WO2000031546A1 - Anticorps diriges contre les peptides ciblant les recepteurs du tractus gastro-intestinal, et methodes associees - Google Patents

Anticorps diriges contre les peptides ciblant les recepteurs du tractus gastro-intestinal, et methodes associees Download PDF

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Publication number
WO2000031546A1
WO2000031546A1 PCT/IE1999/000116 IE9900116W WO0031546A1 WO 2000031546 A1 WO2000031546 A1 WO 2000031546A1 IE 9900116 W IE9900116 W IE 9900116W WO 0031546 A1 WO0031546 A1 WO 0031546A1
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WO
WIPO (PCT)
Prior art keywords
antibody
targeting agent
git
git targeting
antibody fragment
Prior art date
Application number
PCT/IE1999/000116
Other languages
English (en)
Inventor
Daniel Joseph O'mahony
Michela Seveso
Original Assignee
Elan Corporation, Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Elan Corporation, Plc filed Critical Elan Corporation, Plc
Priority to JP2000584309A priority Critical patent/JP2002535246A/ja
Priority to CA002347700A priority patent/CA2347700A1/fr
Priority to AU11743/00A priority patent/AU1174300A/en
Priority to EP99972739A priority patent/EP1131636A1/fr
Publication of WO2000031546A1 publication Critical patent/WO2000031546A1/fr

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • 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

