Patent Application: US-41749595-A

Abstract:
disclosed is a method of directing a cellular response in a mammal by expressing in a cell of the mammal a chimeric receptor which causes the cells to specifically recognize and destroy an infective agent , a cell infected with an infective agent , a tumor or cancerous cell , or an autoimmune - generated cell . also disclosed are cells which express the chimeric receptors and dna encoding the chimeric receptors .

Description:
fig1 a - c characterization of cd4 chimeras . fig1 a presents the amino acid sequence about the site of fusion between cd4 ( residues 1 - 369 ) and the different receptor chains . the underlined sequence shows the position of the amino acids encoded within the bamhi site used for fusion construction . the beginning of the transmembrane domain is marked with a vertical bar . the η sequence is identical to the ζ sequence at the amino terminus , but diverges at the carboxyl terminus ( jin et al ., proc . natl . acad . sci . usa , 87 : 3319 - 3323 ( 1990 )). fig1 b presents flow cytometric analysis of surface expression of cd4 , cd4 : ζ , cd4 : γ and cd4 : η in cv1 cells . cells were infected with virus expressing cd4 chimeras or cd16 pi , incubated for 9 hours at 37 ° c ., and stained with phycoerythrin - conjugated anti - cd4 mab leu3a . fig1 c presents immunoprecipitation of labeled cd4 : ζ , cd4 : γ , or native cd4 expressed in cv1 cells . lanes were run with reducing ( r ) or without reducing ( nr ) agent . molecular mass standards in kd are shown at left . fig2 surface expression of cd16 tm following coinfection of cd16 tm alone ( dense dots ), or coinfected with virus expressing cd4 : γ ( dashes ) or cd4 : ζ ( solid line ). sparse dots , cells infected with cd4 : ζ alone , stained with 3g8 ( fleit et al ., proc . natl . acad . sci . usa , 79 : 3275 - 3279 ( 1982 )) ( anti - cd16 mab ). fig3 a - b mutant cd4 : ζ chimeric receptors lacking ζ asp - 15 do not support the coexpression of cd16 tm . fig3 a is an autoradiogram of immunoprecipitated mutant chimeras electrophoresed either with reduction ( r ) or without reduction ( nr ). fig3 b details surface expression of cd16 tm following coinfection by viruses expressing cd16 tm and the following ζ chimeras : cd4 : ζ ( thick line ), cd4 : ζ c11g ( solid line ); cd4 : ζ ( dashed line ); cd4 : ζ c11g / d15g ( dense dots ); no coinfection ( cd16 tm alone , sparse dots ). cells were incubated with anti - cd16 mab 3g8 and phycoerythrin - conjugated fab &# 39 ; 2 goat antibodies to mouse igg . the level of expression of the ζ chimeras was essentially identical for the different mutants analyzed , and coinfection of cells with viruses expressing cd16 tm and ζ chimeras did not appreciably alter surface expression of the chimeras ( data not shown ). fig4 a - 4d increased intracellular free calcium ion follows crosslinking of mutant ζ chimeras in a t cell line . jurkat e6 cells ( weiss et al ., j . immunol ., 133 : 123 - 128 ( 1984 )) were infected with recombinant vaccinia viruses and analyzed by flow cytometry . the results shown are for the gated cd4 + population , so that only cells expressing the relevant chimeric protein are analyzed . the mean ratio of violet to blue indo - 1 fluorescence reflects the intracellular free calcium concentration in the population as a whole and the percentage of responding cells reflects the fraction of cells which exceed a predetermined threshold ratio ( set so that 10 % of untreated cells are positive ). fig4 a and fig4 b show jurkat cells expressing cd4 : ζ ( solid line ) or cd16 : ζ ( dashed line ) which were exposed to anti - cd4 mab leu3a ( phycoerythrin conjugate ), followed by crosslinking with goat antibody to mouse igg . the dotted line shows the response of uninfected cells to anti - cd3 mab okt3 . fig4 c and 4d show jurkat cells expressing cd4 : ζd15g ( solid line ); cd4 : ζc11g / d15g ( dashes ); or cd4 ; ζc11g ( dots ) which were treated and analyzed as in fig4 a and 4b . fig5 a - c cd4 : ζ , cd4 : η , and cd4 : γ receptors allow cytolytic t lymphocytes ( ctl ) to kill targets expressing hiv - 1 gp120 / 41 . fig5 a : solid circles , ctl expressing cd4 : ζ incubated with hela cells expressing gp120 / 41 ; open circles , ctl expressing cd4 : ζ incubated with uninfected hela cells ; solid squares , uninfected ctl incubated with hela cells expressing gp120 / 41 ; open squares , uninfected ctl incubated with uninfected hela cells . fig5 b : solid circles , ctl expressing cd4 : η incubated with hela cells expressing gp120 / 41 ; open circles , ctl expressing cd4 : γ incubated with hela cells expressing gp120 / 41 ; open squares , ctl expressing the c11g / d15g double mutant cd4 : ζ chimera incubated with hela cells expressing gp120 / 41 . fig5 c : flow cytometric analysis of cd4 expression by the ctl used in fig5 b . to correct the target to effector ratios the percent of cells expressing cd4 chimera was determined by subtracting the scaled negative ( uninfected ) population by histogram superposition ; for comparative purposes in this figure the uninfected cells were assigned an arbitrary threshold which gives roughly the same fraction positive for the other cell populations as would histogram subtraction . fig6 a - b specificity of the cd4 - directed cytolysis . fig6 a : solid circles , ctl expressing cd4 : ζ incubated with hela cells expressing cd16 pi ; open circles , ctl expressing cd4 incubated with hela cells expressing gp120 ; solid squares , ctl expressing cd16 : ζ incubated with hela cells expressing gp120 / 41 ; open squares , ctl expressing cd16 pi incubated with hela cells expressing gp120 / 41 . fig6 b : solid circles , ctl expressing cd4 : ζ incubated with raji ( mhc class ii + ) cells ; open circles , uninfected ctl cells incubated with rj2 . 2 . 5 ( mhc class ii - raji mutant ) cells ; solid squares , uninfected ctl incubated with raji ( mhc class ii + ) cells ; open squares , ctl expressing cd4 : ζ incubated with rj2 . 2 . 5 ( mhc class ii - ) cells . the ordinate scale is expanded . fig7 a - b characterization of the cd16 : ζ chimeric receptor . fig7 a is a schematic diagram of the cd16 : ζ fusion protein . the extracellular portion of the phosphatidylinositol - linked form of monomeric cd16 was joined to dimeric ζ just external to the transmembrane domain . the protein sequence at the fusion junction is shown at the bottom . fig7 b shows a flow cytometric analysis of calcium mobilization following crosslinking of the cd16 : ζ chimera in either a tcr positive or tcr negative cell line . the mean ratio of violet to blue fluorescence ( a measure of relative calcium ion concentration ) among cell populations treated with antibodies at time 0 is shown . solid squares , the response of jurkat cells to anti - cd3 mab okt3 ; solid triangles , the response of cd16 : ζ to anti - cd16 mab 3g8 crosslinking in the rex33a tcr - mutant ; open squares , the response to cd16 : ζ crosslinking in the jurkat tcr - mutant line jrt3 . t3 . 5 ; open triangles , the response to cd16 : ζ crosslinking in jurkat cells ; crosses , the response to nonchimeric cd16 in jurkat cells ; and dots , the response to nonchimeric cd16 in the rex33a tcr - cell line . fig8 a - b deletion analysis of cytolytic potential . fig8 a shows the locations of the ζ deletion endpoints . here as elsewhere mutations in ζ are represented by the original residue - location - mutant residue convention , so that d66 *, for example , denotes replacement of asp - 66 by a termination codon . fig8 b shows cytolysis assay results of undeleted cd16 : ζ and salient ζ deletions . hybridoma cells expressing surface antibody to cd16 were loaded with 51 cr and incubated with increasing numbers of human cytolytic lymphocytes ( ctl ) infected with vaccinia recombinants expressing cd16 : ζ chimeras . the percent of 51 cr released is plotted as a function of the effector ( ctl ) to target ( hybridoma ) cell ratio ( e / t ). solid circles , cytolysis mediated by cells expressing cd16 : ζ ( mfi 18 . 7 ); solid squares , cytolysis mediated by cells expressing cd16 : ζ asp66 * ( mfi 940 . 2 ); open squares , cytolysis mediated by cells expressing cd16 : ζglu60 * ( mfi 16 . 0 ); open circles , cytolysis mediated by cells expressing cd16 : ζtyr51 * ( mfi 17 . 4 ); solid triangles , cytolysis mediated by cells expressing cd16 : ζphe34 * ( mfi 17 . 8 ); and open triangles , cytolysis mediated by cells expressing nonchimeric cd16 ( mfi 591 ). although in this experiment the expression of cd16 : ζasp66 * was not matched to that of the other fusion proteins , cytolysis by cells expressing cd16 : ζ at equivalent levels in the same experiment gave results essentially identical to those shown by cells expressing cd16 : ζasp66 * ( not shown ). fig9 a - d elimination of the potential for transmembrane interactions reveals a short ζ segment capable of mediating cytolysis . fig9 a is a schematic diagram of the monomeric bipartite and tripartite chimeras . at the top is the cd16 : ζ construct truncated at residue 65 and lacking transmembrane cys and asp residues . below are the cd16 : cd5 : ζ and cd16 : cd7 : ζ constructs and related controls . the peptide sequences of the intracellular domains are shown below . fig9 b shows the cytolytic activity of monomeric chimera deletion mutants . the cytolytic activity of cells expressing cd16 : ζ ( solid circles ; mfi 495 ) was compared to that of cells expressing cd16 : ζasp66 * ( solid squares ; mfi 527 ) or the mutants cd16 : ζcys11gly / asp15gly / asp66 *, ( open squares ; mfi 338 ) and cd16 : ζcys11gly / asp15gly / glu60 * ( filled triangles ; mfi 259 ). fig9 c shows the cytolytic activity mediated by tripartite fusion proteins . solid triangles , cd16 : ζasp66 *; open squares , cd16 : 5 : ζ ( 48 - 65 ); solid squares cd16 : 7 : ζ ( 48 - 65 ); open triangles , cd16 : 7 : ζ ( 48 - 59 ); open circles , cd16 : 5 ; solid circles , cd16 : 7 . fig9 d shows calcium mobilization by mutant and tripartite chimeras in the tcr negative jurkat jrt3 . t3 . 