|Title of Invention||
"AN ISOLATED NEUTRALLZING ANTIBODY"
|Abstract||The present invention relates to a neutralizable epitope of hepatocyte growth factor (HGF) which inhibits HGF from binding to a receptor thereof. The common technical feature of epitope is that it is neutralizable by the isolated neutralizing antibody by binding to it and inhibits the binding of HGF to cMet and so prevent and treat intractable disease and cancers.|
NEUTRALIZABLE EPITOPE OF HGF AND NEUTRALIZING ANTIBODY BINDING TO THE SAME
Field of the Invention
The present invention relates to a neutralizable epitope of HGF (hepâtocyte growth factor) inhibiting the binding of HGF to a receptor thereof and a neutralizing antibody against HGF wbich is capable of neutralizing HGF as a single agent by binding to said neutralizable epitope of HGF.
Background of the Invention
HGF (hepâtocyte growth factor) is a multifuncţional heterodimeric polypeptide produced by mesenchymal cells. HGF is composed of an alpha-chain containing an N-terminal domain and four kringle domains (NK4) covalently linked to a serine protease-like beta-chain C-terminal domain (see Fig. 1). Human HGF is synthesized as a biologically inactive single chain precursor consisting of 728 amino acids with a 29 amino acid signal peptide which is not present in the mature protein. Biologically active HGF is achieved through cleavage at the R494 residue by a specific, extracellular serum serine protease. The active HGF thus achieved is a rully active heterodimer which is composed of disulfîde linked 69 kDa alpha-chain and 34 kDa beta-chain. However, the overall tertiary structure of HGF is still unknown and it has not yet been clarified which of these domains is responsible for the specific functions of HGF (Maulik et al, Cytokine & Growth F.actor Reviews 13(1): 1-59,2002).
The binding of HGF to its receptor, Met, induces the growth and scattering of various cell types, mediates the epithelial mesenchymal transitions and the formation of tubules and lumens, and promotes angiogenesis. Both Met and HGF knockout mice are embryonic lethal and show developmental defects in placenta, fetal liver and limb/muscle formation (Cao et al., PNAS 98(13): 7443-7448, 2001; Gmyrek et al., American Journal of Pathology 159(2): 579-590, 2001).
Met was originally isolated as a product of a human oncogene, trp-met, which encodes a constitutively active altered protein kinase with transforming activity. Met activation has also been shown to remarkably enhance the metastastic spread of cancer stemming from its stimulatory influence of processes such as angiogenesis, cell motility, and cell surface protease regulation (Wielenga
et al., American Journal ofPathology 157(5): 1563-1573, 2000). Since Met was reported to be over-expressed in various human cancers of liver, prostate, colon, breast, brain and skin (Maulik et al, supra), it has been regarded as an important target factor for the prevention and treatment of cancer. Further, it has been reported that malaria infection depends on activation of the HGF receptor by secreted HGF, and accordingly, HGF and its receptor are identified as potenţial targets for ne w approaches to malaria prevention (Carrolo M,' et al., Nat. Med. 9(11): 1363-1369, 2003). It has been also discovered the possibility that HGF may be found in association with the pathologic changes which occur in Alzheimer's disease (Fenton H, et al., Brain Res. 779(1-2): 262-270, 1998). Furthermore, it has been found that HGF is definitely involved in enhancing cutaneous wound healing processes, including re-epithelialization, neovascularization and granulation tissue formation (Yoshida S, et al., J. Invest. Dermatol. 120(2): 335-343,2003).
Meanwhile, selective neutralization of tumor-associated growth factors or cytokines and their receptors, which play crucial roles hi the development and spread of cancer, has always been an attractive strategy for the development of anti-cancer drugs. Recently, numerous therapeutic monoclonal antibodies (mAbs) for these targets, e.g., herceptin, and anti-angiopoietin human mAbs have been developed using recombinant antibody technologies such as phage display of combinational antibody library.
It is well known that polyclonal antibodies against HGF block many of HGF biological functions. In addition, it has .been recently reported that mixtures of neutralizing mAbs against HGF display anti-tumor activity in animal models (Cao et al., PNAS 198(13): 7443-7448, 2001). In particular, Cao et al. disclosed that three or more of the epitopes, possibly two for the Met receptor and one for heparin, need to be blocked hi order to inhibit HGF activity in vivo and in vitro, and a mixture of at least 3 mAbs is capable of neutralizing HGF in an in viîro. experiment.
