CA2175482A1 - Process for generating specific antibodies - Google Patents

Abstract

The present invention relates to a method for generating an antibody which is specific for an immunorecessive epitope, and nucleic acid encoding the antibody. The Subject method generally comprises the steps of generating a variegated display library of antibody variable regions and selecting from the library those antibody variable regions which have a desired binding specificity for the immunorecessive epitope. The antibody variable regions used to generate the display library are cloned from an immunotolerance-derived antibody repertoire.

Description

W~ 95/15982 1 2 1 7 5 4 8 2 PCT/US94114106 Processfor Gener~ting SpecificAntibodies , un/~d ofthe Invention In an antibody-producing animal, such as a mammal, antibodies are synthesized and 5 secreted into bodily fluids by plasma cells, a type of terminally di~~ d B-lylll~hocytc.
Exposure of the animal to a foreign molecule (i.e. via ;.I.III...~;~A ;..II) generally produces multiple plasma cell clones resulting in a l~ ub~ vua mixture of antibodies (polyclonal antibodies) in the blood and other fluids. The blood of an immunized animal can be collected, clotted, and the clot removed to leave a sera containing the antibodies produced in 10 response to ;IIIIIIIIII;~ This remaining liquid or serum, which contains the polyclonal antibodies, is referred to as antiserum. However, such antiserum contains many different types of antibodies that are specific for many different antigens. Even in 1,~. . ;.,.." . ,. ~I
animals, seldom are more than one tenth of the circulating antibodies specific for the particular; " " " 1, ..~,,. ., used to immunized the animal. The use of these mixed ~u~ulaiiulla of IS antibodies, though useful in many situations, can create a vaTiety of different problems in r,. 1~ AI techniques. For example, such antiserum will generally be inadequate for use m ~ ;"~" ~ g between the ~, and closely related molecules which share many common .11 ~. . ~I;I.A.I ~ with the ~, Owing to their high specificity for a given antigen, the advent of 1",l ~
20 antibodies (Kohler and Milstein (1975) Nature 256:495) represented a significant technical break-through with imporlant . both i~ lly and commercially.
Mf)nrrlnAAI antibodies (MAbs) are traditionally made by isolating a single antibody secreting cell (e.g. a l~ llo."y ~) from an immunized animal, fusing the l~ o~"y ~ with a myeloma (or other imunortal) cell to form a hybrid cell (called a "hybridoma"), and then culturing the 25 selected hybridoma cell in vivo or in vitro to yield antibodies which are identical in structure and specificity. Because the antibody-secreting cell line is immortal, the ~ of the antibody are ~ udu~,iblf from batch to batch. The usefulness of ~ r.~l....AI antibodies stems from three . l- A- f~ ~ their specificity of binding, their 1 ....-.,,... Iy, and their âbility to be produced in virtually unlimited quantities.
30 While production of .. ~f 1. ,.. 1 arltibodies has resulted in production of antibodies of greater specificity to a particular antigen then polyclonal methods, there are n~,v~ll~,l~,aa a number of limitations associated with these techniques and antibodies produced thereby. For instance, a key aspect in th~ isolation of ~ r.~l- Al antibodies relates to how many antibody producing hybridoma cells with different ~ ;,'; ;1,. can be practically established 35 and sampled in response to ;~ with a particular antigen, compared to how manytheoretically need to be sampled in order to obtain an antibody having specific ~ fl . ;~l ;. `

2 1 7 5 4 8 2 2 PcTlus94ll4lo6 For example, tbe number of different antibody crerifiritil-~ expressed at amy one time by Iy~ o~,y [~,~ of the murine immune system is tbought to be UIJIJI V~llll 8,1y 107 and represents only a small proportion of the potential repertoire of crerifirifi,~c Tmmllni7~ti--n regimens cam provide enrichment of B-cells producing tbe desired 5 antibodies. However, even employing those techniques, typical protocols for isolating amtibody producing B-cells permit sampling of generally less than 500 antibody producing hybridoma cells per immuni_ed animal. Thus, traditional techniques for the production of ""...~ antibodies statistically fa~or generation of ."" ~rl~ antibodies to immllnn~inmin~t molecules, making isolation of antibodies specific for a rare or less ;,,..,..,.,n~ epitope diffcult. This problem cam be further, ' ' by the fact that in many instances pure antigen is not available as an ;l~ L, ." particularly inthe case of cell surface antigens. III.. I.. '~A ;.. I with intact cells frequerltly results in production of antibodies against irrelevamt epitopes, espl cially for xenotypic ;.. ,-- ~;.. ,l To enhance the production of mnnnrlnn ~ antibodies to rare, ";..l...~ ;v~" amtigens, 15 ;..I l- ~ techniques have been employed. Neonatal toleri7ation and chemical illlll....l ..lllllC~,~;vll are most commonly used to reduce clonal expansion of B cells in response to "~.,~.. "' antigen signals, thereby emiching for a population of B cells responsive to the epitopes of interest. However, the practical application of a subtractive ;,..." ~ ,: ,.;;~... technique can be very difficult, as the efficiency of Ar ~,aa;VIl is 20 often not acceptable, or as in the case of ~Y~,Ivll ,' '' ;Il..l. ~ l,l- l.l..~aa;vll, generally results in only a few antibody-producing hybridoma cells per; "" ~ l animal (e.g.
Iess than 100), making it unlikely that ."" ~ l amtibodies can be isolated which are specific to the il~ UlVlCl~ ;V~ epitopes.
Sununary of the Invention The present invention provides a melhod for generating an antibody which is specific for an illlll.ulvlc~ epitope, amd nucleic acid encoding the antibody. The subject method generally comprises the steps of generating a variegated display library of antibody variable regions, and selecting from the library those antibody variable regions which have a desired 30 binding specificity for the ~ ,aa;V~ epitope. The amtibody variable regions used to generate the display library are cloned from an : ' -derived antibody repertoire.
As described herein, the antibody variable regions of the display library are presented by a replicable genetic display package in an ;l, l ~ l;v~ context which permits the antibody to bind to am antigen that is contacted with the display package. Thus, affinity 35 selection tecbniques cam be utili_ed to enrich the population of display packages for those ~=nn a ~ibody v~d~le Ddons ~hdch h ve a dr iDd h_~ s~ocldny for _ wo 9S/15982 2 1 7 5 4 ~3 2 PCTN594~14106 illllllllllUl~ ;Vt: epitope. In exemplary ~ o.~ , the display library can be a phage display library. Alternatively, the display library can be generated on a bacterial cell-surface or a spore.
The subject method can be used to isolate amtibodies which are specific for such5 illllll~lUlC~ >;VC epitopes as, for example, cell-type specific markers, including fetal cell markers such as fetdl nucleated red blood markers, camcer cell markers such as colon carlcer markers or metastatic tumor cell markers, stem cell markers such as markers for precursor nerve cells or l I stem cells.
Likewise, the subject method can be used to generate arltibodies which can 10 .l. . .;Ill;IIA . by binding between a variant form of a protein and other related forms of the protein. The variant protein can differ by one or more amino acid residues from othe} related proteins in order to give rise to the ;llI..lUIIUICi~ l;Yt~ epitope, as well as vary ~ntip,~n;rAlly from the related protein by virtue of ~ ,Vi~yldtiu~l or other post~ ",~Il;ri, ~l.,.l, The variation can arise naturally, as between different isoforms of a protein family, illustrated 15 by the apolipoprotein E family, or carl be generdted by genetic aberration, as illustrated by the neoplastic ~ r~l .,..,.p mutations of oncogenic proteins or tumor suppressor proteins such as p53.
In an illustrative ...llnJ.I.- ....1 of the subject method, a specific antibody to an illllllullUll,-~ l;VC epitope can be generated by affmity pl--ifi~ n of a antibody phage 20 display library derived from an ' -derived antibody repertoire. For example, suitable host cells are l~, r~l,l d with a library of replicable phage vectors encoding a library of phage particles displaying a fusion ~ILil~ody/,~.L protein, where the fusion protein includes a phage coat protein portion and an amtibody variable region portion. The antibody variable region is obtained from the ;..l..l....l,~ -derived antibody repertoire. The 25 l., -r-..l~l cells are cultured, the phage particles are formed, amd the antibody fusion proteins are expressed. Any of resulting phage particles which have an amtibody variable region portion which specifically binds to a an c.,~ ;v~ epitope can be separated from those wmch do not specifically bind the il.... ---.l .. - .. -:vc epitope.
The present invention further pertains to novel ccl_.,a;vt~ amtibody libraries 30 produced by the subject method. From the subject method, for example, an arltibody display library can be isolated which is enriched for antibodies that specifically bind an illllllllllUlC~ ;Vt~ epitope of interest. The display library comprises a population of display packages expressmg a variegated V-gene library which has been cloned from an i ,..,~ -derived antibody repertoire, and which has been further enriched after expression by the display package via affinity separation with the ~., epitope.

wo 95/15982~ ~ 7 5 4 8 ~ 4 PCT/US94/14106 It is also c.. ' ~ by the present invention that indiYidual antibodies, and genes encoding these antibodies, can be isolated from the antibody libraries of tbe subject method.
For instance, after affinity enrichment of the antibody display library for antibodies which specifically bind the iUllllUllUl~ ;V~; epitope, individual display packages cam be obtained, 5 and the antibody gene contained therein subcloned into other appropriate expression vectors suitable for production of the antibody for the desired use.
.
Description of tl~e Dra~ings Figures IA and IB show variable region PCR primers for amplifying the variable 10 regions of both heavy and light chains from murine antibody genes.
Figure 2 shows a schematic I~ iUII of an Fab' expression casseffe.
Figure 3 is a semi-log graph depicting the binding of phage amtibody pools (phab) emiched on the HEL cell line (number indicates the round of emichment). The graph provides additional comparison of the enriched phab pools with the binding of other 15 immlm-glnblllinc (T3, Anti-M and Wilma) to the HEL cells.
