01mg/kg (referring to embodiment 2 and 3).For treating bone The anti-TrkA antibody suitable dose of arthritis ache is at least 0.01mg/kg (referring to embodiment 4).For treating europathology The anti-TrkA antibody suitable dose of property pain is at least 0.01mg/kg, preferably 0.1mg/kg, and most preferably 1mg/kg is (referring to implementation Example 5).These dosage are relevant only with the internal situation of mouse.The present invention relates to the anti-TrkA antibody of humanization in treatment acute inflammation Purposes in pain, wherein hyperalgia can by effectively reverse to apply NSAID observed by degree identical journey Degree.The invention further relates to purposes of the anti-TrkA antibody of humanization in treatment chronic inflam-matory pain, wherein hyperalgia can be by Effectively reverse the degree identical degree arrived with observed by administration NSAID.The invention further relates to the anti-TrkA antibody of humanization Purposes in treatment osteo-arthritic pain, wherein hyperalgia can be arrived with applying Pregabalin and opium by effectively reversing Degree identical degree observed by agent.The invention further relates to the anti-TrkA antibody of humanization in treatment neurogenic pain In purposes, wherein hyperalgia can by effectively reverse arrive and apply Pregabalin observed by degree identical journey Degree.Now particularly as the administration of tumour for, administration can be the direct and local tissue being expelled near tumour or tumor locus In.For Formulations for systemic administration, dosage can be from daily 0.05mg/kg to daily 500mg/kg, but in the lower region of the scope Dosage be preferably as they be easier to apply.Dosage can be calibrated, such as to ensure antibody/derivative in blood plasma Specified level (about 5-30mg/ml, preferably between 10-15mg/ml) and keep one section of preset time of the level until reaching Clinical effectiveness.
It can be based on to prostate spy in blood for measuring or assessing pancreas or the effective ways in tumor of prostate stage The measurement of Specific Antigen (PSA), based on the measurement (for pancreatic neoplasm) to life span, based on to slowing down or suppressing diffusion Measurement is (for the transfer in the case of two kinds of tumor types).
For the direct injection in tumor locus, dosage depends on different factors, includes type, stage and the body of tumour Long-pending and many other variables.According to gross tumor volume, typical therapeutic dose can from 0.01mg/ml to 10mg/ml injection Change between amount, these dosage can be applied with necessary frequency.
Regardless of the property treated, compared with non-humanized antibody, the removing of humanized antibody can more slowly, and Lower dosage is needed to maintain the level of significance in blood plasma.In addition, during using high-affinity antibody, and with compared with low-affinity Antibody compare, using can be carried out with relatively low frequency and less amount.The treatment effective dose of every kind of antibody/derivative can To be determined by skilled practitioners during treating.It is possible if desired to reduce dosage (such as to reduce side effect) or incremental dose (to increase therapeutic effect).
Before administration, the preparation of antibody of the present invention can be stored by freezing or freezing.It is then possible to exist just before use Reconstructed in suitable buffer solution.In view of lyophilized and reconstruct may cause loss of activity, it can be entered by the fertilizing standards to antibody Row is calibrated to compensate.(for conventional immune globulins, IgM antibody tends to than IgG antibody there is bigger activity to damage Lose.) can also specify the pot-life, to avoid using the antibody after certain storage time.
Antibody of the present invention or derivatives thereof can be used for examining for the above-mentioned any disease/illness discussed with regard to medical usage Disconnected or prognosis.For example, can be used for the detection for promoting TrkA positive tumor marks (as the morning of the outbursts such as Alzheimer disease Ripe mark).It can also be used for diagnosis CIPA (" the congenital insensitive merging anhidrosis of the pain sensation ").CIPA is that a kind of heredity is recessive often Chromosome syndrome, it is characterized in that recurrence sexual refractoriness heating, dehydration lacks the reaction to nociception sexual stimulus, feeblemindedness With autotomy tendency.The disease is caused by TrkA gene mutations.In fact, the antibody or derivative of the present invention can be used for being related to TrkA Unconventionality expression (with healthy individuals or health tissues sample TrkA expression compared with) or TrkA relevant abnormalities activity, scope it is wide The diagnosis or prognosis of general various illnesss.Therefore, the present invention includes method in the range of it, and methods described includes obtaining coming from and suffered from The biological sample of person simultaneously makes sample be contacted with the antibody or derivative of the present invention.It is possible if desired to sessile antibody/derivative. Then this method can include the combination that antibody/derivative and the sample are determined in quantitatively or qualitatively mode.If desired, This may be referred to and/or relative to positive control (instruction health status) or negative control (presence/possibility for indicating illness) Carry out.For diagnostic purpose, antibody can use detectable mark substance markers, or can not mark.(art used herein Language " label " includes the part of mark or any other detectable part/triggerable detectable change.)
Product and medicine box
In another embodiment of the disclosure, the antibody or its fragment, composition or immunoconjugates of the present invention is included The product of thing, for treating one or more above-mentioned disease/illnesss.The product can include container and on container or with appearance Label or package insert that device is combined.Suitable container includes such as bottle, bottle or syringe.Container can be by various material shapes Into such as glass or plastics.Container accommodate can effectively sanatory composition, and can have sterile inlet port (example Such as, container can be intravenous solution bag or bottle with the plug that can be penetrated by hypodermic needle).In composition at least A kind of activating agent can be antibody as described herein.Label or package insert may indicate that composition can be used for the shape selected by treatment Condition.
In addition, product can include the first container (a) wherein containing composition, wherein the composition includes this paper's Antibody, and (b) wherein second container containing composition, wherein the composition includes the therapeutic agent outside the antibody.This public affairs Product may further include the package insert for indicating that the first and second compositions can be applied in combination in the embodiment opened. Such therapeutic agent can be described in previous section any auxiliary treatment (for example, thrombolytic agent, anti-platelet agents, chemistry The conditioning agent of therapeutic agent, anti-angiogenic agent, anti-hormonal compound, heart protective agent and/or immune-function in mammals, including Cell factor).Besides or furthermore, product can further include second (or 3rd) container, it is comprising pharmaceutically acceptable Buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution and glucose solution.It can also be wrapped Include the desired other materials of viewpoint from business and user, including other buffer solutions, diluent, filter, syringe needle and syringe.
The medicine box of antibody, composition or immunoconjugates and operation instructions comprising the present invention is also in the scope of the present invention It is interior.The medicine box can be further containing one or more other reagents, such as immunosupress reagent, cytotoxic agent or radiation Toxic agents, or the present invention one or more other antibody (for example, with different epitopes on first antibody combination TrkA antigens, Antibody with complementary activity).
In the case where not further describing, it is believed that those of ordinary skill in the art can use it is described above and with Lower illustrative example, is prepared and the reagent using the disclosure and implementation method claimed.Following examples are provided In order to the implementation of the disclosure, but these embodiments should not be construed in any way as limiting remainder of this disclosure.
Embodiment
The anti-TrkA antibody of humanization described herein represents a new anti-TrkA antibody subgroup, and it is suppressing TrkA work( It is highly effective in terms of energy property activation.Disclosed in WO00/73344 be referred to as MNAC13 anti-TrkA mouse monoclonal antibodies and Its humanization variants disclosed in WO09/098238, it is impossible to reach identical suppression level, and this is the phase in some cases Hope, such as treatment pain and related cancer pain, wherein high-caliber suppression can cause the raising of therapeutic effect.Cause This is, it is necessary to which the TrkA anti-TrkA antibody of humanization can highly be suppressed by developing.
For the antibody-mediated inhibitory action to TrkA functional activations, its appraisal procedure be it is well known in the art that , including TF-1 cell proliferation assays, wherein usage factor dependence human erythroleukemia cell line TF-1, determine antibody resistance Ability (Kitamura T et al., (1989) J.Cellular of the cell propagation of disconnected cell surface TrkA/ β-NGF mediations Physiology 140(2)：323-34).Humanized antibody disclosed in WO09/098238 is in TF-1 proliferation assays Inhibitory activity is shown, wherein BXhVH5VL1 candidates have highest suppression in the humanization variants of MNAC13 mouse antibodies Degree.In addition, using surface plasma body resonant vibration (SPR) technology, it has proved that, the humanization disclosed in WO09/098238 resists Body and TrkA extracellular space directly in conjunction with.Therefore, in order to design the new humanized variant of mouse MNAC13 antibody, make described Variant has improved people TrkA binding affinities and more importantly in TF-1 cells compared with BXhVH5VL1 humanization variants There is the suppression effect improved in proliferation assay, a selected son of the humanized antibody disclosed in WO09/098238 has been used Collect the starting point as engineering.
Letter after " BXh " is VH or VL or both.VH or VL represent respectively with specific heavy-chain variable domains (by Specific numeral is indicated) IGHG1 isotype heavy chains, such as BXhVH5, or with specific light variable domains (by specific numeral Indicate) κ isotype light chains, BXhVL1.The VH of the specific numeral of band is provided after at " BXh " and with specific digital VL When, such as BXhVH5VL1, it represents to be combined the specific antibodies molecule constituted by specific heavy chain and light chain, for example, BXhVH5VL1 Antibody refers to the antibody with BXhVH5 heavy chains and BXhVL1 light chains.Based on the variable knot of VH and VL described in WO09/098238 The humanization variants that the selection subsets newly built constructionization in structure domain is built, are referred to as " GBR " antibody.Identical naming system is anti-for GBR Body is effective.Specific be substituted in bracket implemented in variable heavy chain domain used herein further points out.Unless It is otherwise noted, all GBR antibody are IGHG1 heavy chain isotypes, with κ isotype light chains.
Embodiment 1：Antibody engineering
In 40 kinds of antibody disclosed in WO09/098238,5 kinds of humanized antibodies are selected as the input of engineering：Have Heavy-chain variable domains VH1 (SEQ ID NO：1) with light chain variable district VL1 (SEQ ID NO：6) BXhVH1VL1, with weight Chain variable domains VH3 (SEQ ID NO：3) with light variable domains VL1 BXhVH3VL1, with heavy-chain variable domains VH3 and light variable domains VL3 (SEQ ID NO：8) BXhVH3VL3, with heavy-chain variable domains VH5 (SEQ ID NO：5) with light chain variable district VL1 BXhVH5VL1, and with heavy-chain variable domains VH5's and light chain variable district VL1 BXhVH5VL3.MNAC13 mouse antibodies used herein have SEQ ID NO：20 heavy-chain variable domains MNAC13VH, SEQ ID NO：21 heavy chain and SEQ ID NO：22 light chain.
, it is surprising that all engineered antibodies all benefit from heavy-chain variable domains 37 valines to alanine Simple change (mouse back mutation) (Kabat numberings (Kabat EA et al., ibid quotation))., should as measured by SPR Substitution is with peculiar property of the extensive enhancing to people TrkA affinity in all engineered antibodies.Importantly, for Most of variants, the identical substitution causes the increase of the effect in TF-1 cell proliferating determinings, and unexpectedly, works as introducing Into the VH5 domains matched with VL1 domains when (GBR VH5 (V37A) VL1 antibody), with BXhVH5VL1 humanized antibodies Compare, this is ten times of increase.It is worth noting that, identical GBR VH5 (V37A) VL1 antibody, with MNAC13 mouse antibodies Compare, the effect increase of three times is also show in TF-1 cell proliferating determinings.