However, there has been reported no monoclonal antibody that can neutralize HGF as a single agent and inhibit cell scattering activity in vitro.
Summary of the Invention
Accordingly, it is au object of the present invention to provide a neutralizable epitope of HGF which inhibits the binding of HGF to a receptor thereof.
Other objects of the present invention are to provide:
a polynucleotide encoding said neutralizable epitope;
a neutralizing antibody against HGF which is capable of neutralizing HGF as a single agent by binding to said neutralizable epitope;
use of said neutralizing antibody for preventing and treating intractable diseases and cancers;
a pharmaceutical composition comprising said neutralizing antibody and a pharmaceutically acceptable carrier for preventing and treating intractable diseases and cancers; and
a method for preventing and treating intractable diseases and cancers, which coraprises administering said neutralizing antibody to a patient.
In accordance with one aspect of the present invention, there is provided a neutralizable epitope of HGF having the amino acid sequence of SEQ ID NO: 32 or 33.
In accordance with another aspect of the present invention, there is provided a neutralizing antibody against HGF binding to said neutralizable epitope which comprises VH region having the amino acid sequence of SEQ DD NO: 27 or 29 and VL region having the amino acid sequence of SEQ ID NO: 28 or 30.
Brief Description of the Drawings
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings which respectively show:
Fig. l: the structure of HGF,
Fig. 2: the genetic map of phagemid vector pComb3X used for antibody library construction,
A: the case of displaying Fab on the surface of phagemid, and B: the case of displaying scFv or diabody on the surface of phagemid Fig. 3: enrichment of the phage pool displaying Fab specifîcally binding to HGF through the panning during the culture of HGF-binding clones,
Fig. 4: the result of staining the purified Fab fragments with coomasie blue,
2: non-reduced clone 68 antibody (50,000 Da), and 3: reduced clone 68 antibody (25,000 Da)
Fig. 5: the result of western blotting analysis to determine whether the purifîed Fab fragments are expressed,
Fig. 6: the binding level of a phage containing the inventive neutralizable epitope to c-MET,
1: phage containing the peptide of SEQID NO: 32, 2: phage containing the peptide of SEQ ID NO: 33, and 3 and 4: control phages which do not contain the peptide of SEQ ID NO: 32 or 33
Fig. 7: the specific binding of clone 61 and 68 Fabs to HGF, respectively, Fig. 8: conformation dependency of the inventive neutralizable epitope defined by clones 61 and 68, respectively, A: clone 61, B: clone 68,
Larie l: non-reduced HGF, and Lane 2: reduced HGF
Fig. 9a: the criteria showing the cell scattering level ranging from Grades Ito6,
Fig. 9b: the result of scattering assay showing that the scattering levels of anti-HGF Fab and anti-human Fab antibodies change with the concentrations of HGF added,
Fig. 10: the amount of clone 68 antibody bound to HGF immobilized on CM5 sensor chip increases with the injected amount of clone 68 antibody, I: injection of non-specific Fab, II: injection of 50 nM clone 68 antibody, IU: injection of 100 nM clone 68 antibody, IV: injection of 200 nM clone 68 antibody, V: injection of 400 nM clone 68 antibody, and VI: injection of 600 nM clone 68 antibody Fig. 11: clone 68 antibody inhibits the binding of HGF to c-Met, I: injection of 50 nM HGF,
H: injection of 50 nM HGF mixed with 50 nM clone 68 antibody, III: injection of 50 nM HGF mixed with 250 nM clone 68 antibody, IV: injection of 50 nM HGF mixed with 500 nM clone 68 antibody, V: injection of 50 nM HGF mixed with l uM clone 68 antibody, and VI: injection of 50 nM HGF mixed with 1.5 uM clone 68 antibody
Fig. 12: soluble c-Met inhibits the binding of HGF to c-Met. I: injection of 50 nM HGF,
H: injection of 50 nM HGF mixed with 50 nM soluble c-Met, ffi: injection of 50 nM HGF mixed with 100 nM soluble c-Met, IV: injection of 50 nM HGF mixed with 200 nM soluble c-Met, V: injection of 50 nM HGF mixed with 400 nM soluble c-Met, and VI: injection of 50 nM HGF mixed with 600 nM soluble c-Met
Detailed Description of the Invention
As used herein, the term "neutralizable epitope" includes any protein determinant which is capable of inhibiting the binding of HGF to its receptor, c-Met. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific tertiary structural features, as well as specific charge characteristics. Preferably, the inventive neutralizable epitope is a polypeptide comprising the amino acid sequence of SEQID NO: 32 or 33.