Figure 4 illustrates the percentage of cells (either HEL cells or mature white cells) stained by individual phab isolates generated by the subject method.
Figure 5A shows the results of sequential roumds of pre-adsorption and emichment on fetal liver cells for phab binding. The increase in the percentage of phage amtibodies binding 20 to fetal liver cells is indicative emichment for fetal cell binding phage antibodies. The phage amtibody library was derived using a V-gene library from an immlln~-t~ ri7~-d host animal.
In contrast, Figure 5B compares the results of the ;.., ....,1..1~, ;,. ~1 experiment in Figure 5A
with the results of sequential rounds of panning using phage antibody libraries derived immlmi7~1, but not toleri_ed, host animals.
Figure 6 show variable region PCK primers for amplifying the variable regions ofboth heavy and light chains from human antibody genes.
Figure 7 details the sequences for CDR3 regions of both heavy and light chains for individual phab isolates emiched on fetal cells.
Figures 8A and 8B illustrate the general features of the FB3-2 ~md H3-3 antibodies, I~ ,ly, including the framework regions (double underline; FRs), ~ y ~' ~ regions (CDRs), and constant regions (italics; IgGI CHI or kappa constant).The amino acid residues which differ between the FB3-2 and F4-7 amtibodies are indicated under the FB3-2 sequence in Figure 8A.

Detailed Description of fhe Invention The present invention makes available a powerful directed approæh for isolating specific antibodies which are extremely difficult or impossible to obtain by current S mPthn~ P~ and thereby overcomes the 1~ ti ~ discussed above. One aspect ofthe present invention is the synthesis of a method that combines i.. , ~ ,,. and variegated display libraries to yield a dramatic and surprising synergism in the efficient isolation of antibodies having a desired binding af~mity for an illull~ulc~,~,aa;ve target epitope. Utilizing i,., .... ,1.~l..,,." e techniques such as subtrætive immlmi7~til-n a subset of 10 Iyll.~)llo."y~, producing antibodies against an illUIl_.lulc~,~a;vc target epitope are enriched in an immuni_ed animal. Subsequent isolation of antibody-producing cells from the immuni_ed animal and PCR ~mrlifir~ti~n of at least the variable regions of antibodies expressed by the isolated cells allows the generation of a variegated library of antibody variable region genes (V-genes). From this V-gene library, the subject method selects genes encoding antibodieâ
15 specific for the target epitope by (i) displaying the antibodies encoded by each variable region gene on the outer surface of a replicable genetic display package to create an antibody display library, and (ii) usmg afiinity selection techniques to enrich the population of display packages for those containing V-genes encodmg antibodies which have a desired binding specificity for the target epitope.
In general, most antibodies isolated by lc ' antibody display tPrhn~ gir~
known in the alt are obtained using substantially pure l~lc~al~:Liulla of an antigen of interest, and provide only a few isolates having association constants (KaS) even a~nua~l~lg 1 09M-l .
No phage display method has resulted in isolation of antibodies panning with live cells (i.e., unpurified antigen) which are of the cuu;~ ' in either specificity or affmity to antibodies 25 attainable by cull~.,uLiul~al hybridoma techniques. In contrast, as ~1,,~11 in the Examples provided below, the subject method can be used to generate antibodies which out perform both the ' I and hybridoma-derived antibodies of the prior arL
particularly with respect to binding affinity and degrees of specificity.
For example, antibodies isolated by the subject method can have binding affinities greater than 108M-1, e.g., in the range of 109M-1 to 1012M-I. Moreover, the specificity of these antibodies can be several fold, if not orders of magmitude, better than ..., ,.1, . . " i~l and hybridoma generated antibodies, IJalliuulally with respect to antibodies for cell surfæe epitopes. For instance, the subject method can provide antibodies which have no substantial ba.,~,luulld binding to other related cells, e.g., ~ t~ greater than l O fold binding to the 35 target cells over ba~ 31UU Id binding to the related cells. As ~'~ ' below, antibodies can be generated which do not substann~ly cross-react with other epitopes, preferably having WO 95/15982 2 1 7 ~ 4 8 2 6 PCTIUS94114106 greater than 20 fold over background, more preferably 50, 75 or 100 fold over background, and even more preferably more than 125 fold over 1~..
For the purpose of the present invention, the term "antibody" in its various forms is art-recogniæd and mcludes iullll~ vglvLulill molecules and S ;"", ~ i ,lly active portions of imnm~n~lohlllin molecules, i.e., molecules that contain an amtigen binding site which specifically binds (illull~lV~ ,.,La with) an amtigen.
Structurally, the simplest naturally occurring antibody (IgG) comprises four polr~c~lhlc chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
The light chains exist in two distinct forms called kappa (1c) and lambda (A). Each chairl has 10 a constant region (C) and a variable region (V). Each chain is organiæd mto a series of domains. The light chains have two domains, c~llual~vlld;ll~ to the C region and the other to the V region. The heavy chains have four domains, one ~ ullca~Jvlldillg to the V region and three domains (1,2 and 3) in the C regiorl. The naturaLy occurring amtibody has two arms (each arm being an Fab region), each of ~vhich comprises a VL and a VH region associated 15 with each other. It is this pair of V regions (VL and VH) that differ from one antibody to another (owing to amino acid sequence variations). The variable domains for each of the heavy amd light chains have the same gel1eral structure, including four framework regions (FRs), whose sequences are relatively conserved, comlected by three Lyl.~,~vGfidlle or C....ll,l.,....,~ .;ly d~,i n,, regions (CDRs). The variable region of each chain can typically be represented by the general formula FRI-CDRI-FR2-CDR2-FR3-CDR3-FR4.
The CDRs for a particular variable region are held in close proximity to one and other by the framework regions, and with the CDRs from the other chain arld which together are responsible for l-~ gll;~llg the antigen and providing an antigen binding site (ABS).
Moreover, it has been shown that the fimction of binding antigens can be performed by fragments of a naturally-occurring antibody, and as set out above, these antigen-binding fragments are also intended to be designated by the term "antibody". Examples of binding fragments . ,. ..,.~ within the temm antibody include (i) the Fab fragment consisting of the VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and C~l domains; (iii) the Fv fragment consisting of the VL and VH domams of a single arm of an arltibody, (iv) the dAb fragment (Ward et al., (1989) Natwe 341:544-546 ) which consists of a V~ domain; (v) isolated CDR regions; and (vi) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. Furthermore, although the two domains of the Fv fragment are coded for by separate genes, it has proved possible to make a synthetic linker that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al. (1988) Science 242:423426; and Huston et al.
(1988) PN~lS 85:5879-5883) by I c~ J ~ methods. Such single chain antibodies are also l within the present meaning of the term "antibody".

WO 95/lS982 7 2 1 7 5 4 8 2 PCT~IJ59~14106 The language "antibody variable region" is likewise recogluzed in the art, and includes those portions of an antibody which cam assemble to form an antigen binding site.
For instence, an antibody variable region can comprise each of the framework regions (FRI-FR4) and ~ y .1, ...~ regions (CDRI-CDR3) for one or both chains of an 5 IgG molecule.
The language "a desired binding specificity for an ;llL~llUllUlG~.~aa;Ve epitope", as well as the more general language "antibody specificity", refers to the ability of individual amtibodies to specifically illllll~lul~,dtt with distinct antigens. The desired binding specificity will typically be determmed from the reference point of the ability of the antibody to 10 differentially bind, and therefore distinguish between, two different antigens -palLi~,lJlally where the two antigens have unique epitopes which are present along with many common epitopes. For instance, a desired binding affinity for an illUll~ lUlc~ a;V~ epitope can refer to the ability of an antibody to distinguish between related cells, such as between adult and fetal cells, or between normal and ~ r...,... ~I cells. In other GlllbOU;lll~,llli~, the desired binding 15 affinity can refer to the ability of the antibody to differentially bind a mutant form of a protein versus the wild-type protein, or alt~llali~,ly~ to .' in binding betweendifferent isoforms of a protein. An antibody which binds specifically to an ;llllllllllUlG~,Caa;Vc epitope is referred to as a "specific antibody". The term "relative specificity" refers to the ratio of specific ill~ ~lvlca,Livily to bà,h~lu ' ca~iviLy (e.g., binding to non-20 target antigens). For instance, relative specificity for fetal cells can be expressed as the ratioof the percent binding to fetal cells to the percent binding to maternal cells. Antibodies which have no substantial background binding to a non-target antigen, such as a maternal cell, have large relative ~ r. :~ (e.g., in excess of 10 fold over ba~,h~luullv binding).
The phrases "hllividu~lly selective manner" and "individually selective binding", 25 with respect to viiy of an antibody with a particular cell, refers to the binding of an antibody to a certain cell phenotype which binding, in addition to being ,ul~ ul~,u;~,~lly dependent, is also dependent on the particular individual from which the cell is isolated, e.g.
the source of the cell. Individually selective binding does not refer to inter-species specificity of binding, rather relates to; ~ specificity.
Antibody binding to antigen, though entirely non-covalent, can Il~v~lL}l~L~aa beexquisitely specific for one antigen versus amother, and often very strong. Antibodies can specifically bind different structural f~ of most complex protein, nucleic acid, and pOly.a~ al;l~ antigens. In general, Illa~ are much bigger than the antigen binding site of an antibody. Therefore, an antibody binds to only a particular portion of the 35 Illa~lulllO~ t~ referred to herein as the "fl ' --.1" or "epitope". The total number of antibodies produced by a population of amtibody-producing cells in a particular animal is referred to a the "antibody repertoire". The ~ Ll~lulvillaly diversity of the antibody repertoire WO 9~/15982 ~ 1 7 ~ 4 8 2 8 PCT/ITS94/14106 is a result of variabilily in the structures of the antigen binding sites amongst the individual antibodies which make up the repertoire.