Because the increase of the residue quantity that mouse is originated in humanized antibody can cause the increase of its immunogenicity risk (Harding FA etc., (2010) MAbs 2 (3)：256-65), therefore further study to reduce the mouse in engineered antibody Residue quantity.3 mouse residues lysines, which become, in GBR VH5 (V37A) VL1 antibody turns to glutamine, causes GBR VH5 (K3Q, V37A) VL1 antibody, it has equal number of mouse residues compared with BXhVH5VL1 humanized antibodies, but combines parent Increase with power, and with the FAB heat endurance suitable with parent mouse MNAC13 antibody, and increased suppression effect.
Finally, due to antibody-mediated effector function may be unfavorable for the treatment for wherein only needing TrkA to block, so TrkA but the not anti-TrkA antibody of mediated immunity cell killing TrkA expression cells can be suppressed by wishing to develop.People IGHG4 is of the same race Type does not carry effector function such as ADCC or CDC, therefore when not needing effector function or effector function to be unfavorable for treatment, It is specially suitable antibody formation.However, naturally occurring people IGHG4 isotypes have one disadvantage in that, it is referred to as " FAB exchanges " Phenomenon, wherein heavy chain (Labrijn AF etc., (2009) can be exchanged known to naturally occurring people IGHG4 antibody in vivo Nat.Biotechnol.27(8)：767-71).The method for blocking heavy chain exchange between restructuring and endogenous people IGHG4 is this area It is known, and known exchange can block effectively in the following way：By the serine residue at position in hinge area 228 Proline residue (EU is numbered, Edelman GM et al., ibid quotation) is substituted by, so as to simulate the structure in people's IGHG1 isotypes As hinge arrangement (Lewis KB etc., (2009) Mol.Immunol.46 (16)：3488-94).GBR VH5 (V37A) VL1 and GBR VH5 (K3Q, V37A) VL1, which is formatted as IGHG4 S228P Ig-formats, is used for above-mentioned therapeutic purposes, and shows The effect suitable with its IGHG1 isotype homologue, so that GBR VH5 (V37A) VL1 IGHG4 S228P and GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P antibody is applied to wherein only need the human treatment for blocking TrkA.
Method
Design engineered variant
Use structural homologue Modeling Server SWISS-MODEL (Arnold K et al., (2006) Bioinformatics 22(2)：195-201；http：//swissmodel.expasy.org), automatic mode is set to, calculating VH5-VL1 can The 3D models in structure changes domain pair.The model template retrieved be experiment parsing MNAC13 FAB 3D structures (PDB code 1SEQ, Www.pdb.org, Berman HM et al., (2000) Nucleic Acids Res.28 (1)：235-42, Covaceuszach S Deng (2005) Proteins 58 (3)：717-27).MNAC13VH, VH1, VH3, VH5 and germline VH3-023*01 (SEQ ID NO：23) sequence alignment of domain is shown, position 3,5,19,37,40,42,44,49,50,52A, 53,60,62,74, The different amino acid content in 82A, 83,84,89 and 94 (Kabat numberings).Model analysis allows to based on position to CDR region And/or the presumption of heavy chain-light chain variable domains assembling (packing) influences to select the subset of position.The position subset is： 3,37,40,42,44,49,50,60,62,89 and 94 (Kabat numberings).Thus, engineered antibody has in above-mentioned varistructure Domain sequence is (respectively with SEQ ID NO：1,3,5 VH1, VH3 and VH5) in single or multiple positions comprising substitution heavy chain Variable domains, wherein the position is selected from above-mentioned position subset.More specifically, engineered antibody by with two it is previously mentioned The above-mentioned substituted heavy-chain variable domains composition of one of light variable domains VL1 or VL3 (SEQ ID NO 6 and 8) pairing.
Molecular biology
Using the carrier DNA of BXhVH1, BXhVH3 and BXhVH5 described in WO 09/098238 as pcr template, lead to Standard mutagenesis techniques are crossed, engineering heavy-chain variable domains DNA sequences encoding (cDNA) is generated.Similarly, from WO 09/ The carrier DNA of BXhVL1 and BXhVL3 described in 098238, have directly expanded light variable domains cDNA.Filled using PCR With technology, variable domains cDNA is further assembled in the upstream of its corresponding constant domain cDNA sequence.Finally, will be complete Heavy chain and light chain cdna be respectively connecting to the separate carriers of the pcDNA3.1 carriers (Invitrogen, CA, USA) based on modification In, the carrier carries CMV promoter and bovine growth hormone polyadenylation signal.For light chain idiosyncratic carrier, pass through Purpose light variable domains cDNA is connected to κ light chain constant domains cDNA using BamHI and BsiWI restriction enzyme sites Before, so as to allow κ isotype light chain expressions；And heavy chain idiosyncratic carrier is engineered, use BamHI and SalI restriction enzymes Purpose heavy-chain variable domains cDNA is connected to coding IGHG1CH1, IGHG1 hinge areas, IGHG1CH2 and IGHG1CH3 by site Before the cDNA sequence of constant domain.In both heavy chain and light chain expression vector, before the mouse VJ2C containing BamHI sites Lead peptide driving secretion.BsiWI restriction enzyme sites are located in κ constant domains；And SalI restriction enzyme sites are tied positioned at IGHG1CH1 In structure domain.
By the way that IGHG1CH1, IGHG1 hinge areas, IGHG1CH2 and IGHG1CH3 will be encoded in the special carrier of above-mentioned heavy chain The cDNA sequence of constant domain replace with coding IGHG4CH1, with S228P replace IGHG4 hinge areas, IGHG4CH2 and The cDNA sequence of IGHG4CH3 constant domains, obtains the IGHG4 Ig-formats with substitution S228P.Replace S228P By standard PCR induced-mutation techniques, it is introduced into people's IGHG4 heavy chain cDNA templates.
Antibody producing
For the transient expression of antibody, using polyethyleneimine (Polyplus is transfected, Illkirch Cedex, France), by the heavy chain of equivalent and light chain vector cotransfection enter the adaptation that suspends HEK293-EBNA1 cells (Catalog number (Cat.No.)：CRL-10852 in).Typically, with the expression vector containing 50 μ g encoding heavy chains and 50 μ g encoded lights The DNA- of the expression vector of chainMixture, transfection densities suspend thin for million cells of 0.8-1.2/ml 100ml Born of the same parents.After the recombinant expression carrier of encoding antibody genes is imported into host cell, by further cultivating cell 4 to 5 days, produce Antibody, to allow antibody-secreting to culture medium (EX-CELL 293, HEK293- serum free medium；Sigma, Buchs, Switzerland in), the culture medium is supplemented with 0.1%Pluronic acid, 4mM glutamine and 0.25 μ g/ml heredity Mycin).
Using recombinant protein A Streamline media (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) the antibody purification from cell-free supernatants, and buffering is exchanged into phosphate buffered saline (PBS) before measurement.
Stability test is carried out using differential scanning calorimetry
VP-DSC differential scannings microcalorimeter (GE Healthcare Europe GmbH, Glattbrugg, Switzerland calorimetric measurement is carried out on).Cell volume is 0.128ml, and the rate of heat addition is 200 DEG C/h, and overvoltage is maintained at 65p.s.i.All antibody are used in PBS (pH 7.4) with 1mg/ml concentration.By with eliminate protein contain phase Repeat samples with buffer solution are compared, and have estimated the molar heat capacity of every kind of protein.Inclined mole is analyzed using standardization program Thermal capacitance and melting curve.Origin softwares (v7.0, GE Healthcare Europe GmbH, Glattbrugg, Switzerland before further being analyzed using non-two condition (Non-Two State) model in), baseline school is carried out to thermogram Just normalized with concentration.
SPR affinity measurement
Analyzed using SPR, the association and dissociation speed of the binding kineticses of measurement different antibodies (mouse and humanized antibody) Rate constant.At room temperature, the instruments of BIACORE 2000 (GE Healthcare Europe GmbH, Glattbrugg, Switzerland mouse antibodies and the binding kineticses of humanization variants are measured on), and use BiaEvaluation softwares (v4.1, GE Healthcare Europe GmbH, Glattbrugg, Switzerland) is analyzed.
Because antibody is bivalent molecule, therefore preferably antibody is fixed on sensor chip.If using antibody as Analyte, in addition to affinity, SPR measured values will also carry potency factor.Although BIAcore, which assesses software, to build two with mould Valency simultaneously extracts affinity constant, but preferably evades any valency sexual deviation using unitary analysis thing as much as possible.For this purpose, Digest recombined human TrkA-Fc fusion proteins (the SEQ ID NO prepared in HEK293-EBNA1 cells：24) monovalent shape, is produced People's TrkA extracellular regions of formula.The fusion protein is by being fused to people TrkA extracellular regions (the SEQ ID NO in IGHG1Fc areas：25) constitute, Protease specificity cutting sequence (TEV scinderin enzyme amino acid sequences are included wherein between Liang Ge areas：ENLYFQS).Egg After white cleavage, using standard chromatographic techniques, including protein-step A is to remove Fc fragments, by the people TrkA born of the same parents of monovalent fashion Outskirt is further purified to homogeneous.Finally, it is PBS, Ran Hou by the buffer-exchanged of monovalent people TrkA extracellular region protein matter Dilute and determined for affinity in Biacore running buffers.Sample purity, homogenieity and molecular weight are by SDS-PAGE and greatly Small exclusion assay is confirmed.
Antibody is fixed by capturing its Fc parts, to allow the correct orientation in sensor chip surface.Use Dan Ke Grand mouse anti human IgG (Fc) antibody sensors chip captures all humanized antibodies (no matter its isotype) (human antibody capture examination Agent box, catalog number (Cat.No.) BR-1008-39, GE Healthcare Europe GmbH), and ball is immunized using multi-clone rabbit anti-mouse Albumen sensor chip captures MNAC13 mouse antibodies (mouse antibodies capture agent box, catalog number (Cat.No.) BR-1008-38, GE Healthcare Europe GmbH)。
Data (sensing figure：Fc2-fc1) it is fitted 1:1Langmuir models (have mass transfer, although mass transfer is limited in mass transfer tests Very little processed).Consider the experimental variations on the antibody of capture when measurement starts every time, in all fittings, Rmax values are set To be local.Dissociation time is at least 350 seconds.Triplicate duplicate measurements is carried out, and including zero-dose sample for referring to.Make The quality being fitted between experimental data and each binding model is assessed with Chi2 and residual values.
TF-1 cell proliferating determinings
By the TF-1 cells of suspension adaptation (Number：CRL-2003) culture is containing 10%FCS and 5ng/ml In rhu β NGF (restructuring humanβ-NGF, R＆D Systems Europe Ltd, Abingdon, UK) complete RPMI culture mediums.It is right In measure, TF-1 cells are incubated 5 hours in the complete RPMI of no humanβ-NGF.After the hungry step, by cell from The heart is simultaneously seeded in the recombinant beta-NGF of the various antibody with various concentration and fixed concentration flat 96 orifice plate.TF-1 is thin Born of the same parents are inoculated with the density of 7000 cells/wells, and total antibody-cell volume of mixture is 200 μ l, with 5ng/ml people β- NGF (final concentration).Supplemented at 37 DEG C plus CO 2, be incubated the mixture 4 days.At the 3rd day, by colorimetric dye Alamar blue (AbD Serotec, Morphosys AbD GmbH, D ü sseldorf, Germany) is added in each hole, and incubation conditions do not have There is any change.At the 4th day, in Bio-Tek SynergyTM2 spectrophotometers/microplate reader (BioTek Instruments GmbH, Luzern, Switzerland) on, using 530 to 560nm excitation wavelengths and 590nm launch wavelengths, Read fluorescence.Tested at least twice, and triplicate duplicate measurements is carried out for each antibody concentration.