5 mutant cell line . open circles , response of cells expressing dimeric cd16 : ζasp66 *; solid squares , response of cells expressing cd16 : ζcys11gly / asp15gly / asp66 *; open squares , response of cells expressing cd16 : ζcys11gly / asp15gly / glu60 *; solid triangles , response of cells expressing cd16 : 7 : ζ ( 48 - 65 ); and open triangles , response of cells expressing cd16 : ζ ( 48 - 59 ). fig1 a - f contribution of individual amino acids to the activity of the 18 residue cytolytic signal - transducing motif . fig1 a and 10b show cytolytic activity and fig1 c shows calcium ion mobilization mediated by chimeras bearing point mutations near the carboxyl terminal tyrosine ( y62 ). fig1 a and 10b represent data collected on cells expressing low and high amounts , respectively , of the cd16 : ζ fusion proteins . identical symbols are used for the calcium mobilization and cytolysis assays , and are shown in one letter code at right . solid circles , cells expressing cd16 : ζ ( mfi in a , 21 ; b , 376 ); solid squares , cells expressing cd16 : 7 : ζ ( 48 - 65 ) ( mfi a , 31 ; b , 82 ); open squares , cd16 : 7 : ζ ( 48 - 65 ) glu60gln ( mfi a , 33 ; b , 92 ), crosses , cd16 : 7 : ζ ( 48 - 65 ) asp63asn ( mfi a , 30 ; b , 74 ); solid triangles , cd16 : 7 : ζ ( 48 - 65 ) tyr62phe ( mfi a , 24 ; b , 88 ); open circles , cd16 : 7 : ζ ( 48 - 65 ) glu61gln ( mfi a , 20 ; b , 62 ); and open triangles , cd16 : 7 : ζ ( 48 - 65 ) tyr62ser ( mfi b , 64 ). fig1 d and 10e show cytolytic activity and fig1 f shows calcium ion mobilization by chimeras bearing point mutations near the amino terminal tyrosine ( y51 ). identical symbols are sued for the calcium mobilization and cytolysis assays and are shown at right . solid circles , cells expressing cd16 : ζ ( mfi in d , 21 . 2 ; in e , 672 ); solid squares , cells expressing cd16 : 7 : ζ ( 48 - 65 ) ( mfi d , 31 . 3 ; e , 179 ); solid triangles , cd16 : 7 : ζ ( 48 - 65 ) asn48ser ( mfi d , 22 . 4 ; e , 209 ); open squares , cd16 : 7 : ζ ( 48 - 65 ) leu50ser ( mfi d , 25 . 0 ; e , 142 ); and open triangles , cd16 : 7 : ζ ( 48 - 65 ) tyr51phe ( mfi d , 32 . 3 ; e , 294 ). fig1 a - b alignment of internal repeats of ζ and comparison of their ability to support cytolysis . fig1 a is a schematic diagram of chimeras formed by dividing the ζ intracellular domain into thirds and appending them to the transmembrane domain of a cd16 : 7 chimera . the sequences of the intracellular domains are shown below , with shared residues boxed , and related residues denoted by asterisks . fig1 b shows the cytolytic potency of the three ζ subdomains . solid circles , cells expressing cd16 : ζ ( mfi 476 ); solid squares , cd16 : 7 : ζ ( 33 - 65 ) ( mfi 68 ); open squares , cd16 : 7 : ζ ( 71 - 104 ) ( mfi 114 ); and solid triangles , cd16 : 7 : ζ ( 104 - 138 ) ( mfi 104 ). fig1 a - b calcium mobilization following crosslinking of cd4 : fcrγii and cd16 : fcrγii chimeras . fig1 a shows the ratio of violet to blue fluorescence emitted by cells loaded with the calcium sensitive fluorophore indo - 1 shown as a function of time following crosslinking of the cd16 extracellular domain with antibodies . fig1 b shows a similar analysis of the increase in ratio of violet to blue fluorescence of cells bearing cd4 : fcrγii chimeras , following crosslinking with antibodies . fig1 a - b cytolysis assay of cd4 : fcrγii and cd16 : fcrγii chimeras . fig1 a shows the percent of 51 cr released from anti - cd16 hybridoma ( target ) cells when the cells are exposed to increasing numbers of cytotoxic t lymphocytes expressing cd16 : fcrγii chimeras ( effector cells ). fig1 b shows a similar analysis of cytotoxicity mediated by cd4 : fcrγii chimeras against target cells expressing hiv envelope glycoproteins . fig1 a - e identification of residues in the fcrγii a tail which are important for cytolysis . fig1 a is a schematic diagram of the deletion constructs . fig1 b and 15c shows calcium mobilization and cytolysis by carboxyl - terminal deletion variants of cd16 : fcrγii a . fig1 d and 15e show calcium mobilization and cytolysis by tripartite chimeras bearing progressively less of the amino terminus of the intracellular tail of cd16 : fcrγii a . fig1 ( seq id no : 24 ) shows the amino acid sequence of the cd3 delta receptor protein ; the boxed sequence represents a preferred cytolytic signal transducing portion . fig1 ( seq id no : 25 ) shows the amino acid sequence of the t3 gamma receptor protein ; the boxed sequence represents a preferred cytolytic signal transducing portion . fig1 ( seq id no : 26 ) shows the amino acid sequence of the mb1 receptor protein ; the boxed sequence represents a preferred cytolytic signal transducing portion . fig1 ( seq id no : 27 ) shows the amino acid sequence of the b29 receptor protein ; the boxed sequence represents a preferred cytolytic signal transducing portion . human igg1 heavy chain sequences were prepared by joining sequences in the c h 3 domain to a cdna fragment derived from the 3 &# 39 ; end of the transmembrane form of the antibody mrna . the 3 &# 39 ; end fragment was obtained by polymerase chain reaction using a tonsil cdna library as substrate , and oligonucleotides having the sequences : cgc ggg gtg acc gtg ccc tcc agc agc ttg ggc ( seq id no : 7 ) and cgc ggg gat ccg tcg tcc aga gcc cgt cca gct ccc cgt cct ggg cct ca ( seq id no : 8 ), corresponding to the 5 &# 39 ; and 3 &# 39 ; ends of the desired dna fragments respectively . the 5 &# 39 ; oligo is complementary to a site in the c h 1 domain of human igg1 , and the 3 &# 39 ; oligo is complementary to a site just 5 &# 39 ; of the sequences encoding the membrane spanning domain . the pcr product was digested with bstxi and bamhi and ligated between bstxi and bamhi sites of a semisynthetic igg1 antibody gene bearing variable and constant regions . following the insertion of the bstxi to bamhi fragment , the amplified portions of the construct were replaced up to the smai site in c h 3 by restriction fragment interchange , so that only the portion between the smai site and the 3 &# 39 ; oligo was derived from the pcr reaction . to create a human igg1 : ζ chimeric receptor , the heavy chain gene ending in a bamhi site was joined to the bamhi site of the ζ chimera described below , so that the antibody sequences formed the extracellular portion . flow cytometry of cos cells transfected with a plasmid encoding the chimera showed high level expression of antibody determinants when an expression plasmid encoding a light chain cdna was cotransfected , and modest expression of antibody determinants when the light chain expression plasmid was absent . similar chimeras including human igg1 fused to η or γ ( see below ), or any signal - transducing portion of a t cell receptor or fc receptor protein may be constructed generally as described above using standard techniques of molecular biology . to create a single transcription unit which would allow both heavy and light chains to be expressed from a single promoter , a plasmid encoding a bicistronic mrna was created from heavy and light chain coding sequences , and the 5 &# 39 ; untranslated portion of the mrna encoding the 78 kd glucose regulated protein , otherwise known as grp78 , or bip . grp78 sequences were obtained by pcr of human genomic dna using primers having the sequences : cgc ggg cgg ccg cga cgc cgg cca aga cag cac ( seq id no : 9 ) and cgc gtt gac gag cag cca gtt ggg cag cag cag ( seq id no : 10 ) at the 5 &# 39 ; and 3 &# 39 ; ends respectively . polymerase chain reactions with these oligos were performed in the presence of 10 % dimethyl sulfoxide . the fragment obtained by pcr was digested with noti and hincii and inserted between noti and hpai sites downstream from human igg1 coding sequences . sequences encoding a human igg kappa light chain cdna were then inserted downstream from the grp78 leader , using the hincii site and another site in the vector . the expression plasmid resulting from these manipulations consisted of the semisynthetic heavy chain gene , followed by the grp78 leader sequences , followed by the kappa light chain cdna sequences , followed by polyadenylation signals derived from an sv40 dna fragment . transfection of cos cells with the expression plasmid gave markedly improved expression of heavy chain determinants , compared to transfection of plasmid encoding heavy chain determinants alone . to create a bicistronic gene comprising a heavy chain / receptor chimera and a light chain , the upstream heavy chain sequences can be replaced by any chimeric heavy chain / receptor gene described herein . human ζ ( weissman et al ., proc . natl . acad . sci . usa , 85 : 9709 - 9713 ( 1988b )) and γ ( kuster et al ., j . biol . chem ., 265 : 6448 - 6452 ( 1990 )) cdnas were isolated by polymerase chain reaction from libraries prepared from the hpb - all tumor cell line ( aruffo et al ., proc . natl . acad . sci . usa , 84 : 8573 - 8577 ( 1987b )) and from human natural killer cells , while η cdna ( jin et al ., proc . natl . acad . sci . usa , 87 : 3319 - 3323 ( 1990 )) was isolated from a murine thymocyte library . ζ , η and γ cdnas were joined to the extracellular domain of an engineered form of cd4 possessing a bamhi site just upstream of the membrane spanning domain ( aruffo et al ., proc . natl . acad . sci . usa , 84 : 8573 - 8577 ( 1987b ); zettlmeissl et al ., dna cell biol ., 9347 - 353 ( 1990 )) which was joined to the bamhi site naturally present in the ζ and η cdnas at a similar location a few residues upstream of the membrane spanning domain ( seq id nos : 1 , 3 , 4 and 6 ). to form the fusion protein with γ a bamhi site was engineered into the sequence at the same approximate location ( fig1 ; seq id no : 2 and 5 ). the gene fusions were introduced into a vaccinia virus expression plasmid bearing the e . coli gpt gene as a selectable marker ( m . amiot and b . s ., unpublished ), and inserted into the genome of the vaccinia wr strain by homologous recombination and selection for growth in mycophenolic acid ( falkner et al ., j . virol ., 62 : 1849 - 1854 ( 1988 ); boyle et al ., gene , 65 : 123 - 128 ( 1988 )). flow cytometric analysis showed that the vaccinia recombinants direct the abundant production of cd4 : ζ and cd4 : γ fusion proteins at the cell surface , whereas the expression of cd4 : η is substantially weaker ( fig1 ). the latter finding is consistent with a recent report that transfection of an η cdna expression plasmid into a murine hybridoma cell line gave substantially less expression than transfection of a comparable ζ expression plasmid ( clayton et al ., j . exp . med ., 172 : 1243 - 1253 ( 1990 )). immunoprecipitation of cells infected with the vaccinia recombinants revealed that the fusion proteins form covalent dimers , unlike the naturally occurring cd4 antigen ( fig1 ). the molecular masses of the monomeric cd4 : ζ and cd4 : γ fusion proteins and native cd4 were found to be 63 , 55 and 53 kd respectively . the larger masses of the fusion proteins are approximately consistent with the greater length of the