The process of ";,...,. ~ " refers to the exposure of an animal (that is capable of producing arltibodies) to a foreign antigerl so as to induce active immunity, which includes 5 the production of antibodies to the foreign antigen. Molecules that generate an immune response are called ;I~ nL, ..~
The language ";~ uleC~ vc epitope", which is also substituted from time to time with the terms "rare epitope" or "target epitope", is intended to refOE to epitopes that, in the context that it ordinarily occurs or can be isolated as an ;.. .c,.. are typically not 10efficient for use in generating an amtibody response by ;. ~ . at least so far as polyclonal and ",---, ~ antibody production is concerned. Such ill~ ~lulc~,Ci:~a;vc epitopes will generally be less abundant andlor less antigenic than other epitopes commonly associated with them in the ;.,.. ~ ., Even umder ~,h.,~~ cc~ wherein the h~ u.lule~ ;ve epitope can elicit a strong antibody response, this response can be, for 15 example, statistically masked by the overall number of antibodies produced as a ~
of the antigenic challenge due to other epitopes associated with the i------~v-~ ;v~ epitope in the ;.. ~.. (referred to herein as " ' epitopes" or "ha~u~d epitopes"). I-~ -llulcc~ vc epitopes may be associated with, for example, cell surface antigens that are unique to a particular cell phenotype. In many instances, this cell surface 20 antigen is not in and of itself available as an ;" ' 'L~ ' because no purified form of the antigen has been obtained. This can be especially true in the instance of integral membrane proteinsthatlosetheir ,,.. r.. -~;.. during pl~rifir51tir)n Thus,an;.. ~ .. ,containingthe ;-lul~.h.vl.~,c~;ve epitope will also include many background epitopes which can act to decrease the overall percentage of B-ly~ullo~"~t~,~ activated by the L~ ;ve epitope 25 in the total B-lyll~lJllo~ . population. In an exemplary . I.ll u-l;~ , the ., can comprise the whole cell on which the ;.. ~ ., epitope is expressed. For example, the ;..IIl.~ululc.C:.~;ve epitope can be a cell-type specific marker, such as a camcer cell marker, a fetal cell marker, or a stem cell marker. Likewise, an ve epitope can comprise an epitope unique to a variant form of a protein, such as a variant which differs by 30 only one or two amino acid residues from a related protein. For instance, theUI~C;~ , epitope can be a ,~ of a mutimt p53 which does not arise on the wild-type p53, or an epitope which unique to a particular isoform of human al)ol;uu,ulu~ E, such as ApoE4.
"Tolerization" refers to the process of !" ' ''' " an animal~s ;.. ,.. I~
35 r-,~,uull~ to a potentially antigenic swbstance present in that animal, and the antigenic substance to which tolerance is created is refered to as a "toleragen". Tolerance results from the interætion of toleragen with antigen receptors on 1~ ,. under conditions in which WOgS/15982 9 2 1 75482 PCTI~JS94J14106 the Iymphocytes, instead of becoming activated, are killed or rendered IllllCa~lUll~;Vt Tolerance to particular antigens, ûr more exætly, to particular epitopes of an antigen, can be induced by a number of means, including neonatal tolerization or chernically-induced tc~lr-ri7Afir~n and can be the result of induced clonal deletion or clonal anergy. The route of ;.." of an antigen can also effect the ability of the antigen to act as either an imn..~nng,-n or as a toleragen.
The language " ~ ,, means" relates to a process whereby the antibody response to an illllllllllUI-~ , epitope is unmasked by the deletion of an antibody response to the background epitopes. For instance, as a first step in the imm~motolrri7in~ means, an 10 animal is exposed to a toleragen comprising the; ~ epitopes. The toleragen, however, lacks the illUll~lllUlCl ~ ;VC epitopes. After tolerance to these background epitopes has been induced, an ;~ which includes the illllllllllUl~ a;Ve epitopes, is ",1",;, .~ . ~1 to the animal. Due to the deletion of the antibody response to the background epitopes, the percentage of B-cells activated in response to the rare epitopes are increased 15 relative to the total B-cell population ofthe animal. That is, the ' ~ means can be used to "enrich" for cells producmg antibodies specific for an illllll~lUI..,C~;v~ epitope.
Thus, as used herein, the term "ba~6~,1uulld epitopes" is further defined as those epitopes that are common between the i,. ,~ and the toleragen, while the term "illllll~lvl~,caa;ve epitopes" is further understood to refer to epitopes unique to the ;- - ..,n~,,. .1 (relative to the 20 toleragen). The ~ and the toleragen will typically be closely related, as for example, in the instance of 1 ' ~,v;~ lly related cells, or mut~mt or different isoforms of a protein.
The language "imml-nr~tr lPrAnrr-derived antibody repertoire" refers to the population of antibody-producing cells, and their antibodies, generated by an ' which 25 is intended to emich for antibodies for an ;IIllll~.~Jl..,~aa;VC epitope.
The language "variegated V-gene library" refers to a mixture of 1~ .,..,1.;., - -~ nucleic acid molecules encoding at least the antibody variable regions of one or both of the heavy and light chains of the ~tr~ rAnrr--derived antibody repertoire. A population of display pækages into which the variegated V-gene library has been cloned and expressed on the 30 surface thereof is likewise said to be a "variegated antibody display library" or "antibody display library".
The language "replicable genetic display package" or "display package" describes a biological particle which has genetic ~ r n providing the particle with the ability to replicate. The package cam display a fusion protein including an antibody derived from the 35 variegated V-gene library. The antibody portion of the fusion protein is presented by the display package in am illllll~lvl-a~,l;v~ context which permits the antibody to bind to an WO 95/15982 '21 7 ~ 4 8 ~ l o PCTNS94/14106 antigen that is contacted with the display package. The disp~ay package will generally be derived from a system that allows the sampling of very large variegated V-gene libraries, as well as easy isolation of the IC~ V-genes from purified display packages. The display package can be, for example, derived from vegetative bacterial cells, bacterial spores, 5 and bacterial viruses (especially DNA viruses). A variegated mixture of display packages encoding at least a portion of the V-gene library is also referred to as an "antibody display library".
The language "differential binding meams", as well as "affinity selection" and "affinity ~l~b,lll.l.~.;", refer to the separation of mernbers of the antibody display library based on the 10 differing abilities of antibodies on the surface of each of the display packages of the library to bind to the target epitope. The differential binding of an ;~ ;ve epitope by antibodies of the display can be used in the affinity separation of antibodies which specifically bind the ~ ;ve epitope from antibodies which do not. For example, the same molecule or cell that was used as an, ~ , ,ng.. ~ in the ;.. .~ step can 15 also be used in an afffinity enrichment step to retrieve display packages expressirlg antibodies which specifically bind it. Typically, the affinity selection protocol will also include a pre-emichment step wherein display packages capable of specifically binding tbe background epitopes are removed. Examples of affinity selection means include affinity .LIl _ , ' y, iion, A ~.~,...,e activated cell sorting, ~ and plaque lifts. As 20 described below, the affinity .,1....~ y includes bio-panning techniques using either purified, imr.AnhiliA,~d antigen as well as whole ceLs.
In an exemplary ~ ........ l.o.l;... l of the present invention, the display package is a phage particle which comprises an antibody fusion coat protein that includes the amino acid sequence of an antibody variable region from the variegated V-gene library. Thus, a library 25 of replicable phage vectors, especially phagemids (as defned herein), encoding a library of antibody fusion coat proteins is generated and used to transform suitable host cells. Phage particles formed from the chimeric protein cam be separated by affinity selection based on the ability of the antibody associated with a particular phage particle to specifically bind a target epitopé. In a preferred .. 1",.1; .. 1, each individual phage particle of the library includes a 30 copy of the ~ullc;.l.ullJ;ll~ phagemid encoding the antibody fusion coat protein displayed on the surface of that package. Purification of phage patticles based on the ability of an antibody displayed on an individual paTticle to bind a particular epitope therefore also provides for isolation of the V-gene encoding that amtibody. Exemplary phage for generating the present variegatRd antibody libraries include M13, fl, fd, I~, Ike, Xf, Pfl, Pf3, ~, T4, T7, P2, P4, 35 ~X-I 74, MS2 and f2.
The language "fusion protein" and "chimeric protRin" are art-recognized terms which are used i~ .,L~.~lr herein, amd include contiguous IJolr~iid~ comprising a first WO 95/15982 11 2 1 7 ~ 4 ~ 2 PCT/US94/14106 polypeptide covalently linked via an amide bond to one or more amino acid sequences which define polypeptide domains that are foreign to and not substantially T~nrnnTng,n~l~A with any domain of the first polypeptide. One polypeptide from which the fusion protein is constructed comprises a ~ antibody derived from the cloned V-gene library. A
5 second poly~ portion of the fusion protein is typically derived from am outer surfæe protein or display anchor protein which directs the "display package" (as hereafter defined) to associate the antibody with hs outer surface. As described below, where the display package is a phage, this anchor protein can be derived from a surface protein native to the genetic package, such as a vrral coat protein. Where the fusion protein comprises a viral coat protein 10 and an antibody it will be referred to as an "antibody fusion coat protein". The fusion protein may further comprise a signal sequence, which is a short length of amino acid sequence at the amino terminal end of the fusion protein, that directs at least a portion of the fusion protein to be secreted from the cytosol of a cell and localized on the PYrrAAPll~llAr side of the cell membrane.
Gene constructs encoding fusion proteins are likewise referred to a "chimeric genes"
or "fusion genes".
The term "chimeric antibody" is used to describe a protein including at least the amtigen binding portion of an 1,~,~ ' ' molecule attached by peptide linkage to at least a part of another protein. A chimeric amtibody can be, for example, an ;~
20 chimera, having a variable region derived from a frrst species (e.g. a rodent) and a constant region derived from a second species (e.g. a human), or l~ .,ly, having CDRs derived from a first species and FRs and a constant region from a second species.
The term "vector" refers to a DNA molecule, capable of replication in a host cell, into which a gene can be inserted to construct a 1, ~ ....,I.;..A.,I DNA molecule.