As a result
Engineering builds the anti-TrkA antibody of new humanized based on BXhVH5VL1
3D models based on VH5-VL1 variable domain pairs, have selected different heavy chains variable domains (VH1, VH3 and VH5 one group of common 3D position in), is mutated for mouse to people and people to mouse；This group of position is made up of following position：3, 37,40,42,44,49,50,60,62,89 and 94 (Kabat numberings).On substitution combination used herein, it is engineered strategy What is be based on is that different substituted complementarity (are just substituted in CDR region and/or variable domains assembling (packing) and/or immune For presumption influence in originality).
In first method, in IGHG1 isotype forms, in BXhVH5VL1 candidates being engineered mouse arrives The mutation of people and people to mouse.
The mouse of progress includes following substitution to people's mutation：K3Q, T40A, R44G, A49S, Y50A, P60A, T62S and R94K.Shown respectively based on these monosubstituted or substitution combination some production yields and affinity for being engineered anti-TrkA antibody In tables 1 and 2.Combination is most of or all mouse are to people's mutation, result in that production yields is poor, completely loses the knot with TrkA Close.For example, mouse replaces A49S and Y50A combination completely losing induction of combination to people, and this combine forfeiture can not be by Other mouse replace to people to be saved；And mouse replaces K3Q, T40A, R44G and R94K to people, arrives individually or with other mouse People's substitution combination, any improvement of affinity is not caused.
The people of progress includes following substitution to mouse mutation：V37A, G42E and V89L.People has most to mouse substitution V37A Active influence, causes affinity to increase at least twice (KDReduce at least twice) (table 2 and Fig. 1)；For GBR VH5 (V37A) VL1IGHG1 antibody, recorded 2.5 times of affinity increase.When by people to mouse replace V37A and mouse to people replace P60A and When T62S is combined, this increase of affinity is eliminated, but when being combined with mouse to people's substitution K3Q and/or R44G This increase of affinity is maintained.
In a word, these results determine the importance (allowing to combine aspect) of position 49 and 50 and people replaces to mouse V37A peculiar property (BXhVH5VL1 affinity is improved at least twice).Because antibody-mediated effector function may It is unfavorable for only needing blocking TrkA treatment, so by GBR VH5 (V37A) VL1 and GBR VH5 (K3Q, V37A) VL1 again lattice Formula turns to the above-mentioned IGHG4 isotypes replaced with S228P.Two engineered antibodies are all shown and its IGHG1 isotype pair The affinity that thing is similar is answered, showing that the caused affinity of V37A substitutions improves not changed by isotype is influenceed.
The anti-TrkA antibody of new humanized is built based on BXhVH1VL1, BXhVH3VL1, BXhVH3VL1 and BXhVH5VL3
In the second approach, V37A is replaced and be introduced into the candidate of other selections from WO 09/098238, and And on the basis of BXhVH5VL1 and MNAC13 antibody, the affinity for being engineered anti-TrkA antibody is have detected by SPR.Table 3 and 4 Respectively illustrate yield and affinity that V37A replaces antibody.Although engineered antibody has differences in terms of its production yields, But all engineered antibodies all show the raising of affinity after V37A substitutions are introduced.For BXhVH3VL1, BXhVH1VL1, BXhVH3VL3 and BXhVH5VL3, affinity have been respectively increased 8%, 20%, 30% and 55%.Therefore, people arrives Mouse substitution V37A result in affinity increase when not only being introduced in BXhVH5VL1, and most unexpectedly also all Affinity increase is result in humanization variants.It is worth noting that, GBR VH5 (V37A) VL1 and GBR VH5 (K3Q, V37A) VL1, in IGHG1 or IGHG4 S228P isotype forms, the parent matched with the measure affinity with MNAC13 mouse antibodies With power (table 2 and table 4)；Compared with BXhVH5VL1 antibody, both of which increase at least twice (KD2.5 to 2.7 times are reduced respectively).
Table 3 and Fig. 2 are included in the melting temperature of the FAB parts measured in engineered antibody.Monoclonal antibody melting curve It is its isotype institute characteristic, but the midpoint melting temperature (Tm) of FAB fragments or even can also in full-length immunoglobulin Easily identify (Garber E and Demarest SJ (2007) Biochem.Biophys.Res.Commun.355(3)：751- 7).The stability for carrying out monitoring works candidate is melted using the midpoint of FAB parts.GBR VH5 (V37A) VL1 and GBR VH5 (K3Q, V37A) VL1FAB fragments respectively show the single transition at 73.6 and 73 DEG C, and both have suitable heat endurance, And the shape and amplitude of the FAB transition are consistent with generally directed to the collaboration unfolding observed by compact-folded FAB fragments, It is successful in terms of FAB stability is kept to show the engineering method.When comparing GBR VH5 (V37A) VL1 or GBR VH5 During (K3Q, V37A) VL1 FAB Tm and MNAC13FAB Tm (74 DEG C), this, which further obtains citing, proves that Tm differences are at 1 DEG C Or it is following.
It is engineered the functional test of anti-TrkA antibody
Determine the ability (figure that the anti-TrkA antibody of engineering suppresses TrkA functional activations in TF-1 cell proliferating determinings 3).Calculating half maximum suppression concentration (IC50) of each curve --- compound suppresses measuring for the validity of biological function, As a result it is listed in table 5.Note, GBR VH1 (V37A) VL1 by SRP measurements have relatively low affinity and undetermined due to it.
It was found that GBR VH5 (V37A) VL1 is optimal representation person, be afterwards with identical IC50 GBR VH5 (K3Q, V37A) VL1, GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P and GBR VH3 (V37A) VL1.GBR VH3(V37A)VL3 There is slightly higher IC50 with GBR VH5 (V37A) VL3 antibody, but the performance of the engineered antibody of all tests is superior to BXhVH5VL1, wherein ten times of GBR VH5 (V37A) VL1 are better than BXhVH5VL1.It is worth noting that, with MNAC13 mouse antibodies Compare, GBR VH5 (V37A) VL1 antibody also show the effect increase of three times in TF-1 cell proliferating determinings.When with this When mouse parental antibody is compared, all V37A variants have suitable or more preferable IC50.
Table 1：The yield of the anti-TrkA antibody of engineering based on BXhVH5VL1 from selection；Unless otherwise indicated, institute It is IGHG1 isotypes to have antibody.(*) has the IGHG4 isotypes that S228P replaces.+Including mouse as described in Example 1 It is mutated to people and people to mouse.
Table 2：The affinity of the anti-TrkA antibody of engineering based on BXhVH5VL1 of selection；Unless otherwise indicated, Suo Youkang Body is IGHG1 isotypes.(*) has the IGHG4 isotypes that S228P replaces.N.B. no combination is indicated.
Table 3：The production yields and FAB thermal stability datas of the anti-TrkA antibody of engineering from selection；All antibody are equal For IGHG1 isotypes.
Table 4：V37A is engineered the affinity of anti-TrkA antibody；All antibody are IGHG1 isotypes.
Anti- TrkA antibody kon(1/Ms) koff(1/s) KD(nM)
MNAC13 6.02x104 1.09x10-3 18
BXhVH1VL1 3.13x104 1.33x10-2 426
GBR VH1(V37A)VL1 1.96x104 6.63x10- 3 338
BXhVH3VL1 4.65x104 6.19x10-3 133
GBR VH3(V37A)VL1 3.82x104 4.71x10-3 123
BXhVH3VL3 3.14x104 4.59x10-3 134
GBR VH3(V37A)VL3 3.1x104 2.95x10-3 95
BXhVH5VL1 1.04x105 4.26x10-3 40.9
GBR VH5(V37A)VL1 1.4x105 2.25x10-3 16
GBR VH5 (K3Q, V37A) VL1 1.7x105 2.62x10-3 15.4
GBR VH5VL3 6.23x104 3.95x10-3 63
GBR VH5(V37A)VL3 7.45x104 2.05x10-3 28
Table 5：V37A is engineered the TF-1 cell proliferating determinings IC50 of anti-TrkA antibody；Unless otherwise indicated, all antibody It is IGHG1 isotypes.(*) has the IGHG4 isotypes that S228P replaces.N.D. undetermined is represented.
Anti- TrkA antibody IC50(μg/ml)
MNAC13 0.23
BXhVH5VL1 0.73
GBR VH1(V37A)VL1 N.D.
GBR VH3(V37A)VL1 0.15
GBR VH3(V37A)VL3 0.29
GBR VH5(V37A)VL1 0.075
GBR VH5 (K3Q, V37A) VL1 0.15
GBR VH5 (K3Q, V37A) VL1 (*) 0.15
GBR VH5(V37A)VL3 0.22
Embodiment 2：The anti-TrkA antibody of humanization reverses the pawl acute inflammation pain sensation mistake induced by intraplantar injection CFA It is quick.
Complete Freund's adjuvant (CFA) intraplantar injection is entered to a rear solid end for mouse, causes acute inflammation hyperalgia, its Load-bearing method can be used to be estimated.Body weight is generally equally distributed in two by first (naive) mouse for being used to test Between rear solid end.However, when the CFA rear solid end inflammation injected and pain, weight is reallocated, to mitigate the pawl for being placed on injection Weight on (homonymy), and increase the weight on non-injection (offside) rear solid end.
Method
It is logical using biped balance pain threshold detector (incapacitance tester) (Linton Instruments, UK) measurement Cross the weight that each hind leg is born.Mouse is placed in balance pain threshold detector, rear solid end is separated on single sensor, and And record the mean force applied in two seconds by two hind legs.First (naive) the AMB1 mouse for being used to test are made to be raised at it Operating room is adapted in cage, arbitrarily takes food and drinks water.By the extron that mouse TrkA exons 1 is replaced with to its people's homologue 1 and produce AMB1 mouse, thus the mouse only expresses people's TrkA albumen.Adapted to balance pain threshold detector through several days.Induced damage it Before take baseline load-bearing record.Inflammatory hyperalgesia by intraplantar injection CFA (20ul 1.5mg/ml solution) to left back pawl come Induction.Before processing load-bearing reading is taken to assess the hyperalgia of 23 hours after CFA.Then according to CFA in Latin―Square design Window is ranked up and randomized grouping to animal.24 hours after CFA, pass through single intraperitoneal injection 0.1mg/kg isotypes pair According to or 0.0001,0.001,0.01 and 0.1mg/kg antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P i.p. (10ml/kg dose volumes), handles animal.Non-selective NSAID Indomethacins are used as positive control with 10mg/kg p.o (5ml/kg dose volumes).The 4th after antibody/drug-treated, read load-bearing reading within 8,24,48,72,96 and 120 hours.