intracellular portion , which exceeds that of native cd4 by 75 ( cd4 : ζ ) or 5 ( cd4 : γ ) residues . cell surface expression of the macrophage / natural killer cell form of human fcγriii ( cd16 tm ) on transfectants is facilitated by cotransfection with murine ( kurosaki et al ., nature , 342 : 805 - 807 ( 1989 )) or human ( hibbs et al ., science , 246 : 1608 - 1611 ( 1989 )) γ , as well as by human ζ ( lanier et al ., nature , 342 : 803 - 805 ( 1989 )). consistent with these reports , expression of the chimeras also allowed surface expression of cd16 tm when delivered to the target cell either by cotransfection or by coinfection with recombinant vaccinia viruses fig2 . the promotion of ( cd16 tm ) surface expression by ζ was more pronounced than promotion by γ ( fig2 ) in the cell lines examined , whereas native cd4 ( data not shown ) did not enhance cd16 tm surface expression . to create chimeras which would not associate with existing antigen or fc receptors , mutant ζ fusion proteins which lacked either the intramembranous asp or intramembranous cys residue or both were prepared . flow cytometry showed that the intensity of cell surface expression by the different mutant chimeras was not appreciably different from the unmutated precursor ( data not shown ) and immunoprecipitation experiments showed that total expression by the chimeras was similar ( fig3 ). as expected , the mutant chimeras lacking the transmembrane cysteine residue were found not to form disulfide linked dimers ( fig3 ). the two mutant chimeras lacking asp were incapable of supporting the surface expression of cd16 tm , whereas the monomeric chimeras lacking cys but bearing asp allowed cd16 tm to be coexpressed , but at lower efficiency than the parental dimer ( fig3 ). to determine whether crosslinking of the fusion proteins would allow the accumulation of free intracellular calcium in a manner similar to that known to occur with the t cell antigen receptor , cells of the human t cell leukemia line , jurkat e6 ( atcc accessior number tib 152 , american type culture collection , rockville , md . ), were infected with the vaccinia recombinants and the relative cytoplasmic calcium concentration following crosslinking of the extracellular domain with antibodies was measured . flow cytometric measurements were performed with cells loaded with the calcium sensitive dye indo - 1 ( grynkiewicz et al ., j . biol . chem ., 260 : 3340 - 3450 ( 1985 ); rabinovitch et al ., j . immunol ., 137 : 952 - 961 ( 1986 )). fig4 shows the results of calcium flux experiments with cells infected with cd4 : ζ and the asp - and cys - mutants of ζ . crosslinking of the chimeras , reproducibly increased intracellular calcium . cd4 : η and cd4 : γ similarly allowed accumulation of intracellular calcium in infected cells ( data not shown ). jurkat cells express low levels of cd4 on the cell surface , however , crosslinking of the native cd4 in the presence or absence of cd16 : ζ ( c . r . and b . s . unpublished ) ( fig4 and data not shown ) does not alter intracellular calcium levels . cd4 : ζ , η , and γ chimeras mediate cytolysis of targets expressing hiv gp120 / 41 to determine whether the chimeric receptors would trigger cytolytic effector programs , a model target : effector system based on cd4 recognition of the hiv envelope gp120 / gp41 complex was created . hela cells were infected with recombinant vaccinia viruses expressing gp120 / gp41 ( chakrabarti et al ., nature , 320 : 535 - 537 ( 1986 ); earl et al ., j . virol ., 64 : 2448 - 2451 ( 1990 )) and labeled with 51 cr . the labeled cells were incubated with cells from a human allospecific ( cd8 + , cd4 - ) cytotoxic t lymphocyte line which had been infected with vaccinia recombinants expressing the cd4 : ζ , cd4 : η , or cd4 : γ chimeras , or the cd4 : ζcys11gly : asp15gly double mutant chimera . fig5 shows that hela cells expressing gp120 / 41 were specifically lysed by cytotoxic t lymphocytes ( ctl ) expressing cd4 chimeras . uninfected hela cells were not targeted by ctl armed with cd4 : ζ chimeras , and hela cells expressing gp120 / 41 were not recognized by uninfected ctl . to compare the efficacy of the various chimeras , the effector to target ratios were corrected for the fraction of ctl expressing cd4 chimeras , and for the fraction of hela cells expressing gp120 / 41 , as measured by flow cytometry . fig5 c shows a cytometric analysis of cd4 expression by the ctl used in the cytolysis experiment shown in fig4 a and 4b . although the mean density of surface cd4 : ζ greatly exceeded the mean density of cd4 : η , the cytolytic efficiencies of cells expressing either form were similar . correcting for the fraction of targets expressing gp120 , the efficiency of cytolysis mediated by cd4 : ζ and cd4 : η proteins are comparable to the best efficiencies reported for specific t cell receptor target : effector pairs ( the mean effector to target ratio for 50 % release by t cells expressing cd4 : ζ was 1 . 9 ± 0 . 99 , n = 10 ). the cd4 : γ fusion was less active , as was the cd4 : ζ fusion lacking the transmembrane asp and cys residues . however in both cases significant cytolysis was observed ( fig5 ). to control for the possibility that vaccinia infection might promote artefactual recognition by ctl , similar cytolysis experiments were performed with target cells infected with vaccinia recombinants expressing the phosphatidylinositol linked form of cd16 ( cd16 pi ) and labeled with 51 cr , and with ctl infected with control recombinants expressing either cd16 pi or cd16 : ζ . fig6 a shows that t cells expressing non - cd4 chimeras do not recognize native hela cells or hela cells expressing gp120 / 41 , and similarly that t cells expressing cd4 chimeras do not recognize hela cells expressing other vaccinia - encoded surface proteins . in addition , ctls expressing non - chimeric cd4 do not significantly lyse hela cells expressing gp120 / 41 ( fig6 a ). cd4 is thought to interact with a nonpolymorphic sequence expressed by mhc class ii antigen ( gay et al ., nature , 328 : 626 - 629 ( 1987 ); sleckman et al ., nature , 328 : 351 - 353 ( 1987 )). although a specific interaction between cd4 and class ii antigen has never been documented with purified proteins , under certain conditions adhesion between cells expressing cd4 and cells expressing class ii molecules can be demonstrated ( doyle et al ., nature , 330 : 256 - 259 ( 1987 ); clayton et al ., j . exp . med ., 172 : 1243 - 1253 ( 1990 ); lamarre et al ., science , 245 : 743 - 746 ( 1989 )). next examined was whether killing could be detected against cells bearing class ii antigen . fig6 b shows that there is no specific cytolysis directed by cd4 : ζ against the raji b cell line , which expresses abundant class ii antigen . although a modest (≈ 5 %) cytolysis is observed , a class ii - negative mutant of raji , rj2 . 2 . 5 , ( accolla , r . s ., j . exp . med ., 157 : 1053 - 1058 ( 1983 )) shows a similar susceptibility , as do raji cells incubated with uninfected t cells . sequence requirements for induction of cytolysis by the t cell antigen / fc receptor zeta chain although chimeras between cd4 and ζ can arm cytotoxic t lymphocytes ( ctl ) to kill target cells expressing hiv gp120 , an alternative to cd4 was sought in order to unambiguously compare the properties of zeta chimeras introduced into human t cell lines . such lines can express cd4 , making it difficult to specifically define the relationship between the type or degree of calcium mobilization and the cytotoxic potential of the different chimeras . to circumvent this , chimeras were created between ζ and cd16 in which the extracellular domain of cd16 is attached to the transmembrane and intracellular sequences of ζ ( fig7 a ). the gene fusions were introduced into a vaccinia virus expression plasmid bearing the e . coli gpt gene as a selectable marker and inserted into the genome of the vaccinia wr strain by homologous recombination and selection for growth in mycophenolic acid ( falkner and moss , j . virol . 62 : 1849 ( 1988 ); boyle and coupar , gene 65 : 123 ( 1988 )). t cell lines were infected with the vaccinia recombinants and the relative cytoplasmic free calcium ion concentration was measured following crosslinking of the extracellular domains with antibodies . both spectrofluorimetric ( bulk population ) and flow cytometric ( single cell ) measurements were performed , with cells loaded with the dye indo - 1 ( grynkiewicz et al ., j . biol . chem . 260 : 3440 ( 1985 ); rabinovitch et al ., j . immunol . 137 : 952 ( 1986 )). fig7 b shows an analysis of data collected from cells of the jurkat human t cell leukemia line infected with vaccinia recombinants expressing cd16 : ζ fusion protein . crosslinking of the chimeras reproducibly increased intracellular calcium , while similar treatment of cells expressing nonchimeric cd16 had little or no effect . when the chimera was expressed in mutant cell lines lacking antigen receptor , either rex33a ( breitmeyer et al . j . immunol . 138 : 726 ( 1987 ); sancho et al . j . biol . chem 264 : 20760 ( 1989 )), jurkat mutant jrt3 . t3 . 5 ( weiss et al ., j . immunol . 135 : 123 ( 1984 )), a strong response to cd16 antibody crosslinking was seen . similar data have been collected on the rex20a ( breitmeyer et al ., supra , 1987 ; blumberg et al ., j . biol . chem . 265 : 14036 ( 1990 )) mutant cell line , and a cd3 / ti negative mutant of the jurkat cell line established in this laboratory ( data not shown ). infection with recombinants expressing cd16 : ζ did not restore the response to anti - cd3 antibody , showing that the fusion protein did not act by rescuing intracellular cd3 complex chains ( data not shown ). to evaluate the ability of the chimeras to redirect cell - mediated immunity , ctls were infected with vaccinia recombinants expressing cd16 chimeras and used to specifically lyse hybridoma cells expressing membrane - bound anti - cd16 antibodies ( see below ). this assay is an extension of a hybridoma cytotoxicity assay originally developed to analyze effector mechanisms of cells bearing fc receptors ( graziano and fanger , j . immunol . 138 : 945 , 1987 ; graziano and fanger , j . immunol . 139 : 35 - 36 , 1987 ; shen et al ., mol . immunol . 