Definitions

  • the present invention relates to antibodies to random peptides capable of targeting or specifically binding to gastro-intestinal tract (GIT) transport receptors.
  • this invention relates to methods of using these antibodies as well as specific antibody preparations directed to particular GIT random peptide targeting agents.
  • Antibodies can be produced by using an immunogen to generate antibodies which immunospecifically bind such an immunogen.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • Various procedures known in the art may be useful for the production of polyclonal antibodies to an immunogen.
  • various host animals such as rabbits, mice, rats, fowl etc. can be immunized by injection with the immunogen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, such as Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette- Guerin
  • corynebacterium parvum corynebacterium parvum
  • GIT targeting agents are capable of facilitating transport of an active agent through a human or animal gastro-intestinal tissue and have use, for example, in facilitating transport of active agents from the lumenal side of the GIT into the portal, hepatic or systemic blood system and/or in targeting active agents to the GIT.
  • the active agent can be targeted to specific receptor sites or transport pathways which are known to operate in the human gastrointestinal tract, thus facilitating its absorption into the systemic system.
  • the active agent is a drug or a drug-containing nano- or microparticle.
  • the tissue through which transport is facilitated is of the duodenum, jejunum, ileum, ascending colon, transverse colon, descending colon, or pelvic colon. The tissue is most preferably epithelial cells lining the lumenal side of the GIT.
  • the GIT targeting agents are bound to a material comprising an active agent.
  • Such compositions have use in targeting the active agent to the GIT and/or in facilitating transfer through the lumen of the GIT into the systemic circulation.
  • the active agent is an imaging agent
  • Such compositions can be administered in vivo to image the GIT (or particular transport receptors thereof).
  • Other active agents include but are not limited to: any drug or antigen or any drug- or antigen- loaded or drug- or antigen-encapsulated nanoparticle, microparticle, liposome, or micellar formulation capable of eliciting a biological response in a human or animal.
  • Examples of drug- or antigen-loaded or drug- or antigen-encapsulated formulations include those in which the active agent is encapsulated or loaded into nano- or microparticles, such as biodegradable nano- or microparticles, and which have GIT targeting agents adsorbed, coated or covalently bound, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle. Additionally, the GIT targeting agent can form the nano- or microparticle itself or the GIT targeting agent can be covalently attached to the polymer or polymers used in the production of the biodegradable nano- or microparticles or drug-loaded or drug- encapsulated nano- or microparticles or the peptide can be directly conjugated to the active agent.
  • the GIT targeting agent bound to the active agent can be employed in methods of treatment (and prophylaxis) by administration to a subject of an effective amount of targeting agent/active agent.
  • Any disease or disorder of interest amenable to therapy or prophylaxis by providing a drug in vivo systemically or by targeting a drug in vivo to the GIT (by linkage to a GIT targeting agent) can be treated or prevented by this administration.
  • Any route of administration known in the art may be used, including but not limited to oral, nasal, topical, intravenous, intraperitoneal, intradermal, mucosal, intrathecal, intramuscular, etc.
  • administration is oral.
  • compositions As well as to determine the fate of the compositions following administration to a subject, antibodies to the specific GIT targeting agents are needed.
  • the present invention provides antibodies or antibody fragments specific to a domain of a GIT targeting agent, particularly antibodies to ZElan033 (PAX2 15 mer), ZElan088(HAX42-2 20 mer) and ZElan053 (P31 D-form 16 mer).
  • GIT targeting agent specific antibodies of this invention include methods of detecting, quantitating, and locating the GIT targeting agent either in a pharmaceutical composition or after contact of a GIT targeting agent-containing composition with human or animal gastrointestinal tissue.
  • Figure 1 shows the immuno responses of three groups of 2 rabbits, each group immunized, respectively, with one of KLH conjugated ZElan033, KLH conjugated ZElan088 and KLH conjugated ZElan053, when the fourth bleed samples are tested by ELISA on their respective unconjugated peptides;
  • Figure 2 shows the crossreactivity of rabbit antisera for three goups of 2 rabbits, each group immunized, respectively, with one of KLH conjugated ZElan033, KLH conjugated ZElan088 and KLH conjugated ZElan053, when the fourth bleed samples are tested by
  • Figure 3 shows the immuno-reactivity of anti-HAX42-2 antisera (fifth bleed samples) on a variety of synthetic peptides
  • Figure 4 shows the immuno-reactivity of anti-PAX2 antisera (fifth bleed samples) on a variety of synthetic peptides
  • Figure 5 shows the immuno-reactivity of anti-P3 1 D-form antisera (fifth bleed samples) on a variety of synthetic peptides.
  • a GIT targeting agent may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and a Fab expression library.
  • Particular antibodies provided by this invention include but are not limited to antibodies or antibody fragments, preferably polyclonal antibodies or antibody fragments, specific to a domain of GIT targeting agents ZElan033 (PAX2 1 5 mer), ZElan088 (HAX42-2 20 mer) and ZElan053 (P3 1 D-form 1 6 mer). Additional GIT targeting agents are disclosed throughout the above-referenced WO
  • These antibodies can be used in methods relating to the localization and activity of the GIT targeting agent sequences, e.g., for imaging these peptides after in vivo administration (e.g. , to monitor treatment efficacy), measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
  • antibodies or antibody fragments specific to a domain of a GIT targeting agent can be used to 1 ) identify the presence of the peptide on a nanoparticle or other substrate; 2) quantify the amount of peptide on the nanoparticle; 3) measure the level of the peptide in appropriate physiological samples; 4) perform immunohistology on tissue samples; 5) image the peptide after in vivo administration; 6) purify the peptide from a mixture using an immunoaffinity column, 7) bind or fix the peptide to the surface of nanoparticle or 8) when a tag is also added to either an active-agent containing particle or the active agent itself, track the fate of both the particle/active agent and the GIT targeting agent so as to determine if and/or where they become separated.
  • a GIT targeting agent such as a dansyl group or some other epitope introduced into the peptide
  • Use 7 above envisions attaching the antibody (or fragment of the antibody) to the surface of drug-loaded nanoparticles or other substrates and then incubating this conjugate with the peptide. This procedure results in binding of the peptide in a certain fixed orientation, resulting in a particle that contains the peptide bound to the antibody in such a way that the peptide is fully active.