Blind carries out behavior evaluation.For homonymy and contralateral hindpaw collection load-bearing (g) reading, and it is expressed as % weight bearing differences (%ipsi/contra).By comparing treatment group and isotype controls group, analyze data at each time point.With duplicate measurements Variance analysis (repeated measures ANOVA) carries out statistical analysis, then carries out Planned using InVivoStat Comparing check (p<0.05 is considered as notable).
As a result
CFA intraplantar injections cause 23 hours after significant hyperalgia, such as CFA and detect that load-bearing is from lateral offside Rear solid end is migrated, and causes %ipsi/contra ratios to decline (Fig. 4).The isotype controls processing started for 24 hours after CFA, shows Show does not influence on hyperalgia.Compared with isotype controls, 0.01 and 0.1mg/kg antibody GBR VH5 (K3Q, V37A) are used VL1IGHG4S228P, 4-48 hours upon administration, it was observed that hyperalgesic notable reverse.0.001mg/kg or following dosage Antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P it is invalid in terms of reverse hyperalgesia.Indomethacin (10mg/ Kg) shown at all time points of test to hyperalgesic notable reverse.In a word, antibody GBR VH5 (K3Q, V37A) VL1IGHG4S228P, similar to Indomethacin, result in the hyperalgesic notable reverse of pawl acute inflammation.In addition, apply GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P effective dose (0.01 and 0.1mg/kg), than the effective agent of Indomethacin Measure (10mg/kg), much lower times, show that anti-TrkA antibody can be applied with the much lower dosage of the standard care than the illness.
Embodiment 3：The anti-TrkA antibody of humanization reverses the knee joint chronic inflammatory pain sensation mistake as caused by intra-articular injection CFA It is quick
Intra-articular injection CFA is one of to mouse hind leg knee joint, inducing chronic Inflammatory hyperalgesia, and it can use load-bearing Method is estimated.When the CFA Joint Inflammations injected and pain, weight is reallocated, and injection is placed on (together to mitigate Side) weight on the limbs of joint, and increase do not inject the weight on the limbs of (offside) joint.These marks in this animal model The appearance of elephant is considered as clinically relevant, because they reflect chronic inflammatory arthralgia, (pain is closed with such as bone Section is scorching related to the potential illness of rheumatoid arthritis etc) symptom shown by patient.
Method
The first AMB1 mouse for being used to test is adapted to operating room in rearging cage, arbitrarily take food and drink water.By will be small Mouse TrkA exons 1 is replaced and produces AMB1 mouse for the exons 1 of people's homologue, and thus these mouse only express people TrkA Albumen.Custom balance pain threshold detector.Face and take baseline load-bearing reading before CFA injections.Use aseptically 3:The different fluorine of 1 mixing Alkane and oxygen anesthetized animal.Shaving knee area and with dilution Hibiscrub solvent cleans.Left knee injects 10 μ l 10mg/ mlCFA.Allow animal to recover in warm environment, be then back to rearging cage.Use load-bearing side within the 4,7th and 10 day after CFA Method assesses the Inflammatory hyperalgesia development of animal.According to the measurement of the 10th day after CFA, according to the CFA windows of animal, animal is carried out Sort and be randomly assigned to treatment group.13 days after CFA, when hyperalgia has been established, single injection isotype controls resist Body (10mg/kg, intraperitoneal) or antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P (0.01,1 and 10mg/kg, peritonaeum It is interior), handle animal.COX-2 selective N SAID celecoxibs are using 60mg/kg p.o as positive control, twice daily.For Antibody treatment group, upon administration measurement load-bearing in 4,8,24 and 96 hours.For celecoxib treatment group, after CFA the 13rd day Measurement load-bearing in 1 and 8 hour after administration, then the measurement load-bearing in 1 hour upon administration in 14-17 days after CFA.Blind carries out behavior and commented Estimate.For homonymy and contralateral hind limb, load-bearing (g) reading is taken, and be expressed as % weight bearing differences (%ipsi/contra).By Each time point compares treatment group and isotype controls group, analyze data.Statistical analysis is carried out with duplicate measurements variance analysis, so Afterwards Planned comparing checks (p is carried out using InVivoStat<0.05 is considered significant).
As a result
As detected by shifting by the load-bearing with lateral contralateral hind limb and (cause %ipsi/contra ratios to decline), Since the 3rd day, CFA injection knee joints caused obvious and lasting hyperalgia (Fig. 5).Isotype control antibodies processing pair Hyperalgia is without influence.Using celecoxib (60mg/kg), in processing procedure, hyperalgia is observed within 1 hour upon administration Notable reverse.Single injection 0.01,1 and 10mg/kg antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P, in administration Hyperalgesic notable reverse, at 96 hours compared with isotype controls, only two agent of highest are observed within 4-72 hours afterwards Amount is significant.In a word, single dose 0.01mg/kg or more antibody GBRVH5 (K3Q, V37A) VL1 IGHG4 S228P, similar In multi-agent celecoxib, the hyperalgesic notable and lasting reverse of knee joint chronic inflammatory result in.Moreover, and multi-agent 60mg/kg celecoxib is compared, it is only necessary to single dose 0.01mg/kg antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P Effect is can reach, this shows that antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P can be with than NSAID celecoxib (current standard care) much lower dosage and low frequency are applied.
Embodiment 4：The anti-TrkA antibody of humanization reverses the knee joint arthroxerosis pain as caused by intra-articular injection MIA Feel allergy
Intra-articular injection sodium iodoacetate (MIA) causes to occur related with part arthritis chronic to mouse knee joint Hyperalgia is with cartilage degradation.Therefore, it is ache related that the hyperalgia measured in MIA models is very similar to osteoarthritis. In addition, cartilage degradation causes the chronic neuronal damage with europathology sample feature.Pregabalin (a kind of anticonvulsant drug) It is the first-line drug for the treatment neurogenic pain recommended, therefore is used as comparative compound herein.C16H25NO2 (a kind of weak μ-Ah Piece sample receptor stimulating agent) it is increasingly being used for treating OA, because different from NSAID, C16H25NO2 does not cause stomach and intestine to be said Blood or kidney problems, nor affect on articular cartilage.Therefore, together with Pregabalin, antibody GBR VH5 are used as using C16H25NO2 (K3Q, V37A) VL1 IGHG4 S228P comparative.
Method
The first AMB1 mouse for being used to test is adapted to operating room in rearging cage, arbitrarily take food and drink water.By will be small Mouse TrkA exons 1 replaces the exons 1 for people's homologue, produces AMB1 mouse, and thus these mouse only express people TrkA Albumen.Pain threshold detector is balanced through several days customs.Take baseline load-bearing reading.Use aseptically 3:The isoflurane and oxygen of 1 mixing Gas anesthetized animal.Shaving knee area and with dilution Hibiscrub solvent cleans.By 5 μ l 100mg/ml (500 μ g) MIA or Salt solution (false mould) is expelled in the knee joint of left back leg.Allow animal to recover in warm environment, be then back to rearging cage. The 3-23 days after MIA, using load-bearing, regular intervals of time assesses the hyperalgesic development of animal knee joint.The 14th day after MIA, enter Row load-bearing is measured, and animal is ranked up according to its MIA Process window and is randomly assigned to treatment group, then single intraperitoneal injection 1st, 10,100 μ g/kg antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P or 100 μ g/kg Isotype control antibodies (5ml/kg dose volumes) is handled.Compareed as comparative, animal after MIA the 14th day with 10mg/kg C16H25NO2s or 30mg/kg Pregabalins p.o. (5ml/kg dose volumes) is handled, then after MIA the 16-22 days every other day with 30mg/kg C16H25NO2s or 100mg/kg Pregabalins (5ml/kg dose volumes) processing.The 14th day upon administration 4,8 after MIA All animals were assessed using load-bearing with 24 hours, antibody treatment group is assessed for every 24 hours afterwards, C16H25NO2 and Pregabalin processing Group is assessed for 1 and 24 hour upon administration.Blind carries out behavior evaluation.For homonymy and contralateral hind limb collection load-bearing (g) reading, and It is expressed as % weight bearing differences (%ipsi/contra).By comparing treatment group and vacation at each time point (after MIA the 3-14 days) Module, analyze data understands MIA to hyperalgesic influence.By comparing treatment group and isotype controls at each time point Group, data after analysis administration.Statistical analysis is carried out with duplicate measurements variance analysis, then carried out using InVivoStat Planned comparing checks (p<0.05 thinks notable).
As a result
As detected by shifting by the load-bearing with lateral contralateral hind limb and (cause %ipsi/contra ratios to decline), 500 μ g MIA is expelled in knee joint, since the 3rd day, obvious hyperalgia is caused.At each time point and together The type control of kind is compared, single injection 10 and 100 μ g/kg antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P, in administration 4 hours to 7 days afterwards, it was observed that hyperalgesic notable reverse (Fig. 6 A).1 μ g/kg antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P are on hyperalgia without influence.C16H25NO2 (10mg/kg) and Pregabalin (30mg/kg) after MIA the 14th day Do not act within 1 hour after administration, but show within 4 hours notable reverse hyperalgesia (Fig. 6 B) upon administration.Due to after MIA Lack effect within 1 hour upon administration, therefore C16H25NO2 is increased into 30mg/kg within 14 days, Pregabalin increases to 100mg/kg, both Show notable reverse hyperalgesia within 1 hour upon administration within the 16th, 18 and 20 day after MIA.In a word, the μ g/kg of single dose 10 or Antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P of above dosage, similar to multi-agent C16H25NO2 and Pregabalin, lead Cause knee joint chronic osteoarthritic is hyperalgesic significantly and persistently to reverse.In addition, with (distinguishing in remote much higher dosage 30mg/kg and 100mg/kg) more divided doses of two kinds of medicines (C16H25NO2 and Pregabalin) that use compare, and only need the μ of single dose 10 G/kg antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P are that can reach effect.Therefore, anti-TrkA antibody can with than C16H25NO2 and the much lower dosage of Pregabalin and low administration frequency are applied.
Embodiment 5：The anti-TrkA antibody of humanization reverses peripheral nerve pathology caused by chronic constriction injury of sciatic nerve Property hyperalgia and allodynia
Peripheral nerve pathologic pain is the damage by peripheral nervous system, chronic ache caused by dysfunction or disease Form.It generally includes hyperalgia (the reaction increase to pain stimulation) and allodynia (to the pain of non-pain stimulation Pain reaction).Neurogenic pain can be induced by the experimental injury of peripheral nerve in animal, and this can generally pass through (Bennett＆Xie (1998) Pain, 33 are realized in loose ligation by the chronic constriction injury (CCI) of sciatic nerve：87- 107).Because Pregabalin is a line neurogenic pain medicine of recommendation, therefore it is used as comparing chemical combination herein Thing.