26 : 959 , 1989 ; fanger et al ., immunol . today 10 : 92 , 1989 ). fig8 b shows that expression of cd16 : ζ in cytotoxic t lymphocytes allows the armed ctl to kill 3g8 ( anti - cd16 ; fleit et al ., proc . natl . acad . sci . usa 79 : 3275 , 1982 ) hybridoma cells , whereas ctl expressing the phosphatidylinositol - linked form of cd16 are inactive . ctl armed with cd16 : ζ also do not kill hybridoma cells expressing an irrelevant antibody ( data not shown ). to identify the minimal ζ sequences necessary for cytolysis , a series of deletion mutants were prepared in which successively more of the ζ intracellular domain was removed from the carboxyl terminus ( fig8 a ). most of the intracellular domain of zeta could be removed with little consequence for cytolytic potential ; the full length chimera cd16 : ζ was essentially equal in efficacy to the chimera deleted to residue 65 , cd16 : ζasp66 * ( fig8 b ). a substantial decrease in cytotoxicity was observed on deletion to ζ residue 59 ( chimera cd16 : ζglu60 *), and further deletion to residue 50 resulted in slightly less activity . however complete loss of activity was not observed even when the intracellular domain was reduced to a three residue transmembrane anchor ( fig8 b ). because ζ is a disulfide linked dimer , one explanation for the retention of cytolytic activity was that endogenous ζ was forming heterodimers with the chimeric ζ deletion , thereby reconstituting activity . to test this idea , ζ residues 11 and 15 were changed from asp and cys respectively to gly ( cys11gly / asp15gly ), and immunoprecipitations were carried out as follows . approximately 2 × 10 6 cv1 cells were infected for one hour in serum free dme medium with recombinant vaccinia at a multiplicity of infection ( moi ) of at least ten . six to eight hours post - infection , the cells were detached from the plates with pbs / 1 mm edta and surface labeled with 0 . 2 mci 125 i per 2 × 10 6 cells using lactoperoxidase and h 2 o 2 by the method of clark and einfeld ( leukocyte typing ii , pp 155 - 167 , springer - verlag , new york , 1986 ). the labeled cells were collected by centrifugation and lysed in 1 % np - 40 , 0 . 1 % sds , 0 . 15m nacl , 0 . 05m tris , ph 8 . 0 , 5 mm mgcl 2 , 5 mm kcl , 0 . 2m iodoacetamide and 1 mm pmsf . nuclei were removed by centrifugation , and cd16 proteins were immunoprecipitated with antibody 3g8 ( fleit et al ., supra , 1982 ; medarex ) and anti - mouse igg agarose ( cappel , durham , n . c .). samples were electrophoresed through an 8 % polyacrylamide / sds gel under non - reducing conditions or through a 10 % gel under reducing conditions . these immunoprecipitations confirmed that the cd16 : ζcys11gly / asp15gly chimera did not associate in disulfide - linked dimer structures . the cytolytic activity of the mutant receptors was also tested . the mutated chimera deleted to residue 65 ( cd16 : ζcys11gly / asp15gly / asp66 *) was , depending on the conditions of assay , two to eight fold less active in the cytolysis assay than the comparable unmutated chimera ( cd16 : ζasp66 *), which was usually within a factor of two of , or indistinguishable in activity from , cd16 : ζ ( fig9 b ). the reduction in activity of the mutant chimeras is comparable to the reduction seen with cd4 chimeras of similar structure ( see above ) and is most likely attributable to the lower efficiency of ζ monomers compared to dimers . in contrast , the asp - , cys - mutated chimera deleted to residue 59 had no cytolytic activity ( fig9 b ), supporting the hypothesis that association with other chains mediated by the transmembrane cys and / or asp residues was responsible for the weak persistence of cytolytic activity in deletions more amino terminal than residue 65 . flow cytometric studies showed that the deletion mutants lacking transmembrane asp and cys residues could still promote an increase in free intracellular calcium ion in response to antibody crosslinking in a tcr - mutant jurkat cell line ( fig9 d ). similar results were obtained for chimeras expressed in the parental jurkat line ( not shown ). in the case of cd16 : ζcys11gly / asp15gly / glu60 *, these data demonstrate that the ability to mediate calcium responsiveness can be mutationally separated from the ability to support cytolysis . to definitively eliminate the possible contribution of ζ transmembrane residues , the transmembrane and first 17 cytoplasmic residues of ζ were replaced by sequences encoding the membrane spanning and first 14 or first 17 cytoplasmic residues of the cd5 or cd7 antigens , respectively ( fig9 a ). the resulting tripartite fusion proteins cd16 : 5 : ζ ( 48 - 65 ) and cd16 : 7 : ζ ( 48 - 65 ) did not form disulfide - linked dimers as do the simpler cd16 : ζ chimeras , because they lacked the cysteine residue in the ζ transmembrane domain . both tripartite chimeras were able to mobilize calcium in jurkat and tcr negative cell lines ( fig9 d ) and to mount a cytolytic response in ctl ( fig9 c and data not shown ). however truncation of the ζ portion to residue 59 in chimera cd16 : 7 : ζ ( 48 - 59 ) abrogates the ability of tripartite fusion to direct calcium responsiveness in tcr positive or negative jurkat cells or cytolysis in mature ctl ( fig9 c and 9d and data not shown ). to examine the contributions of individual residues within the 18 - residue motif , we prepared a number of mutant variants by site - directed mutagenesis , and evaluated their ability to mediate receptor - directed killing under conditions of low ( fig1 a and 10d ) or high ( fig1 b and 10e ) expression of chimeric receptor . fig1 shows that while a number of relatively conservative substitutions ( i . e ., replacing acidic residues with their cognate amides , or tyrosine with phenylalanine ) which spanned residues 59 to 63 yielded moderate compromise of cytolytic efficacy , in general the variants retained the ability to mobilize calcium . however collectively these residues comprise an important submotif inasmuch as their deletion eliminates cytolytic activity . conversion of tyr 62 to either phe or ser eliminated both the cytotoxic and calcium responses . at the amino terminus of the 18 residue segment , replacement of tyr 51 with phe abolished both calcium mobilization and cytolytic activity , while substitution of leu with ser at position 50 eliminated the calcium response while only partially impairing cytolysis . without being bound to a particular hypothesis , it is suspected that the inability of the leu50ser mutant to mobilize calcium in short term flow cytometric assays does not fully reflect its ability to mediate a substantial increase in free intracellular calcium ion over the longer time span of the cytolysis assay . however , calcium - insensitive cytolytic activity has been reported for some cytolytic t cell lines , and the possibility that a similar phenomenon underlies the results described herein has not been ruled out . replacement of asn48 with ser partially impaired cytotoxicity in some experiments while having little effect in others . to investigate the potential role of redundant sequence elements , the intracellular domain of ζ was divided into three segments , spanning residues 33 to 65 , 71 to 104 , and 104 to 138 . each of these segments was attached to a cd16 : cd7 chimera by means of a mlui site introduced just distal to the basic membrane anchoring sequences of the intracellular domain of cd7 ( see below ; fig1 a ). comparison of the cytolytic efficacy of the three elements showed they were essentially equipotent ( fig1 b ). sequence comparison ( fig1 a ) shows that the second motif bears eleven residues between tyrosines , whereas the first and third motifs bear ten . although a precise accounting of the process of t cell activation has not been made , it is clear that aggregation of the antigen receptor , or of receptor chimeras which bear ζ intracellular sequences , triggers calcium mobilization , cytokine and granule release , and the appearance of cell surface markers of activation . the active site of ζ , a short linear peptide sequence probably too small to have inherent enzymatic activity , likely interacts with one or at most a few proteins to mediate cellular activation . it is also clear that mobilization of free calcium is not by itself sufficient for cellular activation , as the ability to mediate cytolysis can be mutationally separated from the ability to mediate calcium accumulation . as shown herein , addition of 18 residues from the intracellular domain of ζ to the transmembrane and intracellular domain of two unrelated proteins allows the resulting chimeras to redirect cytolytic activity against target cells which bind to the extracellular portion of the fusion proteins . although chimeras bearing the 18 residue motif are approximately eight - fold less active than chimeras based on full length ζ , the reduced activity can be attributed to the loss of transmembrane interactions which normally allow wild - type ζ to form disulfide linked dimers . that is , ζ deletion constructs which have the same carboxyl terminus as the motif and lack transmembrane cys and asp residues typically show slightly less activity than chimeras bearing only the 18 residue motif . the cytolytic competency element on which we have focused has two tyrosines and no serines or threonines , restricting the possible contributions of phosphorylation to activity . mutation of either tyrosine destroys activity , however , and although preliminary experiments do not point to a substantial tyrosine phosphorylation following crosslinking of chimeric surface antigens bearing the 18 reside motif , the possible participation of such phosphorylation at a low level cannot be excluded . in addition to the effects noted at the two tyrosine residues , a number of amino acid replacements at the amino and carboxyl terminus of the motif weaken activity under conditions of low receptor density . sequences similar to the ζ active motif can be found in the cytoplasmic domains of several other transmembrane proteins , including the cd3 δ and γ molecules , the surface igm associated proteins mb1 and b29 , and the β and γ chains of the high affinity ige receptor , fcεri ( reth , nature 338 : 383 , 1989 ). although the function of these sequences is uncertain , if efficiently expressed , each may be capable of autonomous t cell activation , and such activity may explain the residual tcr responsiveness seen in a zeta - negative mutant cell line ( sussman et al ., cell 52 : 85 , 1988 ). ζ itself bears three such sequences , approximately equally spaced , and a rough trisection of the intracellular domain shows that each is capable of initiating a cytolytic response . η , a splice isoform of ζ ( jin et al ., supra , 1990 ; clayton et al ., proc . natl . acad . sci . usa 88 : 5202 , 1991 ), lacks the carboxyl half of the third motif . because removal of the carboxyl half of the first motif abolishes activity , it appears likely that the majority of the biological effectiveness of η can be attributed to the first two motifs . although by different measures η is equally as active as ζ in promoting antigen - mediated cytokine release ( bauer et al ., proc . natl . acad . sci . usa 88 : 3842 , 1991 ) or redirected cytolysis ( see above ), η is not phosphorylated in response to receptor stimulation ( bauer et al ., supra , 1991 ). thus either the presence of all three motifs is required for phosphorylation , or the third motif represents a favored substrate for an unidentified tyrosine kinase . to evaluate the actions of different human fc receptor subtypes , chimeric molecules were created in which the extracellular domain of the human cd4 , cd5 or cd16 antigens were joined to the transmembrane and intracellular domains of the fcriiγa , b1 , b2 , and c subtypes ( nomenclature of ravetch and kinet , ann . rev . immunol . 