The term "neutralizing antibody" refers to an antibody which is capable of specifically binding to the neutralizable epitope of HGF and substantially inhibiting or eliminating the biologically activity of HGF. Typically, a neutralizing antibody will inhibit such biologically activity of HGF at least by about 50%, and preferably by greater than 80%. The neutralizing antibody of the invention is especially useful in therapeutic applications: to prevent or treat intractable diseases and cancers.
The present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 32 or 33 which functions as a neutralizable epitope of HGF.
In order to prepare the neutralizable epitope of HGF in accordance with the present invention, an ELISA study is conducted to examine whether antisera from the immunized animals with HGF bind to a recombinant human HGF; and the study has shown that antisera from the HGF immunized animals specifically bind to HGF. Then, total RNA is extracted from the HGF immunized animals and subjected to cDNA synthesis.
To amplify the variable region comprising rabbit light chain (VL) (VK , Vx ) and heavy chain (Vn) and the constant region comprising human CK and CHI, PCRs are performed by using the synthesized cDNA as a template and primer
combinations of SEQ ID NOs: l to 20, and then, light and heavy chains of rabbit/human chimeric antibody are amplified by using the PCR products obtained above as templates. After the amplified rabbit VL aud VH sequences are combined with the amplified human CK and CHi sequences, final PCR products encoding a library of antibody fragments (Fab) are cloned into an expression vector, and the resulting vector is transformed into a host cell, e.g., E. coli,to construct a chimeric rabbit/human Fab library. The vector and host cell employable in the present invention include all expression vectors and E. coli strains conventionally used in the art without limit, but it is preferable to use phagemid vector pCombSX (the Scripps Research Institute, CA, USA) as an expression vector and E. Coli ER2537 (NEB) as a host cell.
Phage clones containing anti-HGF Fab are selected by EIA using HGF-coated ELISA plates and anti-human goat Fab polyclonal antibodies. Phage clones selected above are designated H61 (clone 61) and H68 (clone 68).
H61 and H68 clones are subjected to nucleotide sequencing and their amino acid sequences are determined from the analyzed nucleotide sequences, respectively. In a preferred embodiment of the present invention, nucleotide sequencing is performed according to the dye-labeled primer sequencing method (Chung et al., J. Cancer Res. Clin. Oncol. 128: 641-649, 2002). As a result, it has been found that H61 clone is composed of VH and VL regions having the nucleotide sequences of SEQ ID NOs: 23 and 24, respectively; and H68 clone comprises VH and VL regions having the nucleotide sequences of SEQ ID NOs: 25 and 26, respectively.
The amino acid sequences of the respective VH and VL regions of H61 and H68 clones from the analyzed nucleotide sequences suggest that H61 clone is composed of VH region having the amino acid sequence of SEQ ID NO: 28 and VL region having the amino acid sequence of SEQ ID NO: 28; and H68 clone, VH region having the amino acid sequence of SEQ ID NO: 29 and VL region having the amino acid sequence of SEQ ID NO: 30.
Analysis of the framework region (FR) and complementarity determining region (CDR) in the amino acid sequences of H61 and H68 clones has shown that each of VH and VL regions of H61 and H68 clones has 4 FRs and 3 CDRs (see Table 2).
To define a neutralizable epitope of HGF, H61 and H68 clones are enriched through the panning by using phage display of combinatorial peptide library, and the phage pools so amplified are subjected to EIA using anti-HGF H61. Fab or anti-HGF H68 Fab and anti-HGF H61 Fab- or anti-HGF H68 Fab-
coated ELISA plates. Phage clones showing the binding affinity to anti-HGF H61 and H68 Fabs are thus selected. In a preferred embodiment of the present invention, the PHD peptide library (New England Biolob) is employed as a peptide library.
Selected phage clones are subjected to nucleotide sequencing, and amino acid sequences deduced from the analyzed nucleotide sequences have the amino acid sequences of SEQ ID NOs: 32 and 33, which are found to bind to c-MET (see Fig. 6). These results suggest that an antigen binding site of anti-HGF antibody H61 or H68 mimics a HGF binding site of c-MET and the peptides of SEQ ID NOs: 32 and 33 binding to anti-HGF antibody H61 or H68 mimic a c-MET binding site of HGF. Accordingly, the inventive peptides of SEQ ED NOs: 32 and 33 are capable of functioning as a neutralizable epitope of HGF.