Method
Under Ketamine-Xylazine (100 and 10mg/kg/10ml, i.p.) anesthesia, in male and female AMB1 mouse Carry out CCI operations.By the way that mouse TrkA exons 1 to be replaced to the exons 1 for people's homologue, AMB1 mouse are produced, thus These mouse only express people's TrkA albumen.After hair is shaved off, in the left sciatic nerve of the sterile exposure of stock stage casing level.In nerve point Support the front at 1-2 millimeters and to place three loose ligatures (use 4-0 black threads) around sciatic nerve.After skin closure, with poly- Vinyl pyrrolidone iodine solution handles operative site.Animal is allowed to be recovered at ensuing seven days.In experiment the previous day, make Animal adapts to measurement apparatus (cold drawing), and records the pawl withdrawal threshold (0 hour) before administration.Then animal is divided into six again Group.Second day, the antibody GBR VH5 of single intraperitoneal injection Isotype control antibodies (1000 μ g/kg/10ml) or various dose (K3Q, V37A) VL1 IGHG4 S228P (10,100 and 1000 μ g/kg/10ml), handle animal.The period after the administration of 7 days, Pregabalin (30mg/kg/10ml, p.o.) or salt solution are applied once a day.In 4h, 24h, then after every other day record administration Reading, until the 7th day after administration.Have a fixed day applied in Pregabalin or salt solution after reading after the administration of 1 hour record.First Reading after the hyperalgesic administration of mechanicalness is recorded, the reading of cold allodynia is recorded after 5 minutes.Use dynamic plantar tactile Measuring appliance (Plantar Von Frey Instrument；Ugo Basile Srl, Italy), mechanical hyperalgesia is surveyed Measure as the threshold force (g) needed for triggering pawl retraction.The time spent in cold allodynia is measured as bouncing back pawl from cold drawing in seconds (IITC Life Science Inc., USA).
As a result
CCI Post operations 7 days, the drastically decline for the time delay that bounced back respectively by pawl withdrawal threshold and pawl, it was observed that mouse Show significant mechanical hyperalgesia (Fig. 7 A) and cold allodynia (Fig. 7 B).Antibody GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P all dosage have resulted in the reverse of mechanical hyperalgesia (such as by the increase finding of pawl withdrawal threshold) The reverse (the increase finding for the period of delay that such as bounced back by pawl) (Fig. 7 B) of (Fig. 7 A) and cold allodynia.The degree of reverse and Duration is substantially dose dependent, and for two readings, the Puri with 1mg/kg (1000 μ g/kg) maximum dose level Bahrain is suitable.Compared with multi-agent 30mg/kg Pregabalins, antibody GBR VH5 (K3Q, V37A) the VL1 IGHG4 of 1mg/kg dosage S228P is effective when being applied with single dose, is illustrated again compared with Pregabalin standard care, anti-TrkA antibody can with compared with Low dosage and relatively low administration frequency are applied.
Embodiment 6：Influence of the anti-TrkA antibody to sensation and sympathetic neuron during neonatal development
NGF/TrkA signal transductions be feel in growth course and sympathetic neuron survival institute it is crucial necessary to (Bibel and Barde, 2000Genes Dev 14 (23)：2919-37).Correspondingly, by actively or passively between embryonic period, embryonic phase or neonatal period It is immune to block NGF, notable loss and neuron cell body and the related neural meta structure such as friendship of these neurons can be caused Feel ganglionic obvious atrophy (Cattaneo, (2013) Proc Natl Acad Sci USA.2013 March 26；110 (13)：4877-85).On the contrary, causing the hypertrophy and hyperplasia of sympathetic neuron with NGF processing neonate animals, cause sympathetic nerve Section increase (Levi-Montalcini and Booker, 1960a Proc Natl Acad Sci U S A.Mar；46(3): 373-84.；Levi-Montalcini and Cohen,1960Ann N Y Acad Sci.1960Mar 29；85:324-41； Banks et al.,1975 J Physiol.May；247(2):289-98).
In order to determine compared with anti-ngf antibodies, between neonatal period the processing of anti-TrkA antibody to the survival of sympathetic neuron and The influence of form, after the processing in 4 weeks of (AMB1) mouse is knocked in people TrkA, has carried out the friendship being located in superior cervical ganglion (SCG) Feel the quantitative and morphometric analysis in detail of neuron.It is because GBR VH5 (K3Q, V37A) that people TrkA, which knocks in mouse and is required, VL1IGHG4S228P and rodent TrkA has weak cross reactivity.Neonate mouse was used from after birth the 1st day 100mg/kg GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P, a kind of anti-NGF (antibody based on tanezumab sequences There is complete intersection reaction with mouse NGF) (Cattaneo, 2010 Curr Opin Mol Ther, 12,94-106) or PBS, Weekly intraperitoneal (i.p.) is handled 4 weeks.
At the end of processing, by mouse ketamine [(Parke-Davis) 75mg/kg body weight] and Xylazine The mixture anesthesia of [(Streuli) 10mg/kg body weight], then irrigates 0.9%NaCl and afterwards in 0.1M phosphate buffers Freshly prepared 4% paraformaldehyde in (pH 7.3).
The right superior cervical ganglion of neck (superior cervical ganglion, SCG) is dissected, is used in plastic embedding box Foam protect, and at 4 DEG C in identical fixative after fix 24 hours.SCG peripheral arterial is dissected, to allow in tissue Preferably recognized during.Then it is embedded in paraffin (TCA 44-720Medite automate), and is cut with card is come Piece machine (3 μm of slices；Leica RM2135) section.Serial section band is prepared, every 10th (adult) or the 20th is (newborn Youngster) cut into slices to be locked in and " on Superfrost plus " (Menzel) slide, and dyed with h and E (H＆E).With Hamamatsu (Nanozoomer) slide scanning system scan slice, and amplified with 40x object lens.Picture (.ndpi) is in NDP- Amplified in Viewer2 softwares with 10x or 20x object lens, then exported with jpeg forms, determined for volume.Ganglionic volume According to the Cavalieri methods (volumetric estimate in biological structure；Cold Spring Harb Protoc 2012 Nov1；2012 (11)：1129-39；doi：10.1101/pdb.top071787) determine.Using with appropriate plug-in unit (" grid " and " cytometer Number devices ") ImageJ softwares carry out stereometry.Selection size of mesh opening saves 100 points or more to obtain each nerve.
Volume is calculated as follows：Between the structural points x grids area × thickness × section of neuromere volume=neuromere Distance.According to nucleator principle (The Nucleator, J Microsc.1988 Jul；151(Pt 1)：3-21), it is determined that carefully The average external volume of born of the same parents.The area (A) and radius (R) of object are related to its volume (V), and this is also applied for the irregular of such as cell Object.Therefore, by measuring the area of a large amount of cells, the extraordinary approximate of mean corpuscular volume in neuromere can be obtained Value.The area of each ganglionic 100 or more the randomly selected cells of measurement.For each region (A), radius is calculated (r=√ (A/ π)), then calculates volume (volume=(4/3) π * r3).The average value of all volumes is taken as ganglionic average Cell volume.Cell number in one neuromere is calculated according to below equation：Ganglionic total cell volume/ganglionic average Cell volume.Cell density is calculated according to the following formula：Gangliocyte number/neuromere volume.
Compared with PBS processing, SCG neuronal cell diameters are caused to substantially reduce with Tanezumab processing newborn mices, it is whole Individual SCG atrophy is substantially reduced (Fig. 8) with each ganglionic neuronal cell quantity.By contrast, phase is handled with PBS Than GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P processing causes SCG neuronal cell diameters to dramatically increase, whole SCG Hypertrophy, and each ganglionic neuronal cell quantity dramatically increases.
Embodiment 7：Influence of the anti-TrkA antibody to the sympathetic neuron survival of manhood
After neonatal period, sensory neuron survival is unrelated with NGF.However, for sympathetic neuron, NGF the manhood after It is continuous to adjust its form.In adult rodent, NGF is blocked to cause sympathetic neuronal cells by being actively or passively immunized Body atrophy, generally facilitates atrophy (Levi-Montalcini and Booker, the 1960b Proc Natl of sympathetic ganglion Acad Sci U S A.Mar；46(3):384-91；Angeletti et al.,1971a.Brain Res.Apr 2；27(2): 343-55；Bjerre et al.,1975Brain Res.Jul 11；92(2):257-78；Goedert et al, 1978Brain Res.Jun 9；148(1):264-8；Otten et al.,1979Brain Res.Oct26；176(1):79- 90.；Gorin and Johnson,1980Brain Res.1980Sep29；198(1):27-42.；Johnson et al., 1982；Ruit et al.,1990J Neurosci.Jul；10(7):2412-9.) on the other hand, adult grinding tooth is handled with NGF Class animal causes sympathetic neuron loose, but different from neonatal processing, and hyperplasia (Levi-Montalcini and are not caused Booker,1960b Proc Natl Acad Sci U S A.Mar；46(3):384-91；Angeletti et al.,1971b J Ultrastruct Res.1971b Jul；36(1):24-36.；Bueker and Schenkein,1964Ann N Y Acad Sci.Oct 9；118:183-205.).
In order to which GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P are handled compared with anti-NGF to sympathetic during determining adult The influence of neuronal survival and form, with 100mg/kg GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P, tanezumab Or PBS intraperitoneals processing adult (2.5 monthly age) mouse, once in a week, carry out 4 weeks, and analyze left and right as described in Example 6 SCG。
Adult mice handled with tanezumab cause SCG neuronal cell diameters to substantially reduce, entirety SCG atrophy, but It is different from neonate, each ganglionic neuronal cell number (Fig. 9) is not influenceed.GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P processing causes SCG neuronal cell diameters to dramatically increase, entirety SCG hypertrophy but again, each ganglionic god It is unaffected through first cell number.Tanezumab atrophy, which is acted on, reaches similar degree in neonate and adult, and GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P loose effect is more much more obvious than in adult in neonate (to compare Fig. 8 With 9).
Although influences of GBR VH5 (K3Q, V37A) the VL1 IGHG4 S228P to SCG neuronal cell diameters is small in adult Be in mouse it is significant, but GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P processing mouse cell dia scope and PBS The animal of processing is completely overlapped, and the SCG neuronal cells diameter of significantly ratio is located at PBS in the animal of tanezumab processing Outside the scope of the animal of processing and less than the scope (Figure 10).
Visually, the SCG neuron cell bodies of the animal of GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P processing are seen It is similar to the animal of PBS processing up, and there is shrinkage and condensing in the SCG neuron cell bodies of the animal of Tanezumab processing Cytoplasm (Figure 11).
The result obtained with tanezumab is consistent with previous studies, it was demonstrated that NGF blockings can cause sympathetic in neonate The loss of neuronal cell and sympathetic neuronal cells body and ganglionic atrophy in neonate and adult.GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P to SCG loose effect and the proliferative effect in neonate to SCG, with for It is similar that NGF processing is reported.Therefore, GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P and tanezumab significantly exists Opposite effect, but GBRVH5 (K3Q, V37A) VL1 IGHG4 are produced in both neonate and adults to sympathetic neuron Acting in adults for S228P reduces.
Embodiment 8：Effect of the anti-TrkA antibody to periphery neurogenic pain
Peripheral nerve pathologic pain is the chronic ache as caused by damage/dysfunction/disease of peripheral nervous system Form (Bridges et al., (2001) Br J Anaesth.UL；87(1)：12-26).It generally includes hyperalgia (to pain The reaction increase of stimulation) and allodynia (to the pain reaction of non-pain stimulation).Neurogenic pain can pass through Experimental injury peripheral nerve is induced in animal, and this can generally be damaged by the chronic constriction loosely ligatured by sciatic nerve Hinder (CCI) to realize.