  • antibodies or antibody fragments specific to a domain of a GIT targeting agent 9) can be used in confocal microscopy imaging techniques or other imaging techniques in order to demonstrate or confirm or identify the location or localization of the peptide on the surface of a nano- or microparticle, 10) can be used in confocal microscopy imaging techniques or other imaging techniques in order to demonstrate or confirm or identify the location or localization of the peptide on the surface of a nanoparticle or microparticle which has also been loaded with a fluorescent agent, 1 1 ) in the case of nanoparticles or microparticles coated with the peptide which have been sliced into two halves by a microtone or other suitable techniques, the antibody can be used in suitable quantitative techniques such as confocal microscopy imaging techniques or other quantitative imaging techniques in order to identify or quantitate the relative distribution of the peptide between the surface of the nanoparticle or microparticle and the sub-surface interior matrix of the nanoparticles or microparticles, 12) can be used in confocal micros
  • GIT targeting agents PAX2 15mer, HAX42-2 20mer and P3 I D-form 16mer were raised to allow for, among other uses as discussed above, following the destiny of particles coated with peptides in in vivo models.
  • These three GIT targeting agents were selected for their ability to bind in vitro to Caco-2 PI 00 fraction and, when coated on the surface of insulin loaded nanoparticles, to enhance insulin delivery in in vivo studies (rat model/intra-duodenal).
  • the primary sequences for these three GIT targeting agents are given in Table 1.
  • amino acids indicated in lower case and in bold are D-amino acids; K(dns) refers to a dansyl group
  • the peptides were synthesised (Genosys) and conjugated to KLH protein in preparation to immunise rabbits.
  • KLH protein was conjugated at both N- and C- terminals in order to maximise the probability of obtaining specific antibodies.
  • the immunization protocol provided that two rabbits were immunized for each peptide; Rabbits 122 and 123 were immunized with PAX215 mer, Rabbits 120 and 121 were immunized with HAX42-2 20 mer and Rabbits 141 and 142 were immunized with P3 1 D-form 16mer.
  • the initial immunisation was given in Complete Freund's adjuvant and the remaining boosts in Incomplete Freunds.
  • a pre-immune sample was taken from each animal before immunization.
  • the rabbits were injected at day 0, day 14 and 28, bled a week later at day 35 ( 1 st bleed), boosted a week later at day 42 and bled a week later at day 49; this sequence of injections and bleeds was performed every two weeks.
  • bleed samples were tested by ELISA using the following procedures: 96 well plates were coated with peptide at 50 ⁇ g/ml in 0.05M carbonate/bicarbonate buffer, pH9.6, overnight. The plates were washed twice with PBS + 0.05% Tween20 and the plates were blocked with 2% dried skimmed milk (99% fat free) in PBS for one hour at room temperature. The plates were then washed three times with PBS + 0.05%o Tween20 and anti-sera diluted in 2% dried milk-PBS was added followed by incubation for one hour at room temperature.
  • the plates were then washed three times with PBS + 0.05%) Tween20 and secondary antibody goat anti-rabbit IgG-HRP (Sigma A0545, dilution 1 :20000) in 2% dried milk-PBS was added followed by incubation for one hour at room temperature.
  • the plates were washed three times with PBS + 0.05% Tween20, TMB substrate was added, incubated and the absorbance was read at 650nm.
  • the fourth bleed samples were tested by ELISA on both the peptides used for immunisation (but not conjugated to KLH) and on different (dansylated) peptide batches.
  • Pre-immune serum was included in the assay as negative control and background binding to plastic was also tested.
  • the antisera of the immunised rabbits gave an antibody response compared to pre-immune sera of the same animals.
  • the immuno response of the two rabbits immunised in each protocol was comparable except that Rabbit 120 showed a lower antibody titer with respect to Rabbit 121 .
  • Crossreactivity of each rabbit antiserum on different peptides was also analysed by ELISA as shown in Figure 2 and no significant cross-reactivity was detected.
  • the fifth bleed samples were tested by ELISA as described above and examples of the profiles obtained are shown in Figures 3, 4 and 5. A higher titer of antibody was detected for each rabbit after this longer immunisation period compared to the fourth bleed results.
  • Figure 3 shows the immuno-reaction of anti-HAX42 antisera on synthetic peptides (sequences reported in the Figure).
  • Panel A shows the ELISA results for rabbit # 120 antisera: good immuno-response is obtained on unconjugated peptide used as antigen but no response was obtained for the same peptide conjugated to a dansyl group (Zelan088). No immuno-response is observed for the other peptides analysed.
  • Panel B shows the ELISA results for rabbit # 121 antisera: in this case there also was good immuno-response for the unconjugated peptide used as antigen but no response for Zelan088 dansyl-peptide.
  • Panel C shows the response of anti-dansyl IgG on the peptides used in the assay.
  • Figure 4 shows the immuno-reactivity of anti-PAX2 antisera on synthetic peptides.
  • Rabbit # 123 antisera has also very strong reactivity against Zelanl 03A peptide whereas rabbit # 122 does not bind to the same peptide.
  • Rabbit # 123 antisera has also imuno-response against both Zelanl 04 and ZelanOl ⁇ .
  • Panel C shows the response of anti-dansyl IgG on the peptides used in the assay.
  • Table 2 provides a summary of the fifth bleed results.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un anticorps ou fragment d'anticorps spécifique d'un domaine d'un agent ciblant le tractus gastro-intestinal tel qu'un anticorps polyclonal, un anticorps monoclonal, un anticorps chimérique, un anticorps à chaîne unique, un fragment Fab ou une banque d'expression Fab. L'invention concerne, en particulier, un anticorps ou fragment d'anticorps, l'agent ciblant le tractus gastro-intestinal étant sélectionné dans le groupe constitué de ZElan033 (15 mer PAX2), ZElan088(20 mer HAX42-2) ou ZElan053 (16 mer P31 forme D). L'invention concerne enfin de nombreuses méthodes utilisant ces anticorps spécifiques de l'agent ciblant le tractus gastro-intestinal.
PCT/IE1999/000116 1998-11-19 1999-11-19 Anticorps diriges contre les peptides ciblant les recepteurs du tractus gastro-intestinal, et methodes associees WO2000031546A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2000584309A JP2002535246A (ja) 1998-11-19 1999-11-19 Ｇｉｔレセプターを標的とするペプチドに対する抗体および関連する方法
CA002347700A CA2347700A1 (fr) 1998-11-19 1999-11-19 Anticorps diriges contre les peptides ciblant les recepteurs du tractus gastro-intestinal, et methodes associees
AU11743/00A AU1174300A (en) 1998-11-19 1999-11-19 Antibodies to peptides that target git receptors and related methods
EP99972739A EP1131636A1 (fr) 1998-11-19 1999-11-19 Anticorps diriges contre les peptides ciblant les recepteurs du tractus gastro-intestinal, et methodes associees

