prior to describing the invention in further detail , the terms used in this application are defined as follow unless otherwise indicated . the term “ naturally - occurring ”, as used herein as applied to an object refers to the fact that an object can be found in nature . for example , a combination of heavy and light chain germline immunoglobulins derived from a single human antibody - producing ( b ) cell that is present in a human that can be isolated from a source in nature , e . g ., peripheral blood , and which has not been intentionally modified by man in the laboratory is naturally - occurring . the term “ monoclonal antibody ( mab )” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies , i . e ., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts . monoclonal antibodies are highly specific , being directed against a single antigenic site ( epitope ). the modifier “ monoclonal ” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies , and is not to be construed as requiring production of the antibody by any particular method . the term “ complete human antibody ”, as used herein with regard to an antibody , means that is an antibody not only containing exclusively human sequences but also comprising naturally - occurring configuration of heavy - and - light - chain combination . the antibody is preferably a monoclonal antibody . “ non - immunogenic in a human ” means that upon contacting the polypeptide of interest in a physiologically acceptable carrier and in a therapeutically effective amount with the appropriate tissue of a human , no state of sensitivity or resistance to the polypeptide of interest is demonstrable upon the second administration of the polypeptide of interest after an appropriate latent period e . g ., 8 to 14 days . it will be understood by one of skill in the art that a polypeptide of complete human origin or a polypeptide combination with a naturally - occurring germline configuration typically represents “ non - immunogenic in a human ”. as used herein , the term “ site - directed in vitro immunization ” refers to in vitro antigen - specific immune responses from primary human lymphoid cells ( chin et al ., 2007 )( u . s . pat . nos . 7 , 494 , 779 , 8 , 021 , 860 and 8 , 158 , 386 ). this technology relies on presence of coupled t - and b - cell epitopes as well as depleted starting lymphoid populations containing primed precursors recognizing t - cell epitope . a “ heteromyeloma cell line ” is a cell line derived from fusion of two different myeloma cells . the two different myeloma cells are preferably a human myeloma cell and a murine myeloma cell . heteromyeloma cell lines are known in the art . for example , u . s . pat . no . 6 , 228 , 361 and chin et al ., 2001 describe the preparation , characterization and use of various heteromyeloma cell lines . a “ fusion partner ” is a cell that can be used to fuse with an antibody - producing cell for a beneficial purpose . typically , the fusion leads to prolonged antibody production . thus , without fusion to the fusion partner , the antibody - producing cell ceases to produce antibodies in culture . upon fusion to the fusion partner , however , fused cells can be selected that produce antibodies in culture for at least about 3 months , preferably at least about 6 , 9 , 12 , 18 , 24 months or more . fusion partners include , but are not limited to , myeloma cells and heteromyeloma cells . as used herein , the term “ antigen - binding region ” refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen . the antigen - binding region typically comprises the amino acid residues contributed from the paring of the corresponding heavy and light chains necessary to maintain the proper conformation of the antigen - binding residues . the peculiar configuration of specific paring is inherent in a particular antibody - producing b cell . the term “ original ( vh / vl ) pairing ” has been used in the art and herein to illustrate that although a significant number of the available heavy and light chain germline gene segments are used in vh / vl pairing and a given vh sequence can pair with many light chain sequences of both lambda and kappa light chain classes in human ab generation , the vh / vl pairing is unique in a given b cell survived from peripheral selection . the vh / vl interface between the light chain and heavy chain has been shown to affect the binding kinetics of a peptide ( chatellier et al ., 1996 ), suggesting preference for particular pairings . the interactions between vh / vl contribute significantly to the stability of the antigen - binding region as limited structural changes have significant functional effects on binding affinity . the translation of the human immunoglobulin germline sequences including all known alleles has been provided online such as http :// www . imgt . org /. the present invention is based on the ingenious designs the coupled t - and b - cell epitopes to harness naturally - occurring configurations inherent in a single antibody - producing b cell and simultaneously to isolate antibody - producing b cells with specificity against a self antigen , i . e ., hmgb1 ( seq id no : 1 ). it is a very demanding task because of a phenomenon called tolerance ; healthy human peripheral lymphoid cells do not normally produce antibodies to self antigens . however , because the site - directed in vitro immunization system utilizes a pre - existing helper t cells derived from tetanus immunization to drive b - cell maturation , several advantages are realized over conventional human mab strategies . first , the protocol yields antigen - specific b cells rather than antigen - reactive ig genes , thus eliminating the need of random , non - naturally - occurring recombination required to achieve a detectible ig binder like in phage display and ig transgene introduction . secondly , the rate at which newly formed b cells enter the mature peripheral pool accounts for less than 5 % of the immature cells generated in the bone marrow , suggesting a significant selection pressure ( cancro , 2004 ) and thus a possible mechanism to avoid immunogenicity . the in vivo selection cycles help to circumvent the problems associated with immunogenicity , a process also taken by ivig to establish a general non - immunogenic status in humans . third , using an in vitro stimulation scheme , greater than normal antibody repertoire can be obtained from a population of lymphoid cells after antigen exposure . this system is particular useful for the isolation of complete human antibodies against the self antigens . culture materials and reagents are known in the art and may be obtained commercially . the culture medium used is rpmi - 1640 ( hyclone , logan , utah ), supplemented with 1 × non - essential amino acids ( life technologies , gaithersburg , md . ), 10 % fetal bovine serum ( fbs ; life technologies ) and 50 μg / ml of gentamycin and kanamycin ( sinton chemical & amp ; pharmaceutical , hsinchu , taiwan ). purified bovine hmgb1 ( chondrex inc , redmond , wash .) and recombinant human hmgb1 ( sigma , st . louis , mo .) are used in antigen - specific and competing enzyme - linked immunosorbent assay ( elisa ), together with peroxidase - labeled goat antibodies against human igg and igm ( zymed laboratories , south san francisco , calif .) or avidin horseradish peroxidase ( ebioscience , san diego , calif .) as the reporting system . complete human mabs are produced from in vitro stimulation and culture techniques . generally , plasma and buffy coat samples from healthy routine blood donors , screened negative for hiv - 1 / 2 , htlv - i / ii , hcv , hbsag and containing normal levels of alanine transferase ( alt ), can be obtained from local blood centers . peripheral blood mononuclear cells ( pbmc ) are isolated by density centrifugation on ficoll - paque ( ge healthcare bio - sciences , uppsala , sweden ) as described elsewhere . the resulting pbmc are magnetically labeled with cd45ro macs ® microbeads ( miltenyi ) then separated by a variomacs ™ ( miltenyi ) instrument according to the manufacturer &# 39 ; s instructions . the purified cd45ro + t cells are cultured at a density of 2 × 10 6 cells / ml in the culture medium supplemented with 50 μm 2 - mercaptoethanol and 10 μg / ml pokeweed mitogen ( pwm ; sigma , st . louis , mo .). after 24 h , cells are removed by 400 × g centrifugation to collect cd45ro + t cell replacing factor . removal of cytotoxic cell populations is similarly performed by using colloidal super - paramagnetic microbeads conjugated to monoclonal anti - human cd8 and anti - cd56 antibodies ( miltenyi ). removal of il - 10 - producing cells may be achieved by using rat anti - human il - 10 ( southernbiotech , birmingham , ala .) and goat anti - rat igg microbeads ( miltenyi ). site - directed in vitro immunization is performed by using cytotoxic cell - depleted pbmc based on a two - step principle . primary immunization is performed by incubating the cells for 6 days in a medium containing 10 nm of the heterotopic peptide antigen having amino acid sequence represented by seq id no : 5 ( qyikanskfigitel ( t - helper epitope of seq id no : 2 )- hmg a box ( dkaryeremkty ) ( seq id no : 3 ); yao - hong biotechnology inc ., new taipei city , taiwan ) and seq id no : 6 ( qyikanskfigitel ( t - helper epitope of seq id no : 2 )- rage binding segment ( kdiaayrakgkp ) ( seq id no : 4 ); yao - hong biotechnology inc ., new taipei city , taiwan ), 50 μm 2 - mercaptoethanol , 10 % heat - inactivated human serum , 0 . 