In male and female AMB1 mouse, the threshold force that mechanical hyperalgesia is measured as causing pawl retraction required (g) cold allodynia, is measured as the time (second) from cold drawing retraction pawl.Then in Ketamine-Xylazine (100 and 10mg/ Kg/10ml, i.p.) the lower progress CCI operations of anesthesia.After hair is shaved off, in the left sciatic nerve of the sterile exposure of stock stage casing level. Three loose ligatures are placed before nervous ramification around sciatic nerve at 1-2 millimeters (using the black silks of 4-0).After skin closure, use Betadin solution handles operative site.Animal is allowed to recover at ensuing seven days.Animal is set to adapt to measurement dress Put (cold drawing), and record the pawl withdrawal threshold (0 hour) before administration.Then, single intraperitoneal injection 0.3 and 1mg/kg GBR VH5 (K3Q, V37A) VL1IGHG4S228P, tanezumab or salt solution, handle animal.4h, the 1st day and then every other day Until the 9th day after administration, the reading after record administration, and 14d obtains last reading upon administration.
Reading after the record hyperalgesic administration of mechanicalness, records the reading of cold allodynia first after 5 minutes.CCI Operation (0h) 7 days afterwards, mouse shows obvious mechanical allodynia (Figure 12 A) and cold allodynia (Figure 12 B), point The drastically decline of its pawl withdrawal threshold and pawl retraction time delay are not shown as.The GBR VH5 (K3Q, V37A) of two kinds of dosage VL1IGHG4S228P has resulted in the lasting reverse (such as by the increase finding of pawl withdrawal threshold) of mechanical hyperalgesia and cold The lasting reverse (the increase finding for the period of delay that such as bounced back by pawl) of allodynia.Under suitable dosage, GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P are it is apparent that than the anti-ngf antibodies based on tanezumab, cause mechanical hyperalgesia With the higher reverse of cold allodynia.
Embodiment 9：Anti- TrkA antibody is to abnormal sensory and the influence of sympathetic nerve rudiment
Multiple intra-articular injection complete Freund's adjuvant (CFA) causes lasting gonitis several weeks in mouse, causes to close Alloesthesia and sympathetic neuron rudiment and nociception behavior (Ghilardi et al., 2012Arthritis in section Rheum.Jul；64(7):2223-32).Similar dystopy rudiment is also observed in the mouse model of Bone cancer pain, and It has been suggested maintenance (Jimenez-Andrade the et al., 2011Pain.Nov for participating in pain；152(11):2564- 74).In two models, anti-NGF treatments reduce Heterotopic neurons rudiment and nociception behavior, show NGF in this process Effect (Jimenez-Andrade et al., 2011Pain.Nov；152(11):2564-74；Ghilardiet al., 2012Arthritis Rheum.Jul；64(7):2223-32).
GBR is assessed in order to be compared in multiple CFA gonalgias model with the anti-ngf antibodies based on tanezumab VH5 (K3Q, V37A) VL1 IGHG4 S228P effect, 95% oxygen light anesthesia Adult female is mixed with 3% isoflurane AMB1 mouse.Knee joint injects 10 μ l CFA (H37RA to the left；Difco Laboratories, USA, 2.5mg/ml, in mineral In oil, Sigma).Inject after CFA, animal is returned into rearging cage.Routine observation is carried out to monitor the situation of animal after injection. 7th, repetition CFA injections in 14 and 21 days.
One group of animal (n=6) carries out intra-articular injection with 10 μ l phosphate buffer solutions (PBS), to function as false mould pair According to.By training mouse to stand upright on the pad of Incapacitance Meter (load-bearing instrument, Linton) 2-3 minutes, make small Mouse adapts to experimental situation three days.Record being averaged for the weight loading that left side (homonymy) and right side (offside) rear solid end applied in 5 seconds Value.
Three measurements are carried out, and calculate the average value at each time point.Examined within (the 0th day) on the day of before first time injection CFA Look into the baseline value for the body weight being placed on pad, and the 3rd, assess again within 3 days after subsequent injections every time within 10,17 and 24 days, and 4 after being administered at the 24th day in compound, assess again within 8,24,48,72,96 and 120 hours.
The data that behavioral experiment is obtained be calculated as load-bearing than average value ± S.E.M (WBR=homonymies load-bearing/offside is held Weight × 100).Randomly choose one group of 6 mouse to compare as false mould, receive 1 PBS within every 7 days and be injected to left knee joint, by a definite date 21 My god, totally 4 injections (false mould CFA groups), and receive i.p. excipient on the 24th day after the injection of first time knee joint.
Other mouse are the 0th, receive within 7,14 and 21 days totally 4 CFA injections.At the 24th day, load-bearing was than small less than 70% Mouse assigns to excipient control group, 1mg/kg GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P groups and 1mg/kg at random Tanezumab groups.All equal Intraperitoneal medications of excipient/compound, single dose 5ml/kg.Such as by load-bearing from after with lateral offside Detected by the migration of pawl, multiple intra-articular injection CFA causes lasting hyperalgia, causes %ipsi/contra ratios Drop to about 50% (Figure 13).At the 24th day when hyperalgia is well set up, after single i.p. injections, with excipient pair Photograph ratio, 1mg/kg GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P cause significant hyperalgia and reversed, and base With identical dosage there is no remarkable result in tanezumab anti-ngf antibodies.
Embodiment 10：Influence of the anti-TrkA antibody to knitting
Introduce
Ache related treatment of sufficiently fracturing is critically important to the health of patient.However, being currently available that antalgesic can It can produce sizable side effect.Nausea, calmness are normally resulted in for treating the opioid of moderate to severe pain Effect and drowsiness and respiration inhibition.Furthermore, it is possible to occur opiate addiction, and do not know whether such material can shadow Ring union.Relatively, frequent prescription is used to treat the slight nonsteroid anti-inflammatory drugs to moderate musculoskeletal pain (NSAID) have been demonstrated with sizable gastrointestinal side effect (Dib et al. (2014), Scand J Gastroenterol,49,785-9).NSAID antiinflammatory action is based on the selectivity or non-selection to cyclo-oxygenase (COX) -2 Property suppress, so as to block from arachidonic acid synthesis prostaglandin (PG).However, PGE-2 has pleiotropism to bone, and COX-2 functions are required (Blackwell etc. (2010) Trends Endocrinol Metab, 21,294- for knitting 301；Simon etc., (2002) J BoneMiner Res, 17,963-76).In rodent, there is very strong evidence to show, During healing union (Spiro et al., (2010) J Orthop Res, 28,785-791 can be disturbed using NSAID； Krischak et al.,(2007a)Arch Orthop Trauma Surg,127,453-8:Krischak et al., (2007b)Arch Orthop Trauma Surg,127,3-9).In addition, NSAID is recognized as having knitting in human body There is negative effect.
The ache related another method for the treatment of fracture can be block nerves growth factor (NGF)/neurotrophic junket Histidine kinase receptor type 1 (TrkA) signal transduction.Using anti-NGF monoclonal antibodies blocks signal transduction path be proved to for Treat chronic back pain and arthropathy effectively (Katz etc., (2011) Pain, 152,2248-2258；McKelvey etc., (2013) J Neurochem, 124,276-89).NGF and TrkA combination causes Receptor dimerization and the acceptor for passing through receptor auto-phosphorylation Activation.Can by application selective binding and neutralize NGF specific antibody or by application Trk kinase activations it is small Molecular weight inhibitor come destroy ligand-receptor interaction (Kumar and Mahal, (2012) J Pain Res, 5,279- 87:Watson et al.,(2008).Biodrugs,22,349-359)。
Influence of the NGF/TrkA signal transductions to repair of fractured bones is not yet fully explained.Recently, two researchs are reported, After the application anti-NGF monoclonal antibodies of neutrality or small-molecular-weight Trk kinase inhibitor blocks NGF/TrkA signal transductions, pain Pain corelation behaviour substantially reduces (Koewler etal., (2007) J Bone Miner Res, 22,1732-42.:, Ghilardi et al.,(2011)Bone,48,389-98).However, two are studied the increase for reporting poroma size, and And slight decrease (Koewler etc., (2007) J of the bio-mechanical property of the bone after healing are also indicated that in a report BoneMiner Res, 22,1732-42), may interference of the explanation to fracture healing process.The shadow observed due to two researchs Sound is relatively slight, it is therefore desirable to further clarification.Therefore, we pass through shaft fracture-healing mouse model in machinery definition The middle targeting of application respectively NGF and TrkA neutralizing monoclonal antibody, to solve this problem.
Method
Antibody
The anti-NGF monoclonal antibodies of neutrality are the human IgG2s based on tanezumab, and complete intersection anti-with mouse NGF Answer (Cattaneo, (2010) Curr Opin Mol Ther, 12,94-106).The anti-TrkA antibody of neutrality is GBR VH5 (K3Q, V37A) VL1 IGHG4 S228P.
Animal model is with raising
Zoopery is carried out according to country and International Laboratory Animal nursing and guide for use, and obtains local Ethics Committee (T ü bingen, No. 1144) approval.Male AMB1 mouse are in mouse TrkA genes People TrkA is knocked at seat, and is obtained from Charles River (Charles River Italia, Calco, Italy).By mouse With every group of most four animal packet cage, under 23 DEG C and 55 ± 10% humidity, illumination in 14 hours and 10 hours dark cycles.Mark Accurate rodent chows and water can be obtained arbitrarily.Aseptically carry out surgical operation.
Research and design
In order to study influence of the NGF-TrkA signal blockers to union, as earlier detailed (Deng (2010) J Orthop Res, 28,1456-62), the osteotomy being standardized to the right femur of 13 week old mouse.In short, in whole body fiber crops Liquor-saturated (2vol% isofluranes,Abbott, Wiesbaden, Germany) under, use Gigli scroll saws (RISystem, Davos, Switzerland) produces osteotomy breach in the middle diaphysis of right femur.Using external fixator (rigidity 3N × mm-1；RISystem, Davos, Switzerland) stable osteotomy.Before surgery, mouse receives single dose antibiotic (clindamycin -2- dihydros Phosphate, 45mg × kg-1；Clindamycin, Ratiopharm, Ulm, Germany).For analgesia, perioperatively passes through drink in 1 day Tramadol hydrochloride (25mg × L is provided with water-1,Grünenthal,Aachen,Germany)。
Before surgery, mouse is randomly assigned to three groups, with application of phosphoric acid salt buffer salt solution (PBS, PAA Laboratories, Linz, Austria) (control group, n=24), anti-ngf antibodies (n=25) or anti-TrkA antibody (n=24) (two kinds of antibody are provided by Glenmark Pharmaceuticals Ltd, Switzerland).With 10mg × kg-1Body weight Concentration intraperitoneal gives antibody.These materials after surgery the 1st, apply within 6 and 11 days.
Post operation 7, mouse was euthanized in 14 or 25 days, takes out operation and offside femur.