Aihe terms "phage vector" amd "~ .,l;d are ait-recognized and generally refer to a vector derived by .. A,.I;i~ ~; . of a phage genome, containing an origin of replication for a , amd preferably, though optional, and origin for a bacterial plasmid. The use of phage vectors rather thAn the phage genome itself provides greater flexibility to vary the ratio of chimeric all~ibOdy/~ protein to v~ild-type coat protein, as well as cllrplPm~nt the phage 30 genes with additional genes encoding other variable regions, such as may be useful in the two chain antibody constructs described below.
The language "helper phage" describes a phage which is used to mfect cells contailurlg a defective phage genome or phage vector and which functions to ~;.. 1.l.. ,.. : the defect. The defect can be one which results from removal or inactivation of phage genomic 35 sequence required for production of phage particles. Examples of helper phage are Ml3K07, and M13K07 gene III no. 3.

WO 95/1~982 21 7 5 4 8 2 PCT/US94114106 ~2 The term "isolated" as used herein with respect to nucleic acids, such as DNA orRNA, refers to molecules separated from other DNAs, or RNAs, .~ .,ly, blat are present m bhe natural source of bhe lna~,lu-,-ole.,ul~. For example, an isolated nucleic acid encodmg one of the subject anbibodies preferably il1cludes no more b'nan 10 kilobases (kb) of nucleic 5 acid sequence which naturally ~ ' lS/ flanks bhe anbibodies gene in genomic DNA, more preferably no more bhan Skb of such naturally occurring flanking sequence. The term isolated as used herein also refers to a rlucleic acid or peptide bbat is substanbially free of cellular material, viral material, or culture medium when produced by .,. ~..,.l.;, -- l DNA
techniques, or chemical precursors or obher chemicals when chemically synthesized.
10 Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in bhe natural state.
In one aspect, bhe subject invenbion sets forth a mebhod for rapid and efficientisolation of cell-type specific antibodies. For example, amtibodies b;at specifically bind epitopes unique to fetal cells or, ~ ly, epitopes unique to cancer cells, can be15 generated by bhe subject mebhod~ Likewise, bhe subject mebhod can be employed to generate antibodies to variant forms of a protein, and which can be used, for example, to detect a mutabion of a protein or to ~ir~,l, amongst various isoforms of a protein. Thus, the present invention c~n provide antibodies useful for ~ ~ diagnostic, and therapeubic In anobher aspect, the invention cor~cerns novels ;.. ~ ; v~, anbibody libraries produced by bhe subject mebhod~ as well as individual anbibodies isolated b~erefrom. From bhe subject method, for example, an anbibody display library cam be isolated which is enriched for antibodies that specifically bind an ~c.,.~;ve epitope of interest. The display library comprises a population of display packages expressing a variegated V-gene library 25 which has been cloned from an ill~.l~lUt I -derived antibody repertoire, and which has been further enriched after expression by the display package by affinity separation wibh bhe il~lll~lulf~ iVe epitope. Thus, ambibody display libraties cam be generated which are enriched for specific antibodies to cell su.face markers, such as fetal cell of bumor cell markers, as well as variamt forms of proteins.
As the ve epitope is dependent on bhe difference between bhe ; .. .r ,~. and toleragen used to generate bhe;, ".. ,.. L~J~ derived amtibody repertoire, bhe specificity of the amtibodies enriched for in bhe subject library cam be defined in terms of bhe palticular ~ ~toleragen sets used. For example, where bhe specific antibody is desired for ,1 ~ ' " between various cells of common or similar origin or phenotype, 35 bhe cell to which a specific antibody is des;red is used as bhe ~ while a related cell(s) from which it is to be .l;.l;..",.;~l..J is employed as bhe toleragen. Cell-type specific markers for the cell of interest are represented in bhe ~,c.,~;ve epitopes. Tû illustrate, WO 95/15982 1 3 2 1 7 5 4 ~ 2 PCT/USg4~14tO6 wherein the cell-type specific marker is a marker for fetal nucleated red blood cells, the toleragen can include matemal erythroid cells and the ;~ . Ihy"... cam be fetal erythroid cells.
Likewise, where the marker is for colon camcer, the toleragen can comprise nommal colon cells and the ;.11111.. h~..l can be selected from a colorl carcinoma cell line. Other exemplary 5 ;~ /toleragen sets useful for generating tbe subject antibody libraries, as well as individual antibodies therefrom, are provided in the following description and others will be apparent to those skilled in the art.
Similarly, by choice of the " 'toleragen sets, the subject libraries can be generated so as to be enriched for specific antibodies able to distinguish by binding between a 10 variant fomm of a protein and other related fomms of the protein. The variant protein can differ by one or more amino acid residues from other related proteins in order to give rise to the ir~ ulc~."ivc epitope, as well as vary Anti~Pni~Ally from the toleragen by virtue of ,u~lalivll or otber post-~ mr~r~ifir~tinn The variation can arise naturally, as between different isofomms of a protein family, illustrated by the auOIiuu,ulvt~ l E family, or 15 can be generated by genetic aberration, as illustrated by the neoplastic 1~. r~
mutations of oncogenic proteins or tumor suppressor proteins such as p53.
It is also ~- . ' ' by the present invention that individual antibodies, and genes encoding these antibodies, cam be isolated from the antibody libraries of the subject method.
For instance, after affinity enrichment of the antibody display library for antibodies which 20 specifically bind the c.,~ , epitope, individual display packages can be obtained, and the antibody gene contained therein subcloned into other appropriate expression vectors suitable for production of the antibody for the desired use.
The major aspects of the subject invention will be generally described below andpreferred I ",h~.. l;.. ,l~ will be more specifically described in the attached examples.

1. TmmllnotnlPri7~tinn T. l .. , .. ~thl~,;, l ;".......... can be employed in the present invention to generate an antibody repertoire, for use m subsequent V-gene cloning steps, in which the antibody response to an i..r~ lulc-,-,.,~;v~ epitope(s) has been unmasked. TmmlmntnlPri7Atinn can be carried out in either in vivo or in vitro i.. ---. ^~ ., systems. For instance, immlmothlPri7Atinn can be employed in the present invention to emich the pool of activated B-ly .' ~ in animmuDiæd animal for cells producing antibodies directed to irlllll~lvlcc~ , epitopes of interest. In a typical imm~lnntnlPri7Atinn procedure of the subject method, an ;.."..,....~,,..., is mtroduced to the immune system of an animal some time after exposure to a toleragen. The effect of the toleragen is to reduce or abrogate altogether any;".. ".. ln~ 1 response upon WO95/15982 21 7 5 ~ 8~ 14 PCTIUS94/14106 re-exposure of the animal to /1Pfrrmi~nt~ of the toleragen. As the ~1. f ~ composing the toleragen are generally a portion of tLlose antigenic .~..'....1---.l~ comprising the ;"",....,n~s, .. (i.e. the background epitopes), the reduced antibody response to the background epitopes upon challenge with the i.,. -,.. ~ ..,. can act to unmask the antibody response to the S ;.. Il~l.. r~ V~; epitopes of the ;,.--,., ~.. By unmasked, it is meamt that the population of antibody-producing cells directed to the ~ ul~ ,;ve epitopes effectively becomes a greater percentage of the overall population of antibody-producing cells in the animal (see Williams et al. (1992) Bic.~. ' , 12:842-847).
In the subject method most preferred, imnnlm~-t~lPri7in~ means includes subtractive 10 ; 1 ., 1 . ~. ,; ,,.. ;, ., . for enriching a pool of B-cells for clones producing antibodies specific for rare epitopes. Generally, subtractive ;.,.,,.--,;,-l;.-,. is a two-step procedure. Step one is a suppression step in which a state of tolerance is induced in the immune system of a host animal to a specific set of molecules, the tolerogen. Step two is an ;..".. ~ ,; ;l.~ step in which another set of molecules, the ;ll~ grll is introduced to the immune system. The 15 molecules comprising the tolerogen are generally a subset of those comprising the ;"' "'t ~ Ideally, the only molecules to which the immune system will generate the antibodies after exposure to the ~r~ are tLIose molecules present in the ;~ c~ ~
but not present in the tolerogen. Two rnain approaches have been used for subtractive ;....,. .;,~:;.... neonatal toleri_ation and chemical; .. ~ ;vll.
In one rlllL ' of the invention, neonatal toleri7 tion is utilized to generate an enriched pool of B-cells. Neonatal toleri_ation utilizes the self-tolerization process of the developing immune system. For each species, a discrete d~,..,lvl ' period exists during which the immune system classif es all molecules present in the body as self, resulting in an induced state of ;~ lvy.; Al tolerance to those molecules (Billingham et al. (1953) Nature 172:603-606; ~ P~ki et al. (1986) ~Inal BioclZem 154:373-381; Hasek et al. (1979) Immunol Rev 46:3-26; Readmg (1982) Jlmmunol Methods 53:261-291; and Streilen et al.
(1979) Immunol ~ev 46:125-146). Subsequent exposure to any molecules present during this stage will be met with ;". , ~ U~:~V~ l}~ . Forsubtractive; ~ ; - . mice (or other host animals) are neonatally exposed to the tolerogen. When these animals are '-~, 'ly matv;re, they are exposed to the; ~ ~'L " Theoretically, tne immune system should be ' -" 'ly responsive only to those molecules in the ,, but not in the tolerogen.
In another ~... I.o ~ of the subject method, chemical ,, is the ' ~ means employed to generate an enriched B-cell population for subsequent 35 cloning of variable region genes (V-genes). For example, chemical ;l .- I~v~ ;vl~ via the cytotoxic drug CY~ I'-I.I-''~-I'~ ,;.1P is technique useful for subtractive ;~...-- - ;~-1;.-..