Further, the present invention provides the polynucleotide encoding said neutralizable epitope. hi particular, said neutralizable epitope has the nucleotide sequence of SEQ ID NO: 34 or 35.
Furthermore, the present invention provides a neutralizing antibody against HGF which is capable of neutralizing HGF by binding to the peptide of SEQ ID NO: 32 or 33 as a neutralizable epitope of HGF.
The neutralizing antibody of the present invention may be a chimeric antibody, a monoclonal antibody or a humanized antibody.
The chimeric antibody is an irnmunoglobulin molecule comprising human and non-human portions. Specifically, the antigen combining region •(variable region) of a chimeric antibody is derived from a non-human source (e.g. mouse, rabbit, poultry) and the constant region of the chimeric antibody which confers the biological efifector function to the irnmunoglobulin is derived from a human source. The chimeric antibody should have the antigen binding specifîcity of the non-human antibody molecule and the effector function conferred by the human antibody molecule.
In general, the procedures used to produce chimeric antibodies involve the following steps:
(a) identifying and cloning the correct gene segment encoding the antigen
binding portion of the antibody molecule, which (known as VDJ, variable,
diversity and joining regions for heavy chains or Vj, variable, joining regions for
light chains or simply as V for variable region) may be in either the cDNA or
(b) cloning gene segments encoding the constant region or desired part
(c) ligating the variable region with the constant region so that the
complete chimeric antibody is encoded in a form that can be transcribed and
(d) ligating the constnict into a vector containing a selectable marker
and gene control regions such as promoters, enhancers and poly(A) addition
(e) amplifying the vector and introducing it into eukaryotic cells
(transfection), usually mammalian lymphocytes;
(f) selecting cells expressing the selectable marker;
(g) screening for cells expressing the desired chimeric antibody; and
(h) testing the antibody for appropriate binding specificity and effector functions.
A monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone. The monoclonal antibody may comprise, or consist of, two proteins, i.e., heavy and light chains. The monoclonal antibody can be prepared using one of a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
A humanized antibody refers to a molecule that has its CDRs (complementarily determining regions) derived from a non-human species immunoglobulin and the remainder of the antibody molecule derived mainly from a human immunoglobulin. The terni "antibody" as used herein, unless indicated otheiwise, is used broadly to refer to both an antibody molecule and an antibody-derived molecule. Such an antibody-derived molecule comprises at least one variable region (either a heavy chain or a light chain of variable region) and includes molecules such as Fab 'fragments, Fab' firagments, F(ab').sub.2 fragments, Fd fragments, Fab' fragments, Fd fragments, Fabc fragments, Se antibodies (single chain antibodies), diabodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules.
In particular, the present invention provides a rabbit/human chimeric antibody as a neutralizing antibody against HGF. The inventive neutralizing antibody comprises VH region of SEQ ID NO: 27 and VL of SEQ ID NO: 28 or VH region of SEQ ID NO: 29 and VL of SEQ ID NO: 30.
Whether or not a neutralizing antibody exerts neutralizing activity may be examined by MDCK2 scattering assay (Cao et al., PNAS 98(13): 7443-7448, 2001). In case of treating 2 ng/m£ of HGF (29 pM) to MDCK2 cells, the
inventive neutralizing antibody shows the highest scattering inhibitory activity when the molar ratio of anti-HGF Fab to HGF becomes 50:1, and the molar ratio of anti-human Fab to HGF, ranging from 50:1 to 100:1 (see Fig. 9). These results show for the first tirne that blocking of only one.epitope is sufficient for neutralizing HGF at least in vitro, different from the Cao report that it is necessary to neutralize at least three epitopes to inhibit MDCK2 cell scattering (Cao et al., suprd). Further, shown in the present invention is the fact that the neutralizing antibody exerts its neutralizing activity only when the antibody binding to the neutralizable epitope is divalent or more, which suggests that the same neutralizable epitope may exist at two or more sites of HGF.
The binding affinity of anti-HGF Fab for HGF, inhibitory activity of clone 68 for binding HGF to c-Met, and inhibitory activity of soluble c-Met for binding HGF to c-Met may also be examined by EIA. The amount of clone 68 antibody binding to HGF immobilized on a sensor chip increases vvith the injection amount of clone 68 antibody (see Fig. 10), and the amount of HGF binding to c-Met decreases as the concentration of clone 68 antibody increases (see Fig. 11). Further, the amount of HGF binding to c-Met immobilized on the sensor chip decreases with increasing the concentration of soluble c-Met (see Fig. 12).