Activity and the measurement of ground reaction force
In order to determine the analgesic activity for the antibody applied, the movable and operating limb of Post operation mouse have evaluated vertically Face reaction force (GRF), because pain can cause the load of activity reduction and the osteotomy limbs speculated to reduce.In operation Afterwards the 2nd, 5,7, analyzed within 14 and 20 days.In order to assess vertical GRF, it is allowed to mouse move freely through floor (HE6x6, AMTI, Watertown, MA, USA) in contain strong plate acrylic glass passage.Operating limb is recorded straight by blind observer The Peak vertical GRF of vertical phase, is averaged by minimum four measurements of each every mouse of time point.Using being assembled to the red of Special cage Outside line beam system (ActiMot, TSE Systems GmbH, Bad Homburg, Germany), records the activity of mouse overnight. Postoperative measured value is related to pre-operative measurements.
Biomechanics test
In order to study mechanical performance, carried out on the intact femur and the femur of osteotomy taken out at the 25th day foregoing Lossless three point bending test (Deng (2010) J Orthop Res, 28,1456-62；Wehrle et al., (2014) J Orthop Res, 32,1006-13).In brief, the near-end of femur is fixed on aluminium cylinder, and aluminium cylinder is fixed at 3 points again On the knuckle joint of bending apparatus (Z10, Zwick Roell, Ulm, Germany).Condyle of femur is not added with being fixedly positioned at bending On support.Into femur, diaphysis applies axial load in sagittal plane.According to force deflection curve (force-deflection Curve the slope (k) of the range of linearity), calculates bending stiffness.When the midpoint between fracture callus is not placed exactly in support, Consider load vector (load vector) and the distance between near-end (a) and distal end (b) support (l/2).Bending stiffness (EI) root Calculated according to asymmetric bending formula：EI=k ((a2b2)×3l-1)。
Microcomputer tomoscan (μ CT)
Scanned the 14th day and the 25th day and received using μ CT devices (Skyscan 1172, Skyscan, Kontich, Belgium) The femur obtained, with 50kV voltages and 200 μ A, is scanned with the resolution ratio of every 8 μm of pixel.In each scanning, scanning has Defined hydroxyapatite (HA) density (250 and 750mg × cm-3) two die bodys (phantom), to determine that bone mineral is close Spend (BMD).
At the 14th day, cumulative volume (TV), bone volume (BV), diaphysis fraction (BV/TV) and BMD have been carried out to whole poroma Analysis.At the 25th day, region interested in two, whole poroma and osteotomy breach before have carried out above-mentioned parameter Analysis.In order to distinguish mineralized tissue and non-mineralized tissue, apply corresponding to 641.9mg HA × cm-3Global threshold (Morgan etc., (2009) Bone, 44,335-44).
Histomorphometricall
Resected femur 24 hours in 4% formalin, the decalcification 10- in 20% ethylenediamine tetra-acetic acid (pH 7.2-7.4) 12 days, and be embedded in paraffin (Paraplast, Leica Biosystems, Wetzlar, Germany).Cutting thickness For 6 μm of longitudinal section, and using sarranine-O and fast green dyed.Use light microscope (Leica DMI6000B； Software MetaMorph, LeicaMicrosystems, Mannheim, Germany) carry out tissue group with 50 times of magnifying powers Into assessment.At all time points, analyze the fibr tissue of the whole poroma being made up of periosteum poroma and osteotomy breach, cartilage and The relative quantity of bone.
Statistical analysis
As a result it is expressed as mean+/-standard error (SEM).Examined using Shapiro-Wilk, the normal state point of inspection data Cloth.For data analysis, use SPSS statistical softwares (version 21, IBMCorp, Chicago, IL, USA).Using variance analysis, The comparison organized.Examined using Fisher LSD, carry out ex-post analysis.P≤0.05, is considered as significantly.
As a result
Activity and GRF are assessed
Before surgeryIt and Post operation 2, the movable and vertical GRF for assessing mouse for 5,7,14 and 20 days.As expection As, all groups after surgery activity all declined.At the 2nd day, compared with the animal of PBS processing, at anti-TrkA antibody The mouse of reason shows significantly higher mobility (Figure 14 A), shows the mouse of antibody processing by less pain.PBS with There was no significant difference between anti-ngf antibodies or anti-ngf antibodies and the processing of anti-TrkA antibody.In later point, do not detect To significant group difference (Figure 14 A).
At the 2nd day, in all treatment groups, vertical GRF analysis display was reduced to the 83-90% (Figure 14 B) of preoperative value. Vertically further decline within GRF to the 5th days, then slow rise was up to the 20th day, and GRF reaches the 90- of operation consent value at this moment 100%.It is not significantly different (Figure 14 B) between point at any given time, group.
Influence of the NGF-TrkA signaling interrupters to union
The tissue morphology metric evaluation of poroma composition is the 7th, carry out within 14 and 25 days.Representational section is such as Figure 15 A-I institutes Show.Point at any time, we do not detect any statistical significant difference (figure that poroma between treatment group is constituted 15J-L).Three-dimensional assessment was carried out to the poroma of the 14th day using μ CT, display poroma size, BV, BV/TV or BMD are poor without conspicuousness Different (table 6).When analyzing osteotomy or whole poroma, same situation (table 7) was found at the 25th day.Compared with PBS is applied, resist The bending stiffness that NGF antibody or anti-TrkA antibody do not significantly affect poroma (determines) (Figure 16) using lossless three point bending test.
Table 6：The μ CT analyses of 14th day fracture callus after osteotomy.
TV, total Callus volume；BV, bone volume；BV/TV, bone volume/Callus volume；BMD, bone mineral density.Tables of data It is shown as average value ± SEM；N=7-8.
Table 7：The osteotomy of the mouse handled after the healing stage of 25 days with PBS, anti-ngf antibodies or anti-TrkA antibody and complete The μ CT analyses of fracture callus.
TV, total bone is crazy about volume；BV, bone volume；BV/TV, bone volume/bone is crazy about volume；BMD, bone mineral density.
Data are expressed as average value ± SEM；N=7 8
In a word, as shown by data blocks NGF/TrkA signals not negatively affect union.
Discuss and conclusion
Reduce fracture it is ache related be patient treatment in a major issue.However, using NSAID common processing seemingly It is unfavorable for union.
Herein, investigated and whether blocked NGF/TrkA signal transductions to influence union for analgesia.Our knot Fruit shows that, using antibodies selective, osteanagenesis is not influenceed by NGF/TrkA signaling interrupters, poroma formation and it is ripe with Normally occur in the animal of neutrality anti-NGF or anti-TrkA antibody processing.
The 7th, 14 and 25 days by histology or at the 14th and 25 day by μ CT analysis and evaluations, it was observed that with PBS, Poroma composition is not significantly different in the animal of anti-NGF or anti-TrkA antibody processing.In addition, bending stiffness is unaffected, show The analgesic activity blocked by NGF/TrkA is probably safe for knitting.
Evidence suggests NGF/TrkA signal transductions can adjust bon e formation and healing, because NGF and its acceptor TrkA are Expressed (Asaumi etc., (2000) Bone, 26,625-33) by osteocyte.Vitro data points out NGF/TrkA in preosteoblast These cells after Anti-G value (Mogi etc., (2000) Life Sci, 67,1197-206) and NGF- processing in MC3T3-E1 The induction of alkaline phosphatase, thus promotes osteoblast differentiation (Yada et al., (1994) Biochem Biophys Res Commun, 205,1187-93).Research in mouse shows that TrkA is in hyperplasia, ripe and hypertrophy during union (Asaumi etc., (2000) Bone, 26,625-33) is expressed in cartilage cell and Gegenbaur's cell.In addition, it was confirmed that before ossified Along (ossification front) nearby NGF hyperplasia, ripe and expressed in loose cartilage cell and Gegenbaur's cell. Although these discoveries mean the function of the NGF/TrkA signal transductions during union, definite effect is not yet illustrated. It has been proved that part NGF applications improve union (Grills etc., (1997) J in rat fracture of rib model Orthop Res, 15,25-42).Although potential mechanism is not yet widely studied, author speculates that sympathetic nerve is dominated Increase have stimulated differentiation (Grills et al, (1997) J Orthop Res, 15,25- of the bone ancester cell to cartilage cell 42).NGF is carried out by the application anti-NGF monoclonal antibodies of neutrality or small-molecular-weight Trk kinase inhibitors during union After signaling interrupter, it was recently reported that the formation of notable bigger poroma (Koewler etc., (2007) J Bone Miner Res, 22, 1732-42：Ghilardi etc., (2011) Bone, 48,389-98).These discoveries mean the slight delay of agglutination.So And, our discovery and other results of study (Koewler etc., (2007) J Bone Miner Res, 22,1732-42： Ghilardi et al.(2011) Bone, 48,389-98) to point out together, the signal transduction path is during union in bone There is secondary role in cell and its precursor (if effect).
In order to assess the analgesic activity of antibody processing, we longitudinally determine activity and the GRF of operating limb of mouse. Post operation the 2nd day, compared with the animal of PBS processing, the mouse of anti-TrkA antibody processing is found to have significantly higher activity Degree.There is also the trend of bigger mobility in the mouse handled with anti-ngf antibodies, but it is not reaching to significance,statistical. By contrast, when comparing two kinds of antibody, we do not have found significant difference statistically, therefore illustrate at least suitable Analgesic effect.These action results obtain Koewler et al. J Bone Miner Res, and 22,1732-42 observation result is really Recognize, they report and substantially reduce (Koewler etc., 2007J Bone using neutralizing pain corelation behaviour after anti-ngf antibodies Miner Res, 22,1732-42).Usually, the mouse handled during healing with anti-TrkA antibody is found, with PBS processing Compare, the trend with bigger mobility.This can be shown that receive PBS mouse is influenceed by operation and postoperative pain Animal than receiving one of analgesia antibody is big, shows that sufficient early pain control is good for mouse in whole agglutination Health has positive role.Any time point is supported on by the vertical GRF operating limbs represented not showing between significant group Difference.This is also consistent with other people report (Koewler etc., (2007) J Bone Miner Res, 22,1732-42).We Propose, the pain that GRF slight reduction may be related to fracture is unrelated.It is presumed that, the dissection of muscle changes during performing the operation Function, so as to cause the load of the reduction.
In a word, as a result show, union of the NGF/TrkA signal transductions to rodent is blocked using specific antibody Have no adverse effect, because antibody processing does not change bio-mechanical property and poroma composition.
The stability data of embodiment 11- low concentration aqueous preparations
The present inventor has produced and has tested regulation to the first low-concentration liquid preparation of pH 6.0 anti-TrkA antibody, It is included：
The anti-TrkA antibody of 10mg/ml, it, which is included, contains SEQ ID NO：5 light chain variable sequence and contain SEQ ID NO： 7 weight chain variable district；
25mM citrates；
150mM NaCl；
0.05% Tween 80.
The present inventor is in T0, T1, T2, T3, T6, T9, T12, T18, T24, T30 time point (in terms of the moon), in 5 DEG C, 25 DEG C and 40 DEG C at use multiple standards, characterize the stability of the anti-TrkA antibody.
Specifically, antibody present in preparation is characterized in the following way in correlation time point：Determine preparation or its portion The clarity divided, coloring degree, opacity and particle contamination (visible particle)；By wavelength 280nm optical absorption measurement, it is determined that The concentration of protein present in preparation；Visualized by PAGE gel, determine weight change and/or the degraded of antibody； By ELISA, any change of the binding characteristic of antibody is determined；By HPLC-CEX, positive/negative antibody materials group in preparation is determined Into change；By HPLC-IEF, the change of the isoelectric focusing spectrum of antibody present in preparation is determined by means of group Capillary Electrophoresis；It is logical HPLC-SEC analyses are crossed, the change of antibody in preparation is determined.