(Ahmed et al. (1984) J~m~cad Dermafo/ 11:1115-1126; Matthew et al. (1983) CS~Symp Wo 95/15982 15 2 1 7 ~ 4 8 2 PCTNS94/14106 Quant Biol 48:625-631; Matthew et al. (1987) JImmunol Method~ 100:73-82; and Turk et al.
(1972) Immunolog;v 23:493-501). Application of the chemical ~:y ~ 1P to animals exposed to a foreign antigen selectively kills B-cells that have been stimulated to proliferate irl response to the presence of the foreign antigenic molecules. After cy~ P
5 treatment, subsequent exposure to those molecules results in a reduced immlmnln~
response. As a subtractive immlmi7:~tinn technique, animals are first exposed to the foreign antigenic molecule (i.e. the tolerogen), and are then injected with ~ p~ ul --..;.1~ After the drug has been allowed to clear, the animals are exposed to the imn l~nngrn Tllcvle~ lly, the immune system should be ;l, .... ln~ y responsive only to those epitopes of the 10 ;~ O. .. that are not found in the tolerogen.
Other subtractive ;- ...,l...;,-~;..,. protocols are also available for use in the subject method, arld irlclude, for example, the use of ' ' targeted toxins. For instamce, IL-2-toxin fusion proteins (Kelley et al. (1988) PN~S 85:3980-3984) and IL-4-toxin fusion proteins(Lakkisetal.(1991)EurJlmmunol21:2253-2258)canbeusedtoselectivelyinduce 15 tolerance to the epitopes of a toleragen.
II. Gpnpr~fir~ Arltihn-l,y GPne T ihr~riPe After application of an ;.. 1.. 1.. ;,_1;.. ,. step, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known, and can be applied in the 20 subject method, for directly obtaining the DNA sequence of the variable regions of a diverse population of ~p1nb-11in molecules by using a mixture of oligomer primers and PCR.
For instance, mixed ~ .---.. lrvl;~1~ primers UUII~ 1 '' ,, to the 51 leader (signal peptide) sequerlces and/or framework I (FRI) sequences, as well as primer to a corlserved 3' constant region primer can be used for PCR ~mr1ifi~s-tinn of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Ri .,~. l.. ;.l ~ 11: 152-156). A
similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (Larrick et al. (1991) Methods: Companion to Methods in E~lllolo~;~ 2: 106-110). The ability to clone human ,,' L " V-genes takes on special ci~..;ri.... ~ in light of adv in creatmg human amtibody repertoires in tr~msgenic animals (see, for example, Bruggeman et al. (1993) Year Immunol 7:33-40;
Tuaillon et al. (1993) PNAS 90:3720-3724; Bruggeman et al. (1991) Eur Jlmmunol 21:1323-1326; and Wood et al. PCT publication WO 91/00906).
In an illustrative ~ .l.v.1.. 1 RNA is isolated from mature B cells of, for example, peripheral blood cells, bone marrow, or spleen ~UI~::p~ iU...~, using st~mdard protocols (e.g., U.S. Paterlt No. 4,683,202; Orlandi, et al. PN~IS (1989) 86:3833-3837; Sastry et al., PN~S
(1989) 86 5728-5732; and Huse et al. (lg89) Science 246:1275-1281.) First-strand cDNA is WOg5/lS982 ~ 7 5 16 PCT/US94/14106 synthesized using primers specific for the constant region of the heavy chain(s) and each of the ~c and ~ light chains, as well as prirners for the signal sequence. Using variable region PCR primers, such as those shown in l;igures IA and IB (for mouse) or Figure 6 (for human), the variable regions of both heavy and light chains are amplified, each alone or in S f~ r,."~l;"" and ligated into appropriate vectors for further ~ i..,. in generating the display packages.
Oi;~ .... 1- ,~I;~lr primers useful in ~ ., protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction ....lf. rl-_cr reCOglUtiOn sequences may also be in- , ' ' into the primers to allow for the cloning of the amplified 10 fragment into a vector in a ~ reading frame for expression~
III. Vslfi~ ' Antihnfly Dic~l~,y The V-gene library cloned from the ' -derived antibody repertoire can be expressed by a population of display packages to form an antibody display library. With 15 respect to the display package on which the variegated antibody library is manifest, it will be appreciated from the discussion provided herein that the display package will often preferably be able to be (i) genetically altered to encode at least a variable region of an antibody, (ii) maintained and amplified in culture, (iii) , ' ' to display the antibody gene product in a manner permitting the antibody to interact with a target epitope during an affinity 20 separation step, and (iv) affinity separate~. while retaming the antibody gene such that the sequence of the antibody gene can be obtained. In preferred f..ll.;l.l;ll....l~, the display remains viable after affinity separation.
Ideally, the display package comprises a system that allows the sampling of very large vafiegated antibody display libraries, rapid sorting after each affmity separation round, and easy isolation of the antibody gene from purified display packages. The most attractive candidates for this type of screening are prokaryotic organisms and viruses, as they can be amplified quickly, they are relatively easy to , ' , and large number of clones can be created. Preferred display packages include, for example, vegetative bacterial cells, bacterial spores, and most preferably, bacterial viruses (especially DNA viruses) However, the present invention also ~ the use of eukafyotic cells (other than cells which naturally produce antibodies, i.e. B-cells), including yeast and their spores, as potential display packages.
In addition to commercially available kits for generating phage display libraries (e.g.
the Pharmacia R~ ' . Phage ~ntibody System, catalog no. 27-9400-OI; and the Stratage~e SurJZ4PTM phage display kit, cataiog no. 240612), examples of methods and wo 95115982 l 7 2 1 7 5 4 8 2 PCT/US94~14106 reagents IJ~i. uLuly amenable for use in generating the variegated antibody display library of the present inYention can be found in, for eYample, the Ladner et al. U.S. Patent No.
5,æ3,409; the Kang et al. TntPrnAtinnal Publication No. WO 92/18619; the Dower et al.
Tlllrl "-l;,.,.~l Publication No. WO 91/17271; the Winter et al. TntPrnatinnal Publication WO
92/20791; the Markland et al. TntPrnatinnal Publication No. WO 92/15679; the Breitling et al.
Tlllrll,-li,...~l Publication WO 93/01288; the McCafferty et al. IntPrnatinnal Publication No.
WO 92/01047; the Garrard et al. l.. . " ;" l Publication No. WO 92/09690; the Ladner et al. T~ IIIAI Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) ~um Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) JMol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;Garradetal.(1991)Bio/7echnology9:1373-1377;TTnogPnho~-metal.(l991)NucAcid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.
When the display is based on a bacterial cell, or a phage which is assembled 15 ~ ,;, ^lly, the display means of the package will comprise at least two ~
The frrst component is a secretion signal which directs the l~..",l,;, ~ antibody to be localized on the PYfrarPll~ r side of the cell membrane (of the host cell when the display package is a phage). This secretion signal is . l ~ lly cleaved off by a signal peptidase to yield a processed, "mature" antibody. The second component is a display anchor 20 protein which directs the display package to associate the antibody with its outer surface. As described below, this anchor protein can be derived from a surface or coat protein native to the genetic package.
When the display package is a bacterial spore, or a phage whose protein coating is assembled intrarPlllllarly, a secretion signal directing the antibody to the inner membrane of 25 the host cell is u~ ,C~."~.y. In these cases, the means for arraying the variegated antibody library comprises a derivative of a spore or phage coat protein amenable for use as a fusion protein.
The antibody component of the display will comprise, at a minimum, one of either the VH or VL regions cloned from B cells isolated in the subtractive ;",.,~ 1;.... step. It will 30 be appreciated, however, that the VH regions and/or the VL regions may contain, in addition to the variable portion of the antibodies, all or a portion of the constant regions. Typically, the display library will include variable regions of both heavy and light chains in order to generate at least an Fv fragment. For clarity" ,l"~ll;".. 1~ described herein detail the minimal amtibody display as comprising the use of cloned VH regions to construct the fusion 35 protPin with fhe display anchor protein. HoweYer, it should be readily understood that similar rl ~1 .oll., , '~ are possible in which the role of the VL and VH chains are reversed in the ~;ullaLI ~iUII of the display library.

WO 95/15982 ~ 1 8 Pcrlus94/14106 Under certain .,;~ the VH portion of the antibody display is derived from isolated cells of the subtractive ;II~ step, but the VL chain is either absert or is a "fixed" VL (i.e. the same VL chain for every antibody of the display). Where, for example, the VL portion of the display is fixed, the VL chain can be contributed from a gene construct other tharl the construct encodirlg the VH chain, or from the host cell itself (i.e. a light chain producing myeloma cell), or added CAU~. ,luualy to the packages so as to recombine with VH
chains already displayed on their surface. However, it will generally be preferred that the VL chain is derived from a variegated VL library also cloned from the same pûpulation of B
cells from which the VH gene is cloned, irl which case a preferred t`l~ n~ .I places the VL
I û gene in the same construct as the VH gene such that both may be readily recovered together.
When the desired antibody display is a multi-chain antibody (e.g. VH and VL are separate polypeptide chains), the cDNA encoding the light chain may be cloned directly into an appropriate site of the vector containing the heavy chain-coat protein library-; or, ' ~ ly, the light chain may be cloned as a separate library in a different plasmid vector, amplified, arld ' , '!~ the fragments cloned mto the vector library encodirlg the heavy chain. In such U1l~ , the VL chain is cloned so that it is expressed with a signal peptide leader sequence that will drrect its secretion into the periplasm of the host cell. For example, several leader sequences have been shown to direct the secretion of amtibody sequences in E. coli, such as OmpA (Hsiung et al. Bio/rlAec)molo&v (1986) 4:991-995), and (Bener et al. Science 240:1041-1043), phoA (Skerra amd Pluckthun, Science (1988)240:1038).