05 ng / ml recombinant human ( rh ) il - 2 ( ebioscience ), and 25 % ( v / v ) cd45ro + t cell replacing factor . for secondary immunization , 3 × 10 7 primary - immunized cells are mixed with the peptide in a flask that had been immobilized overnight with 5 mg / ml of cd40l ( cd154 ; ebioscience ) together with 1 × 10 7 qyikanskfigitel ( yao - hong biotechnology inc . )- stimulated cd4 + t cells and 5 ng / ml rh il - 15 ( ebioscience ). the cells are cultured for 3 - 5 days in a medium supplemented with 5 % human serum , 50 mm 2 - mercaptoethanol and 10 nm heterotopic peptide antigens . the concept of using immobilized - metal affinity chromatography ( imac ) to fractionate or purify proteins has first been formulated and its feasibility shown by porath et al . in 1975 . the idea was based on the known affinity of transition metal ions such as zn 2 + , cu 2 + , ni 2 + , and co 2 + to histidine and then strongly fixed metal ions to a support for fractionating protein containing such an amino acid ( his - tag ). the chelating reagents used to fix the metal to a solid support are commercially available and usually involve a tridentate chelator iminodiacetic acid ( ida ), tetradentate chelators nitrilotriacetic acid ( nta ) and carboxymethyl aspartate ( cm - asp ). bound proteins can be eluted and thus fractionated or purified in four following ways : ( 1 ) lower the ph ; ( 2 ) add excess imidazole ; ( 3 ) add edta to remove metal ion from purification resin and / or ( 4 ) add enzymes to cleave his - tag ( gaberc - porekar and menart , 2005 ). it is now well established that imac is a highly efficient procedure to purify his - tagged proteins ; in fact , recombinant proteins are artificially terminally labeled with his tags in a form of oligo - his to serve as an affinity handle for the ease of subsequent purification because naturally occurring proteins are lack of stretches of continuous his residues . therefore , unlike biopolymer chitosan which depends on ph and amine groups for conjugation , virtually any protein can be purified from a cell extract in just one or two steps . in one embodiment , the present invention provides a novel method for the separation of specific b cells by interactions of proteins of said immune cells with antigens . typically , the his - tagged hmgb1 proteins ( sino biological , beijing , china ) are immobilized on nta − ni 2 + magnetic nanobeads ( taiwan advanced nanotech , taoyuan , taiwan ) and the hmgb1 - nanobeads complex is immersed to trap the immune b cells to the complex . specific b cells retained on a magnetic field were then released by citric acid - na 2 hpo 4 buffer containing 1 u / ml thrombin , 1 mm edta and 500 mm imidazole ( sigma ). subsequently , the in vitro immunized cells are infected with epstein - barr virus ( ebv ) by virus - containing supernatant derived from the ebv - producing marmoset cell line b95 - 8 ( american type culture collection , atcc crl 1612 ). the infected cells are seeded at 10 5 / well in 96 - well plates together with mytomycin ( kyowa hakko kogyo , tokyo , japan )- treated pbmc as feeder cells ( 10 4 / well ) for the establishment of lymphoblastoid cells and screened for ab production by elisa . hmgb1 - specific elisa can be performed by coating 0 . 25 μg / ml purified hmgb1 , 1 μg / ml bovine serum albumin ( bsa ; sigma ) or 1 μg / ml tetanus toxoid ( tt ; adlmmune , taichung , taiwan ) onto microtitre plates overnight at 4 ° c . culture supernatants are diluted to the desired level in 10 mm sodium phosphate buffer ( ph 8 . 0 ), containing 0 . 5 m sodium chloride and 0 . 1 % tween - 20 . coated plates are incubated with diluted culture supernatants , washed , incubated with peroxidase - labeled goat antibodies against human igg and igm and developed ( 15 min ) by addition of 100 μl of the chromogenic substrate o - phenylaenediamine ( opd ) ( sigma ). the reaction is stopped after 30 min by adding 1 m sulphuric acid , and the absorbances are read at 490 nm . somatic cell hybridization can be further applied to optimize human antibody and can be generated by electrofusion . briefly , hmgb1 - specific ebv - infected lymphoblastoid cells were fused with heteromyeloma cells ( chin et al ., 2001 ) in an isotonic medium ( 280 mm sorbitol , 0 . 5 mm magnesium acetate , 0 . 1 mm calcium acetate and 1 mg / ml bsa ; ph6 . 9 - 7 . 