The property and antibody therein of preparation are estimated using standard technique.
In each case, at least one or more time points and T0 values and/or for use as standard material (for matter Amount ensures and quality control purpose) the standard previously set up of antibody between be compared.
5 DEG C are directed in Figure 17, Figure 18 is directed to 25 DEG C, and Figure 19 is directed to 40 DEG C, and there is provided stability data.
The stability data of embodiment 12- high concentration aqueous's preparations
The present inventor has been produced and has tested regulation to the liquid preparation of pH 5.75 or 6.0 anti-TrkA antibody, and it is wrapped Contain：
The anti-TrkA antibody of 100mg/ml, it, which is included, contains SEQ ID NO：5 light chain variable sequence and contain SEQ ID NO：7 weight chain variable district；
50mM histidines；
150mM NaCl；
0.05% Tween 80.
The present inventor is in T0, T1, T2, T3, T6, T9, T12, T18, T24, T30 time point (in terms of the moon), in 5 DEG C, 25 DEG C and 40 DEG C at use multiple standards, characterize the stability of the anti-TrkA antibody.
Specifically, antibody present in preparation is characterized in the following way at one or more time points：Determine preparation Clarity, coloring degree, the change of opacity and particle contamination (visible particle)；By wavelength 280nm optical absorption measurement, Determine the concentration of protein present in preparation；Visualized by PAGE gel, determine the weight change and/or drop of antibody Solution；By ELISA, any change of the binding characteristic of antibody is determined；By HPLC-CEX, positive/negative antibody thing in preparation is determined The change of matter composition；By HPLC-IEF, by means of a group Capillary Electrophoresis, the change of the isoelectric focusing spectrum of antibody present in preparation is determined Change；Analyzed by HPLC-SEC, determine the change of antibody in preparation.In each case, in each time point and T0 values and It is compared between the standard previously set up for use as the antibody of standard material (for quality assurance and quality control purpose).
It is 5 DEG C to provide in the stability data of the preparation adjusted to pH 5.75, Figure 20, in 25 DEG C and Figure 22 in Figure 21 For 40 DEG C.Regulation is provided to the stability data of pH 6 preparation, is 5 DEG C in Figure 23, Figure 24 is 25 DEG C, is 40 DEG C in Figure 25.
The stability data of embodiment 13- high concentration aqueous's preparations
The present inventor has been produced and has tested the first low-concentration liquid preparation of anti-TrkA antibody, and it is included：
The anti-TrkA antibody of 150mg/ml, it, which is included, contains SEQ ID NO：5 light chain variable sequence and contain SEQ ID NO：7 weight chain variable district；
50mM histidines；
150mM NaCl；
0.05% Tween 80.
The present inventor is in T0, T1, T2, T3, T6, T9, T12, T18, T24, T30 time point (in terms of the moon), in 5 DEG C, 25 DEG C and 40 DEG C at use multiple standards, characterize the stability of the anti-TrkA antibody.
Specifically, antibody present in preparation is characterized in the following way at one or more time points：Determine preparation Clarity, coloring degree, the change of opacity and particle contamination (visible particle)；By wavelength 280nm optical absorption measurement, Determine the concentration of protein present in preparation；Visualized by PAGE gel, determine the weight change and/or drop of antibody Solution；By ELISA, any change of the binding characteristic of antibody is determined；By HPLC-CEX, positive/negative antibody thing in preparation is determined The change of matter composition；By HPLC-IEF, by means of a group Capillary Electrophoresis, the change of the isoelectric focusing spectrum of antibody present in preparation is determined Change；Analyzed by HPLC-SEC, determine the change of antibody in preparation.
In each case, each time point and T0 values and for use as standard material (for quality assurance and matter Amount control purpose) the standard previously set up of antibody between be compared.
It is 5 DEG C to provide in the stability data of the preparation adjusted to pH 5.75, Figure 26, in 25 DEG C and Figure 28 in Figure 27 For 40 DEG C.Regulation is provided to the stability data of pH 6 preparation, is 5 DEG C in Figure 29, is 25 DEG C in Figure 30, is 40 in Figure 31 ℃。
Embodiment 14：The sub- visible particle (Sub-visible particle) of 100mg/ml and 150mg/ml preparations, glues Degree and syringeability
In order to further characterize the property of high concentration liquid preparation of the invention, carry out another serial experiment to determine that Asia can See the presence of particle and the viscosity of preparation and syringeability.The sample for storing 9 to 12 months at 5 DEG C is tested.
- Asia visible particle
One mode of infringement therapeutic protein effect is the undesired immune response of induction, causes antibody-mediated egg White matter activity in and/or bioavilability change.It is well known that the protein aggregate in therapeutic protein products can increase Strongly immunogenic, therefore, when assessing product quality, this influence is the important risk factor considered.Such aggregation exists Sub- visible particle is shown as in preparation.
Protein is generally assembled from the molecule of part unfolding, and the part unfolding molecule can be the natural shape of molecule A part for state set.Although product formulation is developed to maximize and keep the ratio of protein molecule existed with native state Example, but still the aggregation of significant quantity is likely to form, particularly pharmaceutically in the time scale of correlation and under pressure condition.In the presence of The level and size of protein particulate in given product can be because of the related many of the commodity production to therapeutic protein Factor and change.
It is known that there is the large protein assembly (wherein protein molecule has native conformation) for repeating antigen array in induction It is typically most potent during immune response.Moreover, developing the effort of more effective vaccine it has been shown that antigen protein is adsorbed Onto the nanometer or particulate being made up of other materials (such as colloidal state aluminium salt or polystyrene), immunogenicity can be greatly increased. These are applied to therapeutic protein products, there is such dispute always：Protein molecule containing native-like conformation Big aggregation can turn into the greateset risk for causing unfavorable immune response in patients.
The code test regulation of sub- visible particle analysis, size>10 μm of particle is controlled in or less than 6000 particles/ Ml,>25 μm of particle is limited in equal to or less than 600 particle/ml.
The scheme provided using manufacturer, is carried out sub- using HIAC systems (Beckman Coulter Lifesciences) Visible particle analysis.Measurement is carried out based on photoresistance (light obscuration), and is related to the geometry based on particle Particle sizing.
The sub- visible analyses of the HIAC of table 6
All formulations all fully receiving under level in sub- visible particle, it was observed that particle lack and refer to well The high-level protein stability with antibody processed is shown.
- viscosity
The heavy dose of protein of delivering is usually required using monoclonal antibody as therapeutic agent.This concentrated solution is asked with many Topic is related, including solution viscosity.Although there is no professional standard for the acceptable viscosity for injecting solution, generally by 25- Value between 30mPA.s is considered as the soft upper limit, and viscosity usually requires off-gauge application technique/equipment to allow more than 50mPAs Perform injection.
Viscosity is carried out using Haake Reostress 1 (Thermo Scientific) schemes provided using manufacturer Analysis.
The viscosity of table 7
, it is surprising that 150mg/ml preparations A (50mM histidines, 150mM NaCl, Tween 80-0.05%, PH5.75,150mg/ml) superior characteristic is shown, viscosity of its viscosity close to the 100mg/ml preparations of test.Second test 150mg/ml preparations (50mM histidines, 150mM NaCl, Tween 80-0.05%, pH6,150mg/ml) also show that well Performance, and be better than comparable commercially available 150mg/ml therapeutic antibodies preparation.
- syringeability
Syringeability is the critical product performance parameter of any parenteral dosage forms.This is that pointed injection therapeutic agent is small before injection Bottle is easy to the ability shifted by hypodermic needle.Syringeability includes the easiness such as extracted, blocks and foaming becomes The factor such as gesture and the accuracy of dose measurement.Can be by the geometry of pin, i.e. internal diameter, length, opening shape and note The surface smoothness of emitter (4) influences syringeability.This is for injection device (such as pen equipped with very fine needle and automatic Syringe) it is even more important.In fact, patient can use the pen-type injector using 29-31-G pins.With regard to prefilled syringe Speech, the conventional needles for subcutaneous administration are configured to 27G and 25G (4,5).While injection pain is reduced, fine needle needs to increase The power of medicine is penetrated in filling.At present, any pharmacopeia detection program is not specified by pharmacopeia, but generally, for human injection For, the power less than 5-8N is considered as acceptable.
Syringeability is carried out using TA-XT Plus (the Stable Micro Systems) schemes provided using manufacturer Analysis.
The syringeability of table 8
The syringeability data of 27G pins are good, and the syringeability of 30G pins is excellent.

Claims (10)

1. the people TrkA anti-TrkA antibody of humanization or its fragment is combined, it is ache related for treating bone,
Wherein described anti-TrkA antibody or its fragment include heavy-chain variable domains CDR 1,2 and 3 and light variable domains CDR 1,2 and 3, and heavy-chain variable domains are comprising selected from SEQ ID NOs：1-5 sequence, light variable domains, which are included, is selected from SEQ ID NOs：6-13 sequence,
The non-CDR region of wherein described heavy-chain variable domains is included in the amino acid position selected from the group being made up of 37,42 and 89 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, wherein the amino acid position of each group membership is provided using numbering system in Kabat.
2. the anti-TrkA antibody of the humanization of claim 1 or its fragment, are selected from wherein the bone photo closes pain：By bone injury or Involve pain caused by the lesion of bone.
3. the anti-TrkA antibody of the humanization of claim 1 or its fragment, wherein the bone photo closes pain by fracturing, bone split, bone breaks And/or inflammation causes.
4. the anti-TrkA antibody of the humanization of claim 1 or its fragment, wherein the bone photo closes pain because accident causes, Or because caused by excessive use or the mobile injury region of repetition.
5. the anti-TrkA antibody of the humanization of claim 1 or its fragment, wherein the bone photo close pain by anhormonia, infection, Bone caused by osteocarcinoma or bone blood supply are interrupted weak causes.
6. the anti-TrkA antibody of the humanization of claim 1 or its fragment, wherein the bone photo closes pain by osteocarcinoma, particularly after Hair property osteocarcinoma and/or metastatic carcinoma cell cause.
7. comprising the anti-TrkA antibody of the humanization according to claim 1 for methods described or its fragment and it can pharmaceutically connect The composition for the carrier received.
8. immunoconjugates, it includes the anti-TrkA antibody of the humanization according to claim 1 for methods described or its fragment With the therapeutic agent being attached thereto.
9. the composition of claim 8, it also includes other pharmaceutically active agents.
10. the composition of claim 8, it also includes other pharmaceutically active agents, wherein the other pharmaceutically active agents are One below is a variety of：
A) analgestic,
B) another anti-TrkA antibody,
C) NGF,
D) anticancer,
E) anti-ngf antibodies.
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Publication number Priority date Publication date Assignee Title
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WO2020238998A1 (en) * 2019-05-30 2020-12-03 Sunshine Lake Pharma Co., Ltd. Anti-trka antibodies and uses thereof
CN114230663A (en) * 2019-05-30 2022-03-25 广东东阳光药业有限公司 Antibodies to TrkA and uses thereof

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