In the instance wherein the displa~/ package is a phage, the cloning site for the VL
chain sequences in the phagemid should be placed so that it does not ~u~ llly interfere with normal phage fimction. One such locus is the intergerlic region as described by Zinder and Boeke, (1982) Gene 19:1-10. In an illustrative ~ U~ 1 comprising an M13 phage display library, the VL sequence is preferably expressed at an equal or higher-level than the HL CPIII product (described below) to maintain a sufficiently high VL ~ in the periplasm and provide efficient assembly (ACc~riotil-n) of VL with VH chains. For irlstance, a phagemid can be constructed to encode, as separate genes, both a VH/coat fusion protein and a VL cham. Under the appropriate induction, both chains are expressed and allowed to assemble in the IJ~ alll;l space of the host cell, the assembled amtibody bemg linked to the phage particle by virtue of the VH chain being a portion of a coat protein fusion corlstruct.
The number of possible ~....~,;.. ~;....c of heavy and light chains probably exceeds Iol2. To sample as mamy f.~ as possible depends, in part, on the ability to recover 35 large numbers of ~ For phage with plasmid-like forms (as f~' phage), el~tl'"'- .~r~...., ~;,.l-providesamefficienc~ mr~Ah1f-tothatofphage~ f ~;....with in vi~ro pæka~ing, in addition to a very high capacity for DNA input. Al'his allows large amounts Wo 95/15982 PC. ~uss4~14106 of vector DNA to be used to obtain very large numbers of I~ r~ The method described by Dower et al. (1988) Nucleic Acids Res., 16:6127-6145, for example, may be used to transform fd-tet derived ~ at the rate of about 107 I~ IUg of ligated vector into E. coli (such as strain MC1061), and libraries may be constructed in fd-tet S Bl of up to about 3 x 10 members or more. Increasing DNA input and making mn~lifiAAAtinn.~
to the cloning protocol within the ability of the skilled artisan may produce increases of greater than about 10- fold in the recovery of 1~ , providing libraries of up to 1010 or more ~
In other ( ...I o~l;.,. -'~ the V region domains of heavy amd light chains can be 10 expressed on the same ~oly~ ,Lide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene ~ ly cloned into the desired expression vector or phage genome. As generally described in McCafferty et al, Nature (1990) 348:552-554, complete VH and VL domains of am antibody, joined by a flexible (Gly4-Ser)3 linker can be used to produce a single chain antibody which can render the display package separable based on 15 antigen affinity As will be apparent to those skilled in the art, in e~ wherein high affinity antibodies are sought, an important criteria for the present selection method can be that it is able to ~' between antibodies of different affinity for a particular antigen, and preferentially enrich for the antibodies of highest affrnity Applying the well known 20 principles of antibody affinity and valence (i e. avidity), it is understood that . ' ~ the display package to be rendered effectively IIIVIIU~ I ' can allow affinity erlrichment to be carried out for generally higher binding aff~nities (i.e. binding constAAnts in the range of I o6 to 1010 M-l) as compared to the broader range of affinities isolable using a 1, ~ display package. To generate the IIIUIIO v_ I display, the natural (i.e. wild-type) form of the surface 25 or coat protein used to anchor the antibody to the display can be added at a high enough level that it almost entirely eliminates inclusion of the antibody fusion protein in the display package. Thus, a vast majority of the display packages can be generated to include no more than one copy of the antibody fusion protein (see, for example, Garrad et al. (1991) Bio/Technology 9:1373-1377). In a preferred ~,lllb- ' of a luu~luv~k,~l~ display library, 30 the library of display packages will comprise no more than 5 to 10% polyvalent displays, and more preferably no more than 2% of the display will be polyvalent, and most preferably, no more than 1% polyvalent display packages in the population. The sorlrce of the wild-type anchor protein can be, for example, provided by a copy of the wild-type gene present on the same construct as the antibody fusion protein, or provided by a separate construct altogether.
35 However, it will be equally clear that by similar . :~ " polyvalent displays can be generated to isolate a broader range of bindmg affinities. Such amtibodies can be useful, for example, in ~ ;.... protocols where avidity can be desirable.

woss/lss82 ~ 1 7 5~ zo PCT/IJS94/14106 i) P~lages ~s Display Packages Ba~i~,liulJlla~s~ are ahractive prokaryotic-related organisms for use in the subject method. B~ I- ;u~ rr are exce~lent candidates for proYiding a display system of the variegated antibody library as there is little or no enzymatic activity associated with intact 5 mature phage, and because their genes are inactive outside a bæterial host, rendering the mature phage particles 1ll~, abOlil,ally inert. In general, the phage surface is a relatively simple shuch~re. Phage can be grown easily in large numbers, they are amenable to tbe practical handling involved in many poltential mass screening programs, and they carry genetic inf ~rm~tinn for their o~-vn synthesis within a small, simple package. As the antibody 10 gene is inserted into the phage genome, choosing the appropriate phage to be employed in the subject method will generally depend most on whether (i) the genome of the phage allows udu~.liull of the antibody gene either by tolerating additional genetic material or by having replaceable genetic material; (ii) the vi1ion is capable of packaging the genome after accepting the insertion or s~lhstihltir~n of genetic material, and (iii) the display of the antibody 1~ on the phage surface does not disrupt virion shuch~re sufficiently to interfere with phage IJI U~a~aiiUll.
One concem presented ~-vith the use of phage is that the Illul~llo~ .;ic pathway of the phage detemmines the C:I~Vil~ ' in which the antibody will have opportunity to fold.
rt~;l~l~.~;~lly assembled phage are preferred as the displayed antibodies will generally 20 contain essential disulfides, and such arltibodies may not fold correctly within a cell.
However, in certain ~ 1 " in which the display package forms " ' '~/ (e.g., where ~ phage are used), it has been ~ --~ ' that the antibody may assume properfolding after the phage is released from the ~ell.
Another concern related to the use of phage, but also pertinent to the use of bacterial 25 cells and spores as well, is that multiple infections could generate hybrid displays that carry the gene for one particular antibody yet have at least one or more different antibodies on their surfaces. Therefore, it can be preferable, though optional,to minimize this possibility by infecting cells with phage under condhions resulting in a low multiple-infection. However, there may be l,iu~,uill~lan~ in which bigh '`i ' ~ ,lion conditions would be desirable, 30 such as to mcrease ~ ' ' ' events behween gene conshucts encoding the antibody display in order to further expamd the repertoire of the amtibody display library.
For a given I , ' ~, the preferred display means is a protein that is present onthe phage surface (e.g. a coat protein). F;la ll~ u~ phage cam be described by a helical lattice; isomehic phage, by am icosahedral lattice. Each monomer of each major coat protein sits on a lattice point and makes defined ;,.t.. Ii" - with each of its neighbors. Proteins that fit into the lahice by making some, but not all, of the normal lahice contacts are likely to ~ 1 7548~
WO 95/15982 2 1 PCT/lJ59 ~ 06 destabilize the virion by aborting formation of the virion as well as by leaving gaps in the virion so that the nucleic acid is not protected. Thus in l~ l.A,,æ) unlike the cases of bacteria and spores, it is generally import~mt to retain in the amtibody fusion proteins those residues of the coat protein that interact with other proteins in the virion. For example, when 5 using the M13 cpVIlI protein, the entire mature protein will generally be retained with the amtibody fragment being added to the N-terminus of cpVIIl, while on the other hand it cam suffice to retain only the last 100 carboxy terminal residues (or even fewer) of the M13 cpIlI
coat protein in the amtibody fusion protein.
Under the appropriate induction, the amtibody library is expressed and allowed to 10 assemble in the bacterial cytoplasm, such as when the ~ phage is employed. The induction of the protein(s) may be delayed until some replication of the phage genome, synthesis of some of the phage structural-proteins, and assembly of some phage particles has occurred. The assembled protein chains then interact with the phage particles via the binding of the anchor protein on the outer surface of the phage patticle. The cells are Iysed amd the phage bearing 15 the library-encoded receptor protein (that CUIIC~UUIIda to the specific library sequences carried in the DNA of that phage) are released and isolated from the bacterial debris.
To eririch for and isolate phage which contain cloned library sequences that encode a desired protein, and thus to ultimately isolate the nucleic acid sequences themselves, phage harvested from the bacterial debris are affinity purified. As described below, when an 20 antibody which specifically binds a particular amtigen or antigenic ,1~ is desired, the antigen or ~ cam be used to retrieve phage displaymg the desired antibody. The phage so obtained may then be amplified by infectmg into host cells. Additional roumds of affinity enrichment followed by A~ may be employed umtil the desired level of enrichment is reached.
2~ Aihe enriched antibody-phage cam also be screened with additional detection ",~,luu~u~,~
such as expression plaque (or colony) lift (see, e.g., Youmg and Davis, Science (1983) 222:778-782) whereby a labeled amtigen is used as a probe. The phage obtained from the screening protocol are infected into cells, propagated, amd the phage DNA isolated and sequenced, amdlor recloned into a vector intended for gene expression in ~luLu~ut~ or 30 eukaryotes to obtain larger amolmts of the particular amtibody selected.
In yet amother .~ " L~ the antibody is also tr_nsported to Am extra-~;yLu,ul_auu~, ll.,... of the host cell, such as the bacteril periplasm, but as a fusion protein with a viral CoAt protein. In this, I ' the desired protein (or one of its ,uuly,u.,~JLidc chains if it is a multichain antibody) is expressed fused to a viral coat protein which is processed amd 35 transported to the cell inner membr~me. Other chains, if present, are expressed with a secretion leader and thus are also transported to the periplasm or other " ' by extra-W095115982 Z ~ 7 ~ 48~ 22 PCT/US94/14106 cy~la~lllic location. The chains (e.g. heavy amd light chains) present in the extra-cytoplâsm then assemble into a complete antibody (or binding fragment thereofl, The assembled molecules become ill~,UlL~U~ ,d into the phage by virtue of their attachment to the phage coat protein as the phage extrude through the host membrane and the coat proteins assemble 5 around the phage DNA. The phage bearing Lhe antibody complex may then be screened by affinity enrichment as described below.
a) FilamentousPhage Fila,ll.,llLUU~ l "n ~, which include M13, fl, fd, Ifl, Ike, Xf, Pfl, and Pf3, are a group of related viruses that infect bæteria. They are termed filR nt nto~ because they are 10 long, thin parLicles comprised of an elongated capsule that envelopes the deoxyribonucleic acid (DNA) that forms the l ~ r genome. The F pili fil~n~ ntr~ l,= ,t` ;~ Or (Ff phage) infect only gram-negative bacteria by specifically adsorbing to the tip of F pili, and include fd, fl and M13.