9 : 457 , 1991 ). specifically , cdna sequences corresponding to the transmembrane and cytoplasmic domains of the previously described fcriia , b1 , and b2 isoforms were amplified from the preexisting clone pc23 or from a human tonsil cdna library ( constructed by standard techniques ) using the following synthetic oligonucleotides primers : ccc gga tcc cag cat ggg cag ctc tt ( seq id no : 18 ; fcrii a forward ); cgc ggg gcg gcc gct tta gtt att act gtt gac atg gtc gtt ( seq id no : 19 ; fcrii a reverse ); gcg ggg gga tcc cac tgt cca agc tcc cag ctc ttc acc g ( seq id no : 20 ; fcrii b1 and fcrii b2 forward ); and gcg ggg gcg gcc gcc taa ata cgg ttc tgg tc ( seq id no : 21 ; fcrii b1 and fcrii b2 reverse ). these primers contained cleavage sites for the enzymes bamhi and noti , respectively , indented 6 residues from the 5 &# 39 ; end . the noti site was immediately followed by an antisense stop codon , either cta or tta . all primers contained 18 or more residues complementary to the 5 &# 39 ; and 3 &# 39 ; ends of the desired fragments . the cdna fragment corresponding to the fcriiγc cytoplasmic domain , which differs from the iia isoform in only one amino acid residue ( l for p at residue 268 ) was generated by site directed mutagenesis by overlap pcr using primers of sequence : tca gaa aga gac aac ctg aag aaa cca aca a ( seq id no : 22 ) and ttg ttg gtt tct tca ggt tgt gtc ttt ctg a ( seq id no : 23 ). the pcr fragments were inserted into vaccinia virus expression vectors which contained the cd16 or cd4 extracellular domains respectively and subsequently inserted into wild type vaccinia by recombination at the thymidine kinase locus , using selection for cointegration of e coli gpt to facilitate identification of the desired recombinants . the identities of all isoforms ( shown in fig1 ) were confirmed by dideoxy sequencing . production of the chimeric receptor proteins was further confirmed by immunoprecipitation studies . approximately 10 7 jrt3 . t3 . 5 cells were infected for one hour in serum free imdm medium with recombinant vaccinia at a multiplicity of infection of at least ten . twelve hours post - infection , the cells were harvested and surface labeled with 0 . 5 mci 125 i per 10 7 cells using the lactoperoxidase / glucose oxidase method ( clark and einfeld , supra ). the labeled cells were collected by centrifugation and lysed 1 % np - 40 , 0 . 1 mm mgcl 2 , 5 mm kcl , 0 . 2m iodoacetamide and 1 mm pmsf . nuclei were removed by centrifugation , and cd16 fusion proteins immunoprecipitated with antibody 4g8 and anti - mouse igg agarose . samples were electrophoresed under reducing conditions . all immunoprecipitated chimeric receptor molecules were of the expected molecular masses . to test the ability of the chimeric receptors to mediate an increase in cytoplasmic free calcium ion , the recombinant viruses were used to infect the tcr - mutant jurkat cell line jrt3 . t3 . 5 ( as described herein ) and cytoplasmic free calcium was measured in the cells ( as described herein ) following crosslinking of the receptor extracellular domains with monoclonal antibody 3g8 or leu - 3a ( as described herein ). these experiments revealed that the intracellular domains of fcrγii a and c were capable of mediating an increase in cytoplasmic free calcium ion after crosslinking of the extracellular domains , whereas the intracellular domains of fcrγii b1 and b2 were inactive under comparable conditions ( fig1 a and 13b ). the cd4 , cd5 and cd16 hybrids of fcrγii a shared essentially equal capacity to promote the calcium response ( fig1 and data not shown ). other cell lines , from both monocytic and lymphocytic lineages , were capable of responding to the signal initiated by crosslinking of the extracellular domains ( data not shown ). to explore the involvement of the different fcrγii intracellular domains in cytolysis , human cytotoxic t lymphocytes ( ctl ) were infected with vaccinia recombinants expressing cd16 : fcrγii a , b1 , b2 and c chimeras . the infected cells were then cocultured with 51 cr - loaded hybridoma cells ( i . e ., 3g8 10 - 2 cells ) which expressed cell surface antibody to cd16 . in this assay ctls bearing the cd16 chimera killed the hybridoma target cells ( allowing release of free 51 cr ) if the cd16 extracellular domain of the chimera has been joined to an intracellular segment capable of activating the lymphocyte effector program ; this cytolysis assay is described in detail below . fig1 a shows that ctl armed with cd16 : fcrγiia and c , but not fcrγii b1 or b2 , are capable of lysing target cells expressing cell surface anti - cd16 antibody . to eliminate the possibility that the specific cytolysis was in some way attributable to interaction with the cd16 moiety , cytolysis experiments were conducted in which the fcrii intracellular domains were attached to a cd4 extracellular domain . in this case the target cells were hela cells expressing hiv envelope gp120 / 41 proteins ( specifically , hela cells infected with the vaccinia vector vpe16 ( available from the national institute of allergy and infections disease aids depository , bethesda , md ). as in the cd16 system , target cells expressing hiv envelope were susceptible to lysis by t cells expressing the cd4 : fcrγii a chimera , but not fcrγii b1 or b2 ( fig1 b ). the intracellular domains of fcrγii a and c share no appreciable sequence homology with any other protein , including the members of the extended fcrγ / tcrζ family . to define the sequence elements responsible for induction of cytolysis , 5 &# 39 ; and 3 &# 39 ; deletions of the intracellular domain coding sequences ( described below and shown in fig1 a ) were prepared and were evaluated for efficacy in calcium mobilization and cytolysis assays ( as described herein ). in the experiments in which the amino terminal portion of the intracellular domain was removed , the transmembrane domain of fcrγii was replaced with the transmembrane domain of the unrelated cd7 antigen to eliminate the possible contribution of interactions mediated by the membrane - spanning domain . fig1 b and 15c show that removal of the 14 carboxyl - terminal residues , including tyrosine 298 , resulted in a complete loss of cytolytic capacity and a substantial reduction in calcium mobilization potential . further deletion to just before tyrosine 282 gave an identical phenotype ( fig1 b and 15c ). deletion from the n - terminus of the intracellular domain to residue 268 had no substantial effect on either calcium profile or cytolytic potency , whereas deletion to residue 275 markedly impaired free calcium release but had little effect on cytolysis ( fig1 d and 15e ). further deletion , to residue 282 , gave fcrγii tails which lacked the ability to either mobilize calcium or trigger cytolysis ( fig1 d and 15e ). the ` active element ` defined by these crude measures is relatively large ( 36 amino acids ) and contains two tyrosines separated by 16 residues . other intracellular and transmembrane signal transducing domains according to the invention may be derived from the t cell receptor proteins , cd3 delta and t3 gamma , and the b cell receptor proteins , mb1 and b29 . the amino acid sequences of these proteins are shown in fig1 ( cd3 delta ; seq id no : 24 ), fig1 ( t3 gamma ; seq id no : 25 ), fig1 ( md1 ; seq id no : 26 ) and fig1 ( b29 ; seq id no : 27 ). the portions of the sequences sufficient for cytolytic signal transduction ( and therefore preferably included in a chimeric receptor of the invention ) are shown in brackets . chimeric receptors which include these protein domains are constructed and used in the therapeutic methods of the invention generally as described above . approximately 5 × 10 6 cv1 cells were infected for one hour in serum free dme medium with recombinant vaccinia at a multiplicity of infection ( moi ) of at least ten ( titer measured on cv1 cells ). the cells were placed in fresh medium after infection and labelled metabolically with 200 μci / ml 35 s - methionine plus cysteine ( tran 35 s - label , icn ; costo mesa , calif .) in methionine and cysteine free dmem ( gibco ; grand island , n . y .) for six hours . the labelled cells were detached with pbs containing 1 mm edta , collected by centrifugation , and lysed in 1 % np - 40 , 0 . 1 % sds , 0 . 15m nacl , 0 . 05m tris ph 8 . 