1 ). cell fusion can be induced by high - voltage pulses using a btx electro cell manipulator ecm 2001 ( harvard apparatus , holliston , mass .). hmgb1 - specific hybrids were selected and cloned by limiting dilution . methods for determining whether an antibody or antigen - binding fragment inhibits a hmgb1 - induced pro - inflammatory condition are known to one skilled in the art . for example , inhibition of the release of an inflammatory cytokine from a cell in culture can be measured accordingly . for example , as described and exemplified herein , tnf release from human monocytic populations can be measured using cd14 + cells sorted magnetically with macs ® microbeads ( miltenyi ). tnf that is released from the monocytes is measured by a commercial kit obtained from r & amp ; d systems ( minneapolis , minn .). methods for measuring release of other related cytokines from cells are also known in the art . assays to determine affinity and specificity of binding are known in the art , including competitive and non - competitive assays . a competitive assay is preferred in this analysis . the affinity of the mab can be determined against hmgb1 protein with an affinixqn quartz crystal microbalance ( qcm ) biosensor ( inishiamu inc ., tokyo , japan ) according to the manufacturer &# 39 ; s instructions . different amounts of synthetic peptides can then be incorporated into the reaction for competition to confirm the specificity of human mabs . the exact pathogenic process to neuropathic diseases is currently unknown , but experimental autoimmune / allergic encephalomyelitis ( eae ) is primarily used as an animal model of autoimmune inflammatory diseases of the cns and in many aspects resembles human neuropathic diseases , such as ms and nmo . this demyelinating model can be used to evaluate the effectiveness of human mabs in alleviating diseases and is known to one skilled in the art . immunization with myelin oligodendrocyte glycoprotein ( mog ) induces a chronic form of eae . different amounts of synthetic peptides can then be incorporated into the reaction for competition to confirm the specificity of human mabs . human mabs can then be incorporated into the system for verifying the tendency to resolve inflammation and demyelination . the following examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of the present invention . ag - specific enzyme - linked immunosorbent assay ( elisa ) was performed by coating 1 μg / ml purified bovine hmgb1 , 1 μg / ml recombinant human hmgb1 , 1 μg / ml bovine serum albumin ( bsa ; sigma ) or 1 μg / ml tetanus toxoid ( tt ; adlmmune , taichung , taiwan ) onto microtitre plates overnight at 4 ° c . culture supernatants were diluted to the desired level in 10 mm sodium phosphate buffer ( ph 8 . 0 ), containing 0 . 5 m sodium chloride and 0 . 1 % tween - 20 . coated plates were incubated with diluted culture supernatants from the cell line , washed , incubated with peroxidase - labeled goat antibodies against human igg and igm and developed ( 15 min ) by addition of 100 μl of the chromogenic substrate o - phenylaenediamine ( opd ) ( sigma ). the reaction was stopped after 30 min by adding 1 m sulphuric acid , and the absorbances were read at 490 nm ( fig1 ). the ability of particular human mabs to inhibit hmgb1 - induced tnf release was assessed in human primary monocytes administered only hmgb1 , hmgb1 plus particular hmgb1 mabs , or an isotype control antibody . freshly purified human monocytes cultures were stimulated with 1 μg / ml purified bovine hmgb1 and culture supernatant were sampled four hours later to be assayed for tnf . as depicted in fig2 , two particular human mabs inhibited tnf release , indicating that such antibodies could be used to neutralize the biological activity of hmgb1 and thus for alleviating neuropathic diseases . as shown in fig3 , qcm was used to perform kinetic analysis of interaction between hmgb1 , a representing mab and peptides with the same amino composition . hmgb1 was injected to saturate the chip . the frequency change of mab with immunogen - containing peptide or an irrelevant peptide with the same amino composition was analyzed by aqua ™ software . the results indicated that the mab binding to hmgb1 can be only replaced by the immunogen - containing peptide . chronic experimental allergic encephalomyelitis ( eae ), which represents an animal model of human neuromyelitis , was induced in c57bl / 6 mice by immunization with an emulsion of mog35 - 55 or mog1 - 125 in complete freund &# 39 ; s adjuvant ( cfa ) distributed in three spots on the flank , followed by administration of pertussis toxin ( ptx ) in pbs , first on the day of immunization and then again the following day . eae is scored on scale 0 to 5 , based from no obvious changes in motor function compared to non - immunized mice to mice spontaneously rolling in the cage , respectively . 100 μg of purified anti - hmgb1 antibody or isotype control antibody was injected intraperitoneally at days 5 and 7 post mog injection . from day 15 , human anti - hmgb1 mab demonstrated significant alleviating effect ( fig4 ). although the present invention has been explained in relation to its preferred embodiment , it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed .
the present invention relates to methods of making human anti - hmgb1 antibodies , compositions comprising these antibodies and methods of using the antibodies and compositions in hmgb1 associated - neuropathy .