Compared to other ~ ,, fil ~rn~o~ phage in general are attractive and 15 M13 in particular is especially attrætive because: (i) the 3-D structure of the virion is known;
(ii) the processing of the coat protein is well ,. ~ l (iii) the genome is ~rrRn~ ; (iv) the genome is small; (v) the sequence of the genome is knov~n; (vi) the virion is physically resistant to shear, heat, cold, urea, g " chloride, low pH, and high salt; (vii) the phage is a sequencing vector so that sequencing is especially easy; (viii) antibiotic-resistance 20 genes have been cloned into the genome with predictable results (Hines et al. (1980) Gene 11:207-218); (ix) it is easily cultured and stored, with no unusual or expensive media for the infected cells, (x) it has a high burst size, each infected cell yielding 100 to 1000 M13 progeny after infection; and (xi) it is easily harvested and o .~ 1 (Salivar et al. (1964) rrology 24: 359-371). The entire life cycle of the fi'~ phage M13, a 25 common cloning and sequencing vector, is well understood. The genetic structure of M13 is well known, including the complete sequence (Schaller et al. in The Single-Stranded DN~
Phages eds. Denhardt et al. (NY: CSHL Press, 1978)), the identity and function of the ten genes, and the order of l."l.~ . ;1.1;...~ and location of the promoters, as well as the physical structure of the virion (Smith et al. (1985) Science 228:1315-1317; Raschad et al. (1986) 30 Microbiol Dev 50:401-427; Kuhn et al. (1987) Science 238:1413-1415; 7.imm~rmRn et al.
(1982) J Biol Chem 257:6529-6536; and Banner et al. (1981) Nature 289:814-816). Because the genome is small (6423 bp), cassette ,, is prætical on RF M13 (Current Protocols in Molecular Biolog,v, eds. Ausubel et al. (NY: John Wiley & Sons, 1991)), as is single-stranded ol;~..., l ~.l;~l~ directed ,, (Fritz et al. in DNA Cloning, ed by Glover (Oxford, UK: IRC Press, 1985)). M13 is a plasmid and i r '' system in itself, and an ideal sequencing vector. Ml 3 c~m be grown on Rec- strains of E. coli. The Ml 3 genome is expandable (Messing et al. in The Single-Stranded DNA Phages, eds Denhardt et 21 754~2 al. (NY: CSHL Press, 1978) pages 449-453; and Frit~ et al., supra) and M13 does not Iyse cells. Extra genes can be inserted into M13 and will be maintained in the viral genome in a stable manner.
The mature capsule or Ff phage is comprised of a coat of five phage-encoded geneproducts: cpVIII, the major coat protein product of gene VIII that forms the bulk of the capsule; and four minor coat proteins, cplII and cplV at one end of the capsule and cpVII and cplX at the other end of the capsule. The length of the capsule is formed by 2500 to 3000 copies of cpVIII in an ordered helix array that forms the . l, ~ ;r filament structure. The gene IlI-encoded protein (cpIII) is typically present in 4 to 6 copies at one end of the capsule and serves as the receptor for binding of the phage to its bacterial host in the initial phase of infection. For detailed reviews of Ff phage structure, see Rasched et al., MicrobioL Rev., 50:401-427 (1986); and Model et al.,in The B~t.,,~u, ' ~,,, Vol~lme 2, R. Calendar, Ed., Plenum Press, pp. 375-456 (1988).
The phage particle assembly involves extrusion of the viral genome through the host IS cell's membrane. Prior to extrusion, the major coat protein cpVIII and the minor coat protein cplII are s~nthesized and transported to the host cell's membrane. Both cpVIII amd cpIII are anchored in the host cell membrane prior to their l into the mature particle. Inaddition, the viral genome is produced amd coated with cpV protein. During the extrusion process, cpV-coated genomic DNA is stripped of the cpV coat and ~ ,. vu~ly recoated Z0 with the mature coat proteins.
Both cpIII amd cpVIII protems include two domains that provide signals for assembly of the mature phage particle. The first domain is a secretion signal that directs the newly ~ protein to the host cell membrame. The secretion signal is located at the amino ter,rninus of the poly~ ide and targets the poly,v~ id~ at least to the cell membrane. The second domain is a membrane anchor domain that provides signals for association with the host cell membrane and for association with the phage particle during assembly. This second sigmal for both cpVIII amd cplII comprises at least a l.~u~l~vl,;., region for spanning the membrane.
The S0 amino acid mature gene VIII coat protein (cpVIII) is synthesized as a 73 amino acid precoat (Ito et al. (1979) PN~S 76:1199-1203). cpVIII has been extensively studied as a model membrane protein because it cam integrate into lipid bilayers such as the cell membrane in an ~y orientation with the acidic arnino terrninus toward the outside and the basic carboxy terminus toward the irlside of the membrane. The first 23 ammo acids constitute a typical ,, ' ~ which causes the nascent puly~."v~id~ to be inserted into the inner cell membrane. An E. coli signal peptidase (SP-I) recognizes amino acids 18, 21, and 23, and, to a lesser extent, residue 22, and cuts between residues 23 and 24 Wog~/15982 ~ 1 5482 24 PCT/US94114106 oftheprecoat(Kuhnetal.(l985)J.BioL Chem.260:15914-15918;andKu~metal.(1985)J
Biol. Cl~em. 260:15907-15913). Afterremoval oftbe signal sequence, the amino terminus of the mature coat is located on the ~ l;c side of the inner membrane; tbe carboxy terminus is on the cy~ ~c side. Abo-t 3000 copies of the mature coat protein associate 5 side-by-side in the inner membrane.
The sequence of gene VIII is kno~-vn, and the amino acid sequence can be encoded on a synthetic gcne. Mature gene VIII p}otein makes up the sheath around the circular ssDNA.
The gene VIII protein can be a suitable anchor protein because its location and orientation in the virion are kno~vn (Barmer et al. (1981) Nalure 289:814-816). Preferably, the antibody is 10 attached to the amino terminus of the mat lre M13 coat proteirl to generate the phage display library. As set out above, ~ ... of the ~ ; , of both the wild-type cpVlII and Ab/cpVIII fusion in an infected cell can be utilized to decrease the avidity of the display and thereby enhance the detection of high affinity antibodies directed to the target epitope(s).
Another vehicle for displaying the amtibody is by expressing it as a domain of a15 chimeric gene containing part or all of gene 111. When lllulluv~ displays are required, expressing the V-gene as a fusion protein with gplII can be a preferred ~.,.l,.~.t;.,. ', as of the ratio of vvild-type gplll to chimeric gplll during formation of the phageparticles can be readily controlled. This gel1e encodes one of the minor coat proteins of Ml 3.
Genes Vl, Vll, and IX also encode minor coat proteins. Each of these minor proteins is 20 present in about 5 copies per virion and is related to ~ or infection. In contrast, the major coat protein is present in more than 2500 copies per virion. The gene Vl, Vll, and IX proteins are present at the ends of the virion; these three proteins are not post-,.lly processed (Rasched et al. (1986) Ann Rev. Microbiol. 41:507-541). In particular, the smgle-stranded circular phage DN~ associates with about five copies of the 25 gene 111 protein and is then extruded through the patch of membr~me-associated coat protein in such a way that the DNA is encased in a helical sheath of protein (Webster et al. in The Single-SlrandedDNA Phages, eds Dressler et al. (NY:CSHL Press, 1978).
r ~ . ~ of the sequence of cplll has .l.. ... ~1 that the C-terminal 23 amino æid residue stretch of llydlu~ ulJic amino acids normally responsible for a membrane anchor fumction can be altered in a variety of ways and retain the capacity to associate with mPmhrAnr~ Ff phage-based expression vectors were first described in which the cplll amino acid residue sequence was modified by insertion of p~ ulide "epitopes" (Parmely et al, Gene (1988) 73:305-318; and Cwirla et al., PNAS (1990) 87:6378-6382) or an amino æid residue sequence defining a single chain antibody domain (McCafferty et al., Science (1990) 348:552-554). It has been ~ J that insertions into gene m can result in the production of novel protein domains on th.e virion outer surfæe. (Smith (1985) Science æ8:1315-1317; and de la Cruz et al. (1988) J. Biol. Chem. 263:4318~322). The antibody gene may be fused to gene III at the site used by Smith and by de la Cruz et al., at a codon cullcauui~Jillg to another domain bounda~y or to a surface loop of the protein, or to the amino terminus of the mature protein.
Generally, the successful cloning strategy utili~ing a phage coat protein, such as cpIII
5 of ~ phage fd, will provide: (1) expression of an antibody chain fused to the N-terminus of a coat protein (e.g., cpIlI) and transport to the inner membrane of the host where the Il~ ,ullub;c domain in the C-terminal region of the coat protem anchors the fusion protein in the membrane, with the N-terminus containing the antibody chain protruding into the ,u.,li~laa~ , space and available for interaction with a second or subsequent chain (e.g., 10 VL to form an Fv or Fab fragment) which is thus attached to the coat protein; and (2) adequate expression of a second or subsequent ,uol~yp~,~Lidc chain if present (e.g., VL) and transport of tbis chain to the soluble UUIIIIJ~U Ll.-~,l.L of the periplasm.