0 , 5 mm edta , and 1 mm pmsf . nuclei were removed by centrifugation , and cd4 proteins immunoprecipitated with okt4 antibody and anti - mouse igg agarose ( cappel , durham , n . c .). samples were electrophoresed through 8 % polyacrylamide / sds gels under non - reducing ( nr ) and reducing ( r ) conditions . gels containing 35 s - labelled samples were impregnated with en 3 hance ( new england nuclear , boston , mass .) prior to autoradiography . facilitated expression of the transmembrane form of cd16 , cd16 tm , was measured by comparing its expression in cv1 cells singly infected with cd16 tm with expression in cells coinfected with viruses encoding cd16 tm and ζ or γ chimeras . after infection and incubation for six hours or more , cells were detached from plates with pbs , 1 mm edta and the expression of cd16tm or the chimeras was measured by indirect immunofluorescence and flow cytometry . jurkat subline e6 ( weiss et al ., j . immunol ., 133 : 123 - 128 ( 1984 )) cells were infected with recombinant vaccinia viruses for one hour in serum free imdm at an moi of 10 and incubated for three to nine hours in imdm , 10 % fbs . cells were collected by centrifugation and resuspended at 3 × 10 6 cells / ml in complete medium containing 1 mm indo - 1 acetomethoxyester ( grynkiewicz et al ., j . biol . chem ., 260 : 3340 - 3450 ( 1985 )) ( molecular probes ) and incubated at 37 ° c . for 45 minutes . the indo - 1 loaded cells were pelleted and resuspended at 1 × 10 6 / ml in serum free imdm and stored at room temperature in the dark . cells were analyzed for free calcium ion by simultaneous measurement of the violet and blue fluorescence emission by flow cytometry ( rabinovitch et al ., j . immunol ., 137 : 952 - 961 ( 1986 )). to initiate calcium flux , either phycoerythrin ( pe )- conjugated leu - 3a ( anti - cd4 ) ( becton dickinson , lincoln park , n . j .) at 1 μg / ml was added to the cell suspension followed by 10 μg / ml of unconjugated goat anti - mouse igg at time 0 or unconjugated 3g8 ( anti - cd16 ) monoclonal antibody was added to the cell suspension at 1 μg / ml followed by 10 μg / ml of pe - conjugated fab 2 &# 39 ; goat anti - mouse igg at time 0 . histograms of the violet / blue emission ratio were collected from the pe positive ( infected ) cell population , which typically represented 40 - 80 % of all cells . the t cell antigen receptor response in uninfected cells was triggered by antibody okt3 , without crosslinking . for experiments involving cd16 chimeric receptors , samples showing baseline drift toward lower intracellular calcium ( without antibody ) were excluded from the analysis . histogram data were subsequently analyzed by conversion of the binary data to ascii using write hand man ( cooper city , fla .) software , followed by analysis with a collection of fortran programs . the violet / blue emission ratio prior to the addition of the second antibody reagents was used to establish the normalized initial ratio , set equal to unity , and the resting threshold ratio , set so that 10 % of the resting population would exceed threshold . human t cell line wh3 , a cd8 + cd4 - hla b44 restricted cytolytic line was maintained in imdm , 10 % human serum with 100 u / ml of il - 2 and was periodically stimulated either nonspecifically with irradiated ( 3000 rad ) hla - unmatched peripheral blood lymphocytes and 1 μg / ml of phytohemagglutinin , or specifically , with irradiated b44 - bearing mononuclear cells . after one day of nonspecific stimulation , the pha was diluted to 0 . 5 μg / ml by addition of fresh medium , and after three days the medium was changed . cells were grown for at least 10 days following stimulation before use in cytotoxicity assays . the cells were infected with recombinant vaccinia at a multiplicity of infection of at least 10 for one hour in serum free medium , followed by incubation in complete medium for three hours . cells were harvested by centrifugation and resuspended at a density of 1 × 10 7 cells / ml . 100 μl were added to each well of a u - bottom microtiter plate containing 100 μl / well of complete medium . cells were diluted in two - fold serial steps . two wells for each sample did not contain lymphocytes , to allow spontaneous chromium release and total chromium uptake to be measured . the target cells , from hela subline s3 , were infected in 6 . 0 or 10 . 0 cm plates at an approximate moi of 10 for one hour in serum free medium , followed by incubation in complete medium for three hours . they were then detached from the dishes with pbs , 1 mm edta and counted . an aliquot of 10 6 target cells ( hela , raji , or rj2 . 2 . 5 cells for the cd4 chimeric receptor experiments and 3g8 10 - 2 cells ; shen et al ., mol . immunol . 26 : 959 ( 1989 ) for the cd16 chimeric receptor experiments ) was centrifuged and resuspended in 50 μl of sterile 51 cr - sodium chromate ( 1 mci / ml , dupont wilmington , del .) for one hour at 37 ° c . with intermittent mixing , then washed three times with pbs . 100 μl of labelled cells resuspended in medium at 10 5 cells / ml were added to each well . raji and rj2 . 2 . 5 target cells were labelled in the same manner as hela cells . the microtiter plate was spun at 750 × g for 1 minute and incubated for 4 hours at 37 ° c . at the end of the incubation period , the cells in each well were resuspended by gentle pipetting , a sample removed to determine the total counts incorporated , and the microtiter plate spun at 750 × g for 1 minute . 100 μl aliquots of supernatant were removed and counted in a gamma ray scintillation counter . the percent killing was corrected for the fraction of infected target cells ( usually 50 - 90 %) measured by flow cytometry . for infected effector cells the effector : target ratio was corrected for the percent of cells infected ( usually 20 - 50 % for the cd4 chimeric receptor experiments and & gt ; 70 % for the cd16 chimeric receptor experiments ). to create point mutations in amino acid residues 11 and or 15 of the ζ sequence , synthetic oligonucleotide primers extending from the bamhi site upstream of the ζ transmembrane domain , and converting native ζ residue 11 from cys to gly ( c11g ) or residue 15 from asp to gly ( d15g ) or both ( c11g / d15g ) were prepared and used in pcr reactions to generate mutated fragments which were reinserted into the wild type cd4 : ζ constructs . to create ζ deletions , ζ cdna sequences were amplified by pcr using synthetic oligonucleotide primers designed to create a stop codon ( uag ) after residues 50 , 59 , or 65 . the primers contained the cleavage site for the enzyme noti indented five or six residues from the 5 &# 39 ; end , usually in a sequence of the form cgc ggg cgg ccg cta ( seq id no : 11 ), where the last three residues correspond to the stop anticodon . the noti and stop anticodon sequences were followed by 18 or more residues complementary to the diesired 3 &# 39 ; end of the fragment . the resulting chimeras were designated cd16 : ζy51 *, cd16 : ζe60 * and cd16 : ζd66 * respectively . the bamhi site upstream of the transmembrane domain and the noti site were used to generate fragments that were reintroduced into the wild type cd16 : ζ construct . monomeric ζ chimeras were created by liberating the ζ transmembrane and membrane proximal intracellular sequences by bamhi and saci digestion of the asp - and cys - cd4 : ζ construct described above and inserting the fragment into the cd16 : ζe60 * and cd16 : ζd66 * construct respectively . cd16 : 7 : ζ ( 48 - 65 ) and cd16 : 7ζ ( 48 - 59 ) tripartite chimera construction . to prepare the construct cd16 : ζd66 *, the ζ cdna sequence corresponding to the transmembrane domain and the 17 following residues of the cytoplasmic domain was replaced by corresponding transmembrane and cytoplasmic domain obtained from the cd5 and cd7 cdna . the cd5 and cd7 fragments were generated by a pcr reaction using forward oligonucleotides including a bamhi restriction cleavage site and corresponding to the region just upstream of the transmembrane domain of cd5 and cd7 respectively and the following reverse oligonucleotides overlapping the cd5 and cd7 sequences respectively and the ζ sequence which contained the saci restriction cleavage site . cd5 : ζ : cgc ggg ctc gtt ata gag ctg gtt ctg gcg ctg ctt ctt ctg ( seq id no : 12 ) cd7 : ζ : cgc ggg gag ctc gtt ata gag ctg gtt tgc cgc cga att ctt atc ccg ( seq id no : 13 ). the cd5 and cd7 pcr products were digested with bamhi and saci and ligated to bamhi and saci digested cd16 : ζe60 * and replacing the ζ sequence from bamhi to saci by the cd7 fragment . to make the constructs cd16 : cd5 and cd16 : cd7 , cd5 and cd7 fragments were obtained by pcr using an oligonucleotide containing a noti restriction cleavage site and encoding a stop codon ( uaa ) after the residue gln416 and ala193 of cd5 and cd7 respectively . the cd5 and cd7 pcr fragment were digested with bamhi and noti and inserted in the cd16 : ζasp66 * construct . in vitro mutagenesis of the n - terminal residues within the ζ cytolytic signal - transducing motif synthetic oligonucleotide primers extending from the saci site inside the ζ motif and converting native residue 48 from asn to ser ( n48s ), residue 50 from leu to ser ( l50s ) and residue 51 from tyr to phe ( y51f ) were synthesized and used in a pcr reaction to generate fragments that were reintroduced into the wild type cd16 : 7 : ζ ( 48 - 65 ) construct . in vitro mutagenesis of c - terminal residues within the ζ cytolytic signal - transducing motif synthetic oligonucleotide primers extending from the noti site 3 &# 39 ; to the stop codon and converting native residue 60 from glu to gln ( e60q ), residue 61 from glu to gln ( e61q ), residue 62 from tyr to phe or ser ( y62f or y62s ) and residue 63 from asp to asn ( d63n ) were synthesized and used in pcr to generate fragments that were subcloned into the wild type cd16 : ζd66 * construct from the bamhi site to the noti site . a cd7 transmembrane fragment bearing mlui and noti sites at the junction between the transmembrane and intracellular domains was obtained by pcr using an oligonnucleotide with the following sequence : cgc ggg gcg gcc acg cgt cct cgc cag cac aca ( seq id no : 14 ). the resulting pcr fragment was digested with bamhi and noti and reinserted into the cd16 : 7 : ζ ( 48 - 65 ) construct . ζ fragments encoding residues 33 to 65 , 71 to 104 , and 104 to 137 were obtained by pcr reaction using pairs of primers containing mlui sites at the 5 &# 39 ; end of the forward primers and stop codons followed by noti sites at the 5 &# 39 ; end of the reverse primers . in each case the restriction sites were indented six residues from the 5 &# 39 ; terminus of the primer to insure restriction enzyme cleavage . ζ 33 : cgc ggg acg cgt ttc agc cgt cct cgc cag cac aca ( seq id no : 15 ); ζ 71 : cgc ggg acg cgt gac cct gag atg ggg gga aag ( seq id no : 16 ); and ζ 104 : cgc ggg acg cgt att ggg atg aaa ggc gag cgc ( seq id no : 17 ). carboxyl terminal fcriia deletion mutants were constructed by pcr in the same fashion as for the full length constructs , converting the sequences encoding tyrosine at positions 282 and 298 into stop codons ( taa ). the n - terminal deletions were generated by amplifying fragments encoding successively less of the intracellular domain by pcr , using oligonucleotides which allowed the resulting fragments to be inserted between mlui and noti restriction sites into a previously constructed expression plasmid encoding the cd16 extracellular domain fused to the cd7 transmembrane domain , the latter terminating in a mlui site ant the junction between the