The above results demonstrate that the inventive neutralizing antibody acts as a single agent which is capable of neutralizing HGF.
Accordingly, the present invention further provides a pharmaceutical composition comprising an effective dose of the inventive neutralizing antibody and a pharmaceutically acceptable carrier for preventing and treating intractable diseases • and cancers caused by the binding of HGF to a receptor thereof. Further, the present invention provides a method for preventing and treating intractable diseases and cancers by using the inventive nuetralizable antibody. Preferably, the cancer includes, but are not limited to, various human cancers of liver, prostate, colon, breast, brain and skin, and the intractable diseases encompasses those caused by binding HGF to its receptor, c-Met, and include, not but limited to, malaria, Alzheimer's disease and so on.
The inventive pharmaceutical formulation may be prepared in accordance with any one of the convenţional procedures. In preparing the formulation, the effective ingredient is preferably admixed or diluted with a carrier. Examples of suitable carriers, excipients, or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone,
water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oii. The formulation may additionally include fîllers, anti-agglutinating agente, lubricating agente, wetting agente, flavoring agente, emulsifiers, preservatives and the like. The composition of the invention may be formulated so as to provide a quick, sustained or delayed release of the active ingredient after it is administrated to a patient, by employing any one of the procedures well knovm in the art.
The pharmaceutical formulation of the present invention can be administered by injection (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. The pharmaceutical formulation may also be administered by intratumoral, peritumoral, intralesional or perilesional routes, to exert local as well as systemic therapeutic effects. Local or intravenous injection is preferred injection.
For treating a human patient, a typical daily dose of the inventive neutralizing antibody as an effective ingredient may range from about 0.1 to 100 mg/kg body weight, preferably l to 10 mg/kg body weight, and can be administered in a single dose or in divided doses. However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the irfdividual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usage and conditions,
Example 1; HGF immunization and antibodv librarv construction
Over a period of 4 to 5 months, 2 rabbite of the New Zealland White străin were immunized by 5 cutaneous injections of HGF (R&D systems, USA) dispersed in an emulsion of MPL (monophosphoryl lipid A; highly-refîned non-
toxic lipid A isolated from remutants of S. minnesotd) + TDM (synthetic trehalose dicorynomycolate; an analogue of trehalose dimycolate from the cord factor of the tubercle bacillus) + CWS (cell wall skeleton; from deproteinized and delipidated cell walls of mycobacteria) adjuvant (Sigma) at 3-week intervals. Antisera from the immunized animals were analyzed for their binding to recombinant human HGF (R&D systems or Research Diagnostics, Inc.) by ELISA using horseradish peroxidase-conjugated anti-rabbit Fc goat polyclonal antibodies (Pierce). As a result, it was found that while antisera obtained before HGF immunization almost never bind to HGF, antisera obtained after 5 cutaneous injections specifîcally bound to HGF.
Seven days after the final boost, the spleen and bone marrow were extracted from the immunized animals and used for total RNA preparation with TRI reagent (Molecular Research Center, Cincinnati, USA) and lithium chloride precipitation. First-strand cDNA was synthesized using the SUPERSCRIPT Preamlification System with oligo(dT) priming (Life Technologies, Inc.).
Rabbit/human chimeric antibody library was constructed according to the method described by Rader et al (Rader C. et al., /. Biol. Chem. 275: 13668-13676, 2000).
Example 2; Ampllfication of rabbit-derived Ab variable region and human-
derived Ab constant region
(2-1) Amplification of rabbit-derived Ab variable region
hi order to amplify variable regions of rabbit VL (VK , Vx ) and VH, PCR was performed by using primer combinations described ui Table l.
|Indian Patent Application Number||3385/DELNP/2006|
|PG Journal Number||39/2011|
|Date of Filing||12-Jun-2006|
|Name of Patentee||NATIONAL CANCER CENTER|
|Applicant Address||809, MADU-1-DONG, ILSAN-GU, GOYANG-SI, KYUNGKI-DO 411-351, REPUBLIC OF KOREA.|
|PCT International Classification Number||C07K 14/47|
|PCT International Application Number||PCT/KR2004/002888|
|PCT International Filing date||2004-11-09|