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10903698P 1998-11-19 1998-11-19
US60/109,036 1998-11-19

Publications (1)

Publication Number Publication Date
WO2000031546A1 true WO2000031546A1 (fr) 2000-06-02

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EP (1) EP1131636A1 (fr)
JP (1) JP2002535246A (fr)
AU (1) AU1174300A (fr)
CA (1) CA2347700A1 (fr)
WO (1) WO2000031546A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0210970A2 (fr) * 1985-07-25 1987-02-04 The Regents Of The University Of Minnesota Détection, visualisation et thérapie de carcinomes de cellules rénales utilisant des anticorps monoclonaux
WO1997017613A1 (fr) * 1995-11-10 1997-05-15 Elan Corporation, Plc Peptides favorisant le transport dans les tissus et procedes d'identification et d'utilisation correspondants
WO1998051325A2 (fr) * 1997-05-15 1998-11-19 Cytogen Corporation Peptides aleatoires se liant au recepteurs de transport du tractus gastrointestinal (git) et procedes y relatifs
WO1998051825A1 (fr) * 1997-05-15 1998-11-19 Elan Corporation, Plc Peptides favorisant le transport d'un agent actif dans les tissus, compositions les renfermant et leurs procedes d'utilisation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0210970A2 (fr) * 1985-07-25 1987-02-04 The Regents Of The University Of Minnesota Détection, visualisation et thérapie de carcinomes de cellules rénales utilisant des anticorps monoclonaux
WO1997017613A1 (fr) * 1995-11-10 1997-05-15 Elan Corporation, Plc Peptides favorisant le transport dans les tissus et procedes d'identification et d'utilisation correspondants
WO1998051325A2 (fr) * 1997-05-15 1998-11-19 Cytogen Corporation Peptides aleatoires se liant au recepteurs de transport du tractus gastrointestinal (git) et procedes y relatifs
WO1998051825A1 (fr) * 1997-05-15 1998-11-19 Elan Corporation, Plc Peptides favorisant le transport d'un agent actif dans les tissus, compositions les renfermant et leurs procedes d'utilisation

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EP1131636A1 (fr) 2001-09-12
AU1174300A (en) 2000-06-13
JP2002535246A (ja) 2002-10-22
CA2347700A1 (fr) 2000-06-02

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