transmembrane and the intracellular domain . the examples described above demonstrate that aggregation of ζ , η , or γ chimeras suffices to initiate the cytolytic effector cell response in t cells . the known range of expression of ζ , η , and γ , which includes t lymphocytes , natural killer cells , basophilic granulocytes , macrophages and mast cells , suggests that conserved sequence motifs may interact with a sensory apparatus common to cells of hematopoietic origin and that an important component of host defense in the immune system may be mediated by receptor aggregation events . the potency of the cytolytic response and the absence of a response to target cells bearing mhc class ii receptors demonstrates that chimeras based on ζ , η , or γ form the basis for a genetic intervention for aids through adoptive immunotherapy . the broad distribution of endogenous ζ and γ and evidence that fc receptors associated with γ mediate cytotoxicity in different cells types ( fanger et al ., immunol . today , 10 : 92 - 99 ( 1989 )) allows a variety of cells to be considered for this purpose . for example , neutrophilic granulocytes , which have a very short lifespan (≈ 4h ) in circulation and are intensely cytolytic , are attractive target cells for expression of the chimeras . infection of neutrophils with hiv is not likely to result in virus release , and the abundance of these cells ( the most prevalent of the leukocytes ) should facilitate host defense . another attractive possiblity for host cells are mature t cells , a population presently accessible to retroviral engineering ( rosenberg , s . a . sci . am ., 262 : 62 - 69 ( 1990 )). with the aid of recombinant il - 2 , t cell populations can be expanded in culture with relative ease , and the expanded populations typically have a limited lifespan when reinfused ( rosenberg et al ., n . engl . j . med ., 323 : 570 - 578 ( 1990 )). under the appropriate conditions , hiv recognition by cells expressing cd4 chimeras should also provide mitogenic stimuli , allowing the possibility that the armed cell population could respond dynamically to the viral burden . although we have focused here on the behavior of the fusion proteins in cytolytic t lymphocytes , expression of the chimeras in helper lymphocytes might provide an hiv - mobilized source of cytokines which could counteract the collapse of the helper cell subset in aids . recent description of several schemes for engineering resistance to infection at steps other than virus penetration ( friedman et al ., nature , 335 : 452 - 454 ( 1988 ); green et al ., cell , 58 : 215 - 223 ( 1989 ); malim et al ., cell , 58 : 205 - 214 ( 1989 ); trono et al ., cell , 59 : 113 - 120 ( 1989 ); buonocore et al ., nature , 345 : 625 - 628 ( 1990 )) suggests that cells bearing cd4 chimeras could be designed to thwart virus production by expression of appropriate agents having an intracellular site of action . the ability to transmit signals to t lymphocytes through autonomous chimeras also provides the ability for the regulation of retrovirally engineered lymphocytes in vivo . crosslinking stimuli , mediated for example by specific igm antibodies engineered to remove complement - binding domains , may allow such lymphocytes to increase in number in situ , while treatment with similar specific igg antibodies ( for example recognizing an amino acid variation engineered into the chimeric chain ) could selectively deplete the engineered population . additionally , anti - cd4 igm antibodies do not require additional crosslinking to mobilize calcium in jurkat cells expressing cd4 : ζ chimeras ( data not shown ). the ability to regulate cell populations without recourse to repeated extracorporeal amplification may substantially extend the range and efficacy of current uses proposed for genetically engineered t cells . to create other chimeras consisting of ζ , η or γ intracellular sequences , cdna or genomic sequences encoding an extracellular domain of the receptor can be endowed with a restriction site introduced at a location just preceding the transmembrane domain of choice . the extracellular domain fragment terminating in the restriction site can then be joined to ζ , η , or γ sequences . typical extracellular domains may be derived from receptors which recognize complement , carbohydrates , viral proteins , bacteria , protozoan or metazoan parasites , or proteins induced by them . similarly , ligands or receptors expressed by pathogens or tumor cells can be attached to ζ , η , or γ sequences , to direct immune responses against cells bearing receptors recognizing those ligands . while the invention has been described in connection with specific embodiments thereof , it will be understood that it is capable of further modifications and this application is intended to cover variations , uses , or adaptations of the invention and including such departures from the present disclosure as come within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 27 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 1728 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 1 : atgaaccggggagtcccttttaggcacttgcttctggtgctgcaactggc50gctcctcccagcagccactcagggaaacaaagtggtgctgggcaaaaaag100gggatacagtggaactgacctgtacagcttcccagaagaagagcatacaa150ttccactggaaaaactccaaccagataaagattctgggaaatcagggctc200cttcttaactaaaggtccatccaagctgaatgatcgcgctgactcaagaa250gaagcctttgggaccaaggaaacttccccctgatcatcaagaatcttaag300atagaagactcagatacttacatctgtgaagtggaggaccagaaggagga350ggtgcaattgctagtgttcggattgactgccaactctgacacccacctgc400ttcaggggcagagcctgaccctgaccttggagagcccccctggtagtagc450ccctcagtgcaatgtaggagtccaaggggtaaaaacatacagggggggaa500gaccctctccgtgtctcagctggagctccaggatagtggcacctggacat550gcactgtcttgcagaaccagaagaaggtggagttcaaaatagacatcgtg600gtgctagctttccagaaggcctccagcatagtctataagaaagaggggga650acaggtggagttctccttcccactcgcctttacagttgaaaagctgacgg700gcagtggcgagctgtggtggcaggcggagagggcttcctcctccaagtct750tggatcacctttgacctgaagaacaaggaagtgtctgtaaaacgggttac800ccaggaccctaagctccagatgggcaagaagctcccgctccacctcaccc850tgccccaggccttgcctcagtatgctggctctggaaacctcaccctggcc900cttgaagcgaaaacaggaaagttgcatcaggaagtgaacctggtggtgat950gagagccactcagctccagaaaaatttgacctgtgaggtgtggggaccca1000cctcccctaagctgatgctgagcttgaaactggagaacaaggaggcaaag1050gtctcgaagcgggagaagccggtgtgggtgctgaaccctgaggcggggat1100gtggcagtgtctgctgagtgactcgggacaggtcctgctggaatccaaca1150tcaaggttctgcccacatggtccaccccggtgcacgcggatcccaaactc1200tgctacttgctagatggaatcctcttcatctacggagtcatcatcacagc1250cctgtacctgagagcaaaattcagcaggagtgcagagactgctgccaacc1300tgcaggaccccaaccagctctacaatgagctcaatctagggcgaagagag1350gaatatgacgtcttggagaagaagcgggctcgggatccagagatgggagg1400caaacagcagaggaggaggaacccccaggaaggcgtatacaatgcactgc1450agaaagacaagatgccagaagcctacagtgagatcggcacaaaaggcgag1500aggcggagaggcaaggggcacgatggcctttaccaggacagccacttcca1550agcagtgcagttcgggaacagaagagagagagaaggttcagaactcacaa1600ggacccttgggttaagagcccgccccaaaggtgaaagcacccagcagagt1650agccaatcctgtgccagcgtcttcagcatccccactctgtggagtccatg1700gccacccagtagcagctcccagctctaa1728 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1389 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 2 : 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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 1599 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 3 : atgaaccggggagtcccttttaggcacttgcttctggtgctgcaactggc50gctcctcccagcagccactcagggaaacaaagtggtgctgggcaaaaaag100gggatacagtggaactgacctgtacagcttcccagaagaagagcatacaa150ttccactggaaaaactccaaccagataaagattctgggaaatcagggctc200cttcttaactaaaggtccatccaagctgaatgatcgcgctgactcaagaa250gaagcctttgggaccaaggaaacttccccctgatcatcaagaatcttaag300atagaagactcagatacttacatctgtgaagtggaggaccagaaggagga350ggtgcaattgctagtgttcggattgactgccaactctgacacccacctgc400ttcaggggcagagcctgaccctgaccttggagagcccccctggtagtagc450ccctcagtgcaatgtaggagtccaaggggtaaaaacatacagggggggaa500gaccctctccgtgtctcagctggagctccaggatagtggcacctggacat550gcactgtcttgcagaaccagaagaaggtggagttcaaaatagacatcgtg600gtgctagctttccagaaggcctccagcatagtctataagaaagaggggga650acaggtggagttctccttcccactcgcctttacagttgaaaagctgacgg700gcagtggcgagctgtggtggcaggcggagagggcttcctcctccaagtct750tggatcacctttgacctgaagaacaaggaagtgtctgtaaaacgggttac800ccaggaccctaagctccagatgggcaagaagctcccgctccacctcaccc850tgccccaggccttgcctcagtatgctggctctggaaacctcaccctggcc900cttgaagcgaaaacaggaaagttgcatcaggaagtgaacctggtggtgat950gagagccactcagctccagaaaaatttgacctgtgaggtgtggggaccca1000cctcccctaagctgatgctgagcttgaaactggagaacaaggaggcaaag1050gtctcgaagcgggagaagccggtgtgggtgctgaaccctgaggcggggat1100gtggcagtgtctgctgagtgactcgggacaggtcctgctggaatccaaca1150tcaaggttctgcccacatggtccaccccggtgcacgcggatcccaaactc1200tgctacctgctggatggaatcctcttcatctatggtgtcattctcactgc1250cttgttcctgagagtgaagttcagcaggagcgcagagccccccgcgtacc1300agcagggccagaaccagctctataacgagctcaatctaggacgaagagag1350gagtacgatgttttggacaagagacgtggccgggaccctgagatgggggg1400aaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcaga1450aagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgc1500cggaggggcaaggggcacgatggcctttaccagggtctcagtacagccac1550caaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa1599 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 575 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 4 : metasnargglyvalprophearghisleuleuleuvalleuglnleu151015alaleuleuproalaalathrglnglyasnlysvalvalleuglylys202530lysglyaspthrvalgluleuthrcysthralaserglnlyslysser354045ileglnphehistrplysasnserasnglnilelysileleuglyasn505560glnglyserpheleuthrlysglyproserlysleuasnaspargala65707580aspserargargserleutrpaspglnglyasnpheproleuileile859095lysasnleulysilegluaspseraspthrtyrilecysgluvalglu100105110aspglnlysglugluvalglnleuleuvalpheglyleuthralaasn115120125seraspthrhisleuleuglnglyglnserleuthrleuthrleuglu130135140serproproglyserserproservalglncysargserproarggly145150155160lysasnileglnglyglylysthrleuservalserglnleugluleu165170175glnaspserglythrtrpthrcysthrvalleuglnasnglnlyslys180185190valgluphelysileaspilevalvalleualapheglnlysalaser195200205serilevaltyrlyslysgluglygluglnvalglupheserphepro210215220leualaphethrvalglulysleuthrglyserglygluleutrptrp225230235240glnalagluargalaserserserlyssertrpilethrpheaspleu245250255lysasnlysgluvalservallysargvalthrglnaspprolysleu260265270glnmetglylyslysleuproleuhisleuthrleuproglnalaleu275280285proglntyralaglyserglyasnleuthrleualaleuglualalys290295300thrglylysleuhisglngluvalasnleuvalvalmetargalathr305310315320glnleuglnlysasnleuthrcysgluvaltrpglyprothrserpro325330335lysleumetleuserleulysleugluasnlysglualalysvalser340345350lysargglulysprovaltrpvalleuasnproglualaglymettrp355360365glncysleuleuseraspserglyglnvalleuleugluserasnile370375380lysvalleuprothrtrpserthrprovalhisalaaspprolysleu385390395400cystyrleuleuaspglyileleupheiletyrglyvalileilethr405410415alaleutyrleuargalalyspheserargseralagluthralaala420425430asnleuglnaspproasnglnleutyrasngluleuasnleuglyarg435440445arggluglutyraspvalleuglulyslysargalaargaspproglu450455460metglyglylysglnglnargargargasnproglngluglyvaltyr465470475480asnalaleuglnlysasplysmetproglualatyrsergluilegly485490495thrlysglygluargargargglylysglyhisaspglyleutyrgln500505510aspserhispheglnalavalglnpheglyasnargarggluargglu515520525glysergluleuthrargthrleuglyleuargalaargprolysgly530535540gluserthrglnglnserserglnsercysalaservalpheserile555550565560prothrleutrpserprotrpproproserserserserglnleu565570575 