Patent Application: US-93144207-A

Abstract:
methods and compositions for using the mhc class ii invariant chain polypeptide , ii , as a receptor for macrophage migration inhibitory factor , are disclosed . these include methods and compositions for using this receptor , as well as agonists and antagonists of mif which bind to this receptor , or which otherwise modulate the interaction of mif with cd74 or the consequences of such interaction , in treatment of conditions characterized by locally or systemically altered mif levels , particularly inflammatory conditions and cancer .

Description:
the following abbreviations are used herein : alexa - mif : alexa 488 - mif conjugate , erk : extracellular - signal - regulated kinase , ii : mhc class ii - associated invariant chain ( cd74 ), infγ : interferon - γ , mab : monoclonal antibody , mif : macrophage migration inhibitory factor . utilizing expression cloning and functional analyses , we have identified as a cellular receptor for mif the class ii - associated invariant chain , ii ( cd74 ) 10 . mif binds to the extracellular domain of ii , a type ii membrane protein , and ii is required for mif - induced cellular effects , including for instance , activation of the erk - 1 / 2 map kinase cascade and cell proliferation . these data provide a mechanism for mif &# 39 ; s activity as cytokine and identify it as a natural ligand for ii , which has been previously implicated in signaling and accessory functions for immune cell activation 11 - 13 . we linked the fluorescent dye alexa 488 14 to recombinant mif by standard techniques , verified the retention of biological activity of the conjugate ( fig1 a , b ), and conducted binding experiments with a panel of cell types known to respond to mif . by way of illustration , using flow cytometry , we observed high - affinity binding of alexa - mif to the surface of the human monocytic cell line , thp - 1 . this binding activity was induced by activation of monocytes with interferon - γ ( ifnγ ), and was competed by the addition of excess , unlabeled mif ( fig1 c ). confocal microscopy and direct visualization of ifnγ - treated monocytes at 4 ° c . showed surface binding of alexa - mif , and cell - bound alexa - mif was internalized upon shifting temperature to 37 ° c . ( fig1 d ). quantitative binding studies performed with increasing concentrations of alexa - mif revealed two apparent classes of cell surface receptors ( fig1 e ). the higher affinity binding activity showed a k d of 3 . 7 × 10 − 8 m and 3 . 1 × 10 4 binding sites per cell , and the lower affinity binding showed a k d of 3 . 5 × 10 − 7 m and 4 . 9 × 10 4 sites per cell . to identify the mif receptor , we prepared cdna from ifnγ - activated thp - 1 monocytes and constructed a mammalian expression library in the lambdazap - cmv vector 15 . library aliquots representing a total of 1 . 5 × 10 7 recombinants were transfected into cos - 7 cells , which we had established previously to exhibit little detectable binding activity for mif , and the transfectants were analyzed by flow cytometry for alexa - mif binding . positively - staining cells were isolated by cell sorting , and the cdna clones collected , amplified , and re - transfected into cos - 7 cells for additional rounds of cell sorting ( fig2 a ). after four rounds of selection , single colonies were prepared in e . coli and 250 colonies were randomly picked for analysis . we sequenced 50 clones bearing cdna inserts of ≧ 1 . 6 kb and observed that 10 encoded the class ii - associated invariant chain , ii ( cd74 ), a 31 - 41 kd type ii transmembrane protein 16 . while the isolated clones differed with respect to their total length , each was in the sense orientation and encoded a complete extracellular and transmembrane domain ( fig2 b ). to confirm that ii is a cell surface binding protein for mif , we analyzed the binding of alexa - mif to cos - 7 cells transfected with an ii expression plasmid ( fig2 c ). binding was inhibited by excess , unlabeled mif ( data not shown ), and by an anti - ii mab directed against the extracellular portion of the protein . anti - ii mab also inhibited the binding of alexa - mif to ifn - γ stimulated thp - 1 monocytes . the inhibition by anti - ii mab of alexa - mif binding to thp - 1 monocytes was significant , but partial , consistent with the interpretation that ii represents one of the two classes of cell surface receptors for mif revealed by scatchard analysis ( fig1 e ). [ 35 s ]- ii protein prepared by a coupled transcription and translation reticulocyte lysate system bound to mif in vitro , and the principal binding epitope was localized to a 40 amino acid region contained within the ii extracellular domain ( fig2 d ). to verify the functional significance of mif binding to ii in an exemplary system , we examined the activity of mif to stimulate erk - 1 / 2 activation and cellular proliferation in different ii - expressing cells . we observed an mif - mediated increase , and a dose - dependent , anti - ii mab - mediated decrease , in erk - 1 / 2 phosphorylation in ii - transfected cos - 7 cells ( fig3 ). irrespective of ii gene transfection however , we could not detect any proliferative effect of mif on this monkey epithelial cell line ( data not shown ). we then examined the activity of mif to induce erk - 1 / 2 activation and downstream proliferative responses in the human raji b cell line , which expresses a high level of ii 19 . mif stimulated the phosphorylation of erk - 1 / 2 in quiescent raji cells , and each of two anti - ii mabs blocked this stimulatory effect of mif ( fig4 a , b ). of note , the inhibitory effect of anti - ii on erk - 1 / 2 phosphorylation was associated with a significant decrease in the mif - stimulated proliferation of these cells ( fig4 c ). additionally , we confirmed the role of the mif - ii stimulation pathway in cells outside the immune system . mif extends the lifespan of primary murine fibroblasts 8 , and both mif &# 39 ; s mitogenic effects and its induction of the erk - 1 / 2 signal transduction cascade have been best characterized in this cell type 7 . fibroblasts express low levels of ii 20 , and we observed that anti - ii significantly inhibited both erk - 1 / 2 phosphorylation and the mitogenic effect of mif on cultured fibroblasts ( fig4 d and data not shown ). in prior experiments , we have experienced considerable difficulty in preparing a bioactive , 125 i - radiolabelled mif , and have observed the protein to be unstable to the ph conditions employed for biotin conjugation . by contrast , modification of mif by alexa 488 at a low molar density produced a fully bioactive protein which enabled identification of mif receptors on human monocytes , and the expression cloning of ii as a cell surface mif receptor . these data significantly expand our understanding of ii outside of its role in the transport of class ii proteins , and support recent studies which have described an accessory signaling function for ii in b and t cell physiology 10 - 13 . these findings provide a first insight into the long sought - after mif receptor , although additional proteins are likely involved in some mif - mediated activities . for instance , like mif , ii is a homotrimer 23 , and the ii intracellular domain consists of 30 - 46 amino acids , depending on which of two in - phase initiation codons are utilized 16 . monocyte - encoded ii has been shown to enhance t cell proliferative responses , and this accessory function of ii has been linked to a specific , chondroitin - sulphate - dependent interaction between ii and cd44 11 . we have observed an inhibitory effect of anti - cd44 on erk - 1 / 2 phosphorylation , but not mif binding , in ii - expressing cells . this is consistent with the inference that mif - bound ii is a stimulating ligand for cd44 - mediated map kinase activation . cd44 is a highly polymorphic type i transmembrane glycoprotein 24 , and cd44 likely mediates some of the downstream consequences of mif binding to ii . interference in the signal transduction pathways induced by mif - ii interaction , for instance by providing antagonists or inhibitors of mif - ii interaction , offers new approaches to the modulation of cellular immune and activation responses to mif . agents active in this regard ( agonists and antagonists and other inhibitors ) have predicted therapeutic utility in diseases and conditions typified by local or systemic changes in mif levels . the specific binding interaction between mif and the class ii invariant chain polypeptide , ii , also makes convenient the use of labeled mif reagents as “ trojan horse - type ” vehicles by which to concentrate a desired label or toxin in cells displaying cell surface ii . briefly , a desired label or toxic entity is associated with an mif ligand ( for instance , by covalent attachment ), and the modified mif ligand then is presented to cells displaying cell surface - localized ii , which class ii invariant chain polypeptide binds to and causes the internalization of the modified mif ligand , thus causing the operative cell to become specifically labeled or toxicated . the ii - displaying cells may be exposed to the modified mif ligand in vitro or in vivo , in which latter case ii - displaying cells may be specifically identified or toxicated in a patient . a wide variety of diagnostic and therapeutic reagents can be advantageously conjugated to an mif ligand ( which may be biologically active , full length mif or an ii - binding fragment thereof , or a mutein of either of the preceding and particularly such a mutein adapted to be biologically inactive and / or to be more conveniently coupled to a labeling or toxicating entity ), providing a modified mif ligand of the invention . typically desirable reagents coupled to an mif ligand include : chemotherapeutic drugs such as doxorubicin , methotrexate , taxol , and the like ; chelators , such as dtpa , to which detectable labels such as fluorescent molecules or cytotoxic agents such as heavy metals or radionuclides can be complexed ; and toxins such as pseudomonas exotoxin , and the like . human recombinant mif was purified from an e . coli expression system as described previously 22 and conjugated to alexa 488 14 by the manufacturer &# 39 ; s protocol ( molecular probes , eugene oreg .). the average ratio of dye ligand to mif homotrimer was 1 : 3 , as determined by matrix - assisted laser - desorption ionization mass spectrometry ( kompact probe / seq , kratos analytical ltd , manchester , uk ). anti - human ii monoclonal antibodies ( clones ln2 and m - b741 ) were obtained from pharmingen ( san jose calif .). thp - 1 cells ( 2 . 