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 462 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 5 : metasnargglyvalprophearghisleuleuleuvalleuglnleu151015alaleuleuproalaalathrglnglyasnlysvalvalleuglylys202530lysglyaspthrvalgluleuthrcysthralaserglnlyslysser354045ileglnphehistrplysasnserasnglnilelysileleuglyasn505560glnglyserpheleuthrlysglyproserlysleuasnaspargala65707580aspserargargserleutrpaspglnglyasnpheproleuileile859095lysasnleulysilegluaspseraspthrtyrilecysgluvalglu100105110aspglnlysglugluvalglnleuleuvalpheglyleuthralaasn115120125seraspthrhisleuleuglnglyglnserleuthrleuthrleuglu130135140serproproglyserserproservalglncysargserproarggly145150155160lysasnileglnglyglylysthrleuservalserglnleugluleu165170175glnaspserglythrtrpthrcysthrvalleuglnasnglnlyslys180185190valgluphelysileaspilevalvalleualapheglnlysalaser195200205serilevaltyrlyslysgluglygluglnvalglupheserphepro210215220leualaphethrvalglulysleuthrglyserglygluleutrptrp225230235240glnalagluargalaserserserlyssertrpilethrpheaspleu245250255lysasnlysgluvalservallysargvalthrglnaspprolysleu260265270glnmetglylyslysleuproleuhisleuthrleuproglnalaleu275280285proglntyralaglyserglyasnleuthrleualaleuglualalys290295300thrglylysleuhisglngluvalasnleuvalvalmetargalathr305310315320glnleuglnlysasnleuthrcysgluvaltrpglyprothrserpro325330335lysleumetleuserleulysleugluasnlysglualalysvalser340345350lysargglulysprovaltrpvalleuasnproglualaglymettrp355360365glncysleuleuseraspserglyglnvalleuleugluserasnile370375380lysvalleuprothrtrpserthrprovalhisalaaspproglnleu385390395400cystyrileleuaspalaileleupheleutyrglyilevalleuthr405410415leuleutyrcysargleulysileglnvalarglysalaaspileala420425430serargglulysseraspalavaltyrthrglyleuasnthrargasn435440445glngluthrtyrgluthrleulyshisglulysproprogln450455460462 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 532 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 6 : metasnargglyvalprophearghisleuleuleuvalleuglnleu151015alaleuleuproalaalathrglnglyasnlysvalvalleuglylys202530lysglyaspthrvalgluleuthrcysthralaserglnlyslysser354045ileglnphehistrplysasnserasnglnilelysileleuglyasn505560glnglyserpheleuthrlysglyproserlysleuasnaspargala65707580aspserargargserleutrpaspglnglyasnpheproleuileile859095lysasnleulysilegluaspseraspthrtyrilecysgluvalglu100105110aspglnlysglugluvalglnleuleuvalpheglyleuthralaasn115120125seraspthrhisleuleuglnglyglnserleuthrleuthrleuglu130135140serproproglyserserproservalglncysargserproarggly145150155160lysasnileglnglyglylysthrleuservalserglnleugluleu165170175glnaspserglythrtrpthrcysthrvalleuglnasnglnlyslys180185190valgluphelysileaspilevalvalleualapheglnlysalaser195200205serilevaltyrlyslysgluglygluglnvalglupheserphepro210215220leualaphethrvalglulysleuthrglyserglygluleutrptrp225230235240glnalagluargalaserserserlyssertrpilethrpheaspleu245250255lysasnlysgluvalservallysargvalthrglnaspprolysleu260265270glnmetglylyslysleuproleuhisleuthrleuproglnalaleu275280285proglntyralaglyserglyasnleuthrleualaleuglualalys290295300thrglylysleuhisglngluvalasnleuvalvalmetargalathr305310315320glnleuglnlysasnleuthrcysgluvaltrpglyprothrserpro325330335lysleumetleuserleulysleugluasnlysglualalysvalser340345350lysargglulysprovaltrpvalleuasnproglualaglymettrp355360365glncysleuleuseraspserglyglnvalleuleugluserasnile370375380lysvalleuprothrtrpserthrprovalhisalaaspprolysleu385390395400cystyrleuleuaspglyileleupheiletyrglyvalileleuthr405410415alaleupheleuargvallyspheserargseralagluproproala420425430tyrglnglnglyglnasnglnleutyrasngluleuasnleuglyarg435440445arggluglutyraspvalleuasplysargargglyargaspproglu450455460metglyglylysproargarglysasnproglngluglyleutyrasn465470475480gluleuglnlysasplysmetalaglualatyrsergluileglymet485490495lysglygluargargargglylysglyhisaspglyleutyrglngly500505510leuserthralathrlysaspthrtyraspalaleuhismetglnala515520525leuproproarg530 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 33 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 7 : cgcggggtgaccgtgccctccagcagcttgggc33 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 50 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 8 : cgcggggatccgtcgtccagagcccgtccagctccccgtcctgggcctca50 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 33 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 9 : cgcgggcggccgcgacgccggccaagacagcac33 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 33 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 10 : cgcgttgacgagcagccagttgggcagcagcag33 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 15 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 11 : cgcgggcggccgcta15 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 42 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 12 : cgcgggctcgttatagagctggttctggcgctgcttcttctg42 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 48 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 13 : cgcggggagctcgttatagagctggtttgccgccgaattcttatcccg48 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 33 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 14 : cgcggggcggccacgcgtcctcgccagcacaca33 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 36 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 15 : cgcgggacgcgtttcagccgtcctcgccagcacaca36 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 33 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 16 : cgcgggacgcgtgaccctgagatggggggaaag33 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 33 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 17 : cgcgggacgcgtattgggatgaaaggcgagcgc33 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 26 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 18 : cccggatcccagcatgggcagctctt26 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 42 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 19 : cgcggggcggccgctttagttattactgttgacatggtcgtt42 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 30 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 20 : gcggggggatcccactgtccaagctcccag30 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 32 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 21 : gcgggggcggccgcctaaatacggttctggtc32 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 31 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 22 : tcagaaagagacaacctgaagaaaccaacaa31 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 31 bases ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : nucleic acid ( xi ) sequence description : seq id no : 23 : ttgttggtttcttcaggttgtgtctttctga31 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 171 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : amino acid ( xi ) sequence description : seq id no : 24 : metgluhisserthrpheleuserglyleuvalleualathrleuleu51015serglnvalserprophelysileproileglugluleugluasparg202530valphevalasncysasnthrserilethrtrpvalgluglythrval354045glythrleuleuseraspilethrargleuaspleuglylysargile505560leuaspproargglyiletyrargcysasnglythraspiletyrlys65707580asplysgluserthrvalglnvalhistyrargmetcysglnsercys859095valgluleuaspproalathrvalalaglyileilevalthraspval100105110alailethrleuleuleualaleuglyvalphecysphealaglyhis115120125gluthrglyargleuserglyalaalaaspthrglnalaleuleuarg130135140asnaspglnvaltyrglnproleuargaspargaspaspalaglntyr145150155160serhisleuglyglyasntrpalaargasnlys165170 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 182 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : amino acid ( xi ) sequence description : seq id no : 25 : metgluglnglylysglyleualavalleuileleualaileileleu51015leuglnglythrleualaglnserilelysglyasnhisleuvallys202530valtyrasptyrglngluaspglyservalleuleuthrcysaspala354045glualalysasnilethrtrpphelysaspglylysmetileglyphe505560leuthrgluasplyslyslystrpasnleuglyserasnalalysasp65707580proargglymettyrglncyslysglyserglnasnlysserlyspro859095leuglnvaltyrtyrargmetcysglnasncysilegluleuasnala100105110alathrileserglypheleuphealagluilevalserilepheval115120125leualavalglyvaltyrpheilealaglyglnaspglyvalarggln130135140serargalaserasplysglnthrleuleuproasnaspglnleutyr145150155160glnproleulysasparggluaspaspglntyrserhisleuglngly165170175asnglnleuargargasn180 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 220 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : amino acids ( xi ) sequence description : seq id no : 26 : metproglyglyleuglualaleuargalaleuproleuleuleuphe51015leusertyralacysleuglyproglycysglnalaleuargvalglu202530glyglyproproserleuthrvalasnleuglygluglualaargleu354045thrcysgluasnasnglyargasnproasnilethrtrptrppheser505560leuglnserasnilethrtrpproprovalproleuglyproglygln65707580glythrthrglyglnleuphepheprogluvalasnlysasnthrgly859095alacysthrglycysglnvalilegluasnasnileleulysargser100105110cysglythrtyrleuargvalargasnprovalproargpropheleu115120125aspmetglygluglythrlysasnargileilethralagluglyile130135140ileleuleuphecysalavalvalproglythrleuleuleuphearg145150155160lysargtrpglnasnglulyspheglyvalaspmetproaspasptyr165170175gluaspgluasnleutyrgluglyleuasnleuaspaspcyssermet180185190tyrgluaspileserargglyleuglnglythrtyrglnaspvalgly195200205asnleuhisileglyaspalaglnleuglulyspro210215220 ( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 228 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : amino acid ( xi ) sequence description : seq id no : 27 : metalathrleuvalleusersermetprocyshistrpleuleuphe51015leuleuleuleupheserglygluprovalproalametthrserser202530aspleuproleuasnpheglnglyserprocysserglniletrpgln354045hisproargphealaalalyslysargsersermetvallysphehis505560cystyrthrasnhisserglyalaleuthrtrpphearglysarggly65707580serglnglnproglngluleuvalserglugluglyargilevalgln859095thrglnasnglyservaltyrthrleuthrileglnasnileglntyr100105110gluaspasnglyiletyrphecyslysglnlyscysaspseralaasn115120125hisasnvalthraspsercysglythrgluleuleuvalleuglyphe130135140serthrleuaspglnleulysargargasnthrleulysaspglyile145150155160ileleuileglnthrleuleuileileleupheileilevalproile165170175pheleuleuleuasplysaspaspglylysalaglymetglugluasp180185190histhrtyrgluglyleuasnileaspglnthralathrtyrgluasp195200205ilevalthrleuargthrglygluvallystrpservalglygluhis210215220proglyglnglu225__________________________________________________________________________