5 × 10 5 cells / ml ) were cultured in dmem / 10 % fbs with or without ifnγ ( 1 ng / ml , r & amp ; d systems , minneapolis , minn .) for 72 hrs . after washing , 5 × 10 5 cells were resuspended in 0 . 1 ml of medium and incubated with 200 ng of alexa - mif at 4 ° c . for 45 mins . the cells then were washed with ice - cold pbs ( ph 7 . 4 ) and subjected to flow cytometry analysis ( facscalibur , becton dickinson , san jose , calif .). in selected experiments , thp - 1 monocytes or cos - 7 transfectants were incubated with alexa - mif together with 50 μg / ml of an anti - ii mab or an isotypic control mab . for scatchard analysis , triplicate samples of ifnγ - treated , thp - 1 cells ( 1 × 10 6 ) were incubated for 45 mins at 4 ° c . in pbs / 1 % fbs together with alexa - mif ( 0 - 1 . 5 μm , calculated as mif trimer ), washed 3 × with cold pbs / 1fbs , and analyzed by flow cytometry using cellquest software ( becton dickinson , san jose , calif .) 29 . the specific binding curve was calculated by subtracting non - specific binding ( measured in the presence of excess unlabeled mif ) from total binding . confocal fluorescence microscopy of alexa - mif binding to cells was performed with an lsm 510 laser scanning instrument ( carl zeiss , jena germany ). thp - 1 cells were incubated with infγ for 72 hrs and washed 3 × with pbs / 1 % fbs prior to staining for 30 mins ( 4 ° c .) with 2 g / μl of alexa - mif , or alexa - mif plus 50 ng / μl unlabeled , rmif . cdna was prepared from the poly ( a ) + rna of ifnγ - activated , thp - 1 monocytes , cloned into the lambdazap - cmv vector ( stratagene , la jolla , calif . ), and dna aliquots ( 2 . 5 μg / ml ) transfected into 15 × 10 6 cos - 7 cells by the deae - dextran method 30 . the transfected cells were incubated with alexa - mif for 45 min at 4 ° c ., washed , and the positively - staining cells isolated 31 with a moflo cell sorter ( cytomation , fort collins , colo .). in a typical run , 1 . 5 × 10 7 cells / ml were injected and analyzed at a flow rate of 1 × 10 4 cells / sec . recovery was generally 90 %. plasmid dna was extracted from sorted cells using the easy dna kit ( invitrogen , carlsbad , calif .) and transformed into e . coli xl - 10 gold ( stratagene , la jolla , calif .) for further amplification . purified plasmid dna then was re - transfected into cos - 7 cells for further rounds of sorting . after 4 rounds of cell sorting , 250 single colonies were picked at random and the insert size analyzed by pcr . clones with inserts & gt ; 1 . 6 kb were individually transfected into cos - 7 cells and the mif binding activity re - analyzed by flow cytometry . using a full - length ii cdna clone as template , three truncated ( 1 - 72aa , 1 - 109aa , 1 - 149aa ) and one full - length ( 1 - 232aa ) ii product were generated by pcr and subcloned into the pcdna 3 . 1 / v5 - histopo expression vector ( invitrogen ). the complete nucleotide sequence of an exemplary ii cdna clone and the putative ii polypeptide forms that it encodes are presented in fig5 . the fidelity of vector construction was confirmed by automated dna sequencing and the constructs then used as template for coupled transcription and translation using the t n t reticulocyte lysate system ( promega , madison wis .). the binding of [ 35 s ]- labeled ii to immobilized mif was assessed by a 3 hr incubation at room temperature , as recommended by the tnt protocol . the dose - dependent phosphorylation of erk - 1 / 2 was measured by western blotting of cell lysates using specific antibodies directed against phospho - p44 / p42 or total p44 / p42 following methods described previously 7 . mif - mediated suppression of apoptosis was assessed in serum - deprived , murine embryonic fibroblasts by immunoassay of cytoplasmic histone - associated dna fragments ( roche biochemicals , indianapolis , ind .) 8 . proliferation studies were performed by a modification of previously published procedures 7 , 8 . human raji b cells ( american type tissue culture , rockville , md .) were cultured in rpmi / 10 % fbs , plated into 96 well plates ( 500 - 1000 cells / well ), and rendered quiescent by overnight incubation in rpmi / 0 . 5 % serum . the cells were washed , the rpmi / 0 . 5 % serum replaced , and the mif and antibodies added as indicated . after an additional overnight incubation , 1 μci of [ 3 h ]- thymidine was added and the cells harvested 12 hrs later . fibroblast mitogenesis was examined in normal human lung fibroblasts ( ccl210 , american type tissue culture ) cultured in dmem / 10 % fbs , resuspended in dmem / 2 % serum , and seeded into 96 well plates ( 150 cells / well ) together with rmif and antibodies as shown . isotype control or anti - ii mabs were added at a final concentration of 50 μg / ml . proliferation was assessed on day 5 after overnight incorporation of [ 3 h ]- thymidine into dna . as will be apparent to a skilled worker in the field of the invention , numerous modifications and variations of the present invention are possible in light of the above teachings . it is therefore to be understood that the invention may be practiced otherwise than as specifically described herein . 1 . weiser , et al ., “ molecular cloning of a cdna encoding a human macrophage migration inhibitory factor ”, proc . natl . acad . sci . usa , 86 , 7522 - 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