Similar ~,ull~Lluu~iulla could be made with other filD~nDnt~ phage. Pf3 is a well known l;lA..,. 11~l~ phage that infects P ~ aerugenosa cells that harbor an IncP-I
plasmid. The entire genome has been sequenced ((Luiten et al. (1985) ~ Virol. 56:268-276) and the genetic signals involved in replication and assembly are known (Luiten et al. (1987) DNA 6:129-137). The major coat protein of PF3 is unusual in having no signal peptide to direct its secretion. The sequence has charged residues ASP-7, ARG-37, LYS-40, and PHE44 which is consistent with the amino terminus being exposed. Thus, to cause an antibody to 20 appear on the surface of Pf3, a tripartite gene can be constructed which comprises a signal sequence known to cause secretion in P. ~..,..~,.,.o~u, fused in-frame to a gene fragment encoding the antibody sequence, which is fused in-frame to DNA encoding the mature Pf3 coat protein. Optionally, DNA encoding a flexible linker of one to 10 amino æids is introduced between the antibody gene fragment and the Pf3 coat-protein gene. This tripartite 25 gene is introduced into Pf3 so that it does not interfere with expression of any Pf3 genes.
Once the signal sequence is cleaved off, the antibody is in the periplasm and the mature coat protein acts as an anchor and phage-assembly signal.
b) Bu"f~ .U~ XI 74 The bA~ JPI~ XI74 is a very small icosahedral virus which has been 30 thoroughly studied by genetics, l~;n h- ~.y, and electron llfi~,luacu~ (see 17~e Single Stranded Dl\~,4 Phages (eds. Den hardt et al. (NY:CSHL Press, 1978)). Three gene products of ~X174 are present on the outside of the mature virion: F (capsid), G (major spike protein, 60 copies per virion), and H (minor spike protein, 12 copies per virion). The G protein comprises 175 amino acids, while H comprises 328 amino acids. The F protein interacts with 35 the single-stranded DNA of the virus. The proteins F, G, amd H are translated from a single mRNA in the viral infected cells. As the virus is so tightly çnn~AtrPinD(I because several of its W095/15982 ~1 7 ~48~ PCT/US94/14106 genes overlap, ~X174 is not typically used as a cloning vector due to the fact that it can accept ver~v little additional DNA. However, mutations in the viral G gene (encoding the G
protein) can be rescued by a copy of the wild-type G gene carried on a plasmid that is expressed in the same host cell (Chambers et al. (1982) Nuc Acid Res 10:6465-6473) In one n~ o~ ', one or more stop codons are introduced into the G gene so that no G protein is produced from the viral genome. The variegated antibody gene library can then be fused with the nucleic acid sequence of the H gene. An amount of the viral G gene equal to the siæ of antibody gene fragment is eliminated from the ~X174 genome, such that the siæ of the genome is ultimately unchanged. Thus, in host cells also ~,,.,.~r. ."". ,I with a second plasmid expressing the wild-type G protein, the production of viral particles from the mut~mt virus is rescued by the exogenous G protein source. Where it is desirable that only one antibody be displayed per ~X174 particle, the second plasmid can further imclude one or more copies of the wild-type H protein gene so that a mix of H and Ab/H proteins will be ~ 1 bythe wild-type H upon ;llcul~ul~lioll into phage particles.
c~ ~.arge DNA P)lage Phage such as ~ or T4 have much :larger genomes than do M13 or ~X174, and have more: , ' ' 3-D capsid structures tham M13 or ~PX174, with more coat proteins tochoose from. In ~mho~ nPn~c of the invention whereby the amtibody library is processed and assembled into a functional form and associates with the 1.~ . patticles within the cytoplasm of the host cell, I ' )L ' .~, ~ and derivatives thereof are examples of suitable vectors. The i~fr~ O of phage ~ cam potentially prevent protein domains that ordinatily contain disulfide bonds from folding correctly. Ho~vever, variegated libraries expressing a population of functional am~ibodies, including both heavy amd light chain variable regions, have been generated in ~ phage. (Huse et al. (1989) Science 246:1275-1281; Mullinax et al. (1990) PIVAS 87:8095-8099; and Pearson et al. (1991) PI~AS 88:2432-2436). Such strategies take advantage of the rapid .,U~ u~,~iul, amd effcient ,.~ r~.. ,.. ,;.,.
abilities of ~ phage.
When used for expression of amtibûdy sequences, such as VH, VL, Fv (vatiable region fragment) or Fab, library DNA may be readily inserted into a ~ vector. For inst~mce, 30 variegated antibody libraries have been constructed by ,,,,..I;r;. 1;.~., of ~ ZAP Il (Short et al.
(1988) ~uc Acid Res 16:7583) comprising inserting both cloned heavy and light chain vatiable regions into the multiple cloning site of a ~ ZAP II vector (Huse et al. supra.). To illustrate, a pair of ~ vectors may be desiglled to be asymmettic with respect to resttiction sites that flank the cloning and expression sequences. This asymmetry allows efficient 35 r~ ~ o., l ,;, . - ;.1l l of libraties coding for separate chams of the active protein. Thus, a library expressing antibody light chain vatiable regions (VL) may be combined with one expressing antibody heavy chain vatiable regions (VH), thereby ~;U~-fL~uu~ g ~ ' ' ' antibody or WO95/15982 27 2 1 75482 PCT/US94~14106 Fab expression libraries. For insti~nce, one ~ vector is designed to serve as a cloning vector for antibody light chain sequences, amd another ~ vector is designed to serve as a cloning vector for amtibody heavy chain sequences in the initial steps of library, U~ JII. A
r,~..,.l.'....~.,;~l library is constructed from the two ~ libraries by crossing them at an 5 appropriate restriction site. DNA is first purified from each library, and the right and left arms of eæh respective ~ vector cleaved so as to leave the antibody chain sequences imtact. The DNAs are then mixed and ligated, and only clones that result from proper assembly of reciprocal vectors ,~ as viable phage (Huse et all, supra.) ii) Bacterial Cells as Display Packages RCC~ antibodies are able to cross bacterial membranes after the addition of bacterial leader sequences to the N-terminus of the protein (Better et al (1988) Science 240:1041-1043; and Skerra et al. (1988) Science 240:1038-1041). In addition, l~ ' amtibodies have been fused to outer membrane proteins for surface pl~i For exiample, one strategy for displaying antibodies on bacterial cells comprises generating a 15 fusion protein by inserting the antibody into cell surface exposed portions of an integral outer membrime protein (Fuchs et al. (1991) Bio/~echnolog~ 9:1370-1372). In selecting a bacterial cell to serve as the display package, any well-..l.~ bacterial strain will typically be suitable, provided the bacteria may be grown in culture, engineered to display the antibody lib}ary on its surface, and is compatible with the particular affinity selection process practiced 20 m the subject method. Among bacterial cells, the prefe~red display systems include Salmonella ty~.., Bacillus subtilis, r ~ aeruginosa, Vibrio cholerae, Klebsiella~ . 7 Neisseria~;v,.vr,hv~v~, Neisseria i . ~ I, Bacteroides nodosus, Moraxell~7 bovis, and especially Escherichia coli. Many bacterial cell surface proteins useful in the present invention have been ~ , and works on the Ir~AAAli7AAtirm of these Z5 proteins and the methods of ~ ' ,, their structure include Ben_ et al. (1988) Ann Rev Microbiol 42: 359-393; Balduyck et al. (1985) Biol Chem Hoppe-Seyler 366:9-14; Ehrmarln et al (1990) PNAS 87:7574-7578; Heijne et al. (1990) Protein E.~ ,;.." 4:109-112;
Ladner et al. U.S. Patent No. 5,223,409; Ladner et al. W088/06630; Fuchs et al. (1991) Bio/technology 9:1370-1372; and Goward et al. (1992) TIBS 18:136-140.
To furtber illustrate, the LamB protein of E coli is a well understood surface protem that can be used to generate a variegated library of antibodies on the suriAace of a bacterial cell (see, for example, Ronco et al. (1990) Biochemie 72:183-189; van der Weit et al. (1990) Vaccine 8 269-277; Charabit et al. (1988) Gene 70:181-189; and Ladner U.S. Patent No.
5,Z22,409). LamB of E. coli is a porin for maltose and ' ' trarlsport, and serves as 35 the receptor for adsorption of IJA' ~ ;-J~ and ICI0. LamB is triansported to the outer membrane if a functional N-terminal signal sequence is present (Benson et al. (1984) PNAS
81:3830-3834). As with other cell surface proteins, LamB is synthesi_ed with a typical WO 95/15982 ~ ¦ 7 5 4 8 ~ 28 PCT/US94/14106 signal-sequence which is .,,,1,~.1,.. Iy removed. Thus, the variegated antibody gene library can be cloned into the LamB gene such that the resulting library of fusion proteins comprise a portion of LamB sufficient to anchor the protein to the cell membrane with the antibod~
fragment oriented on the .~trA~ ar si~e of the membr~me. Secretion of the . ~trA~ lAr 5 portion of the fusion protein can be facili~ated by inclusion of the LamB signal sequence, or other suitable signal sequence, as the N-terminus of the protem.
The E. coli LamB has also been expressed in functional form in S. typhimurium (Harkki et al. (1987) Mol Gen Genet 209:607-611), Vl cholerae (Harkki et al. (1986) Microb Pathol 1:283-288), and K pneumonia (Wehmeier et al. (1989) Mol Gen Genet 215:529-536), 10 so that one could display a population of .antibodies in any of these species as a fusion to E.
coli LamB. Moreover, K pneumonia expresses a maltoporin similar to LamB which could also be used. In P. aeruginosa, the Dl protein (a homologue of LamB) can be used (Trias et al. (1988) Biochem Biophys Acta 938:493~96). Similarly, otber bacterial surface proteins, such as PAL, OmpA, OmpC, OmpF, PhoE, pilin, BtuB, FepA, FhuA, IutA, FecA and FhuE, 15 may be used in place of LamB as a portion of the display means in a bacterial cell.
iii) Bacterial Spores as Display Packages Bacterial spores also have desirable properties as display package candidates in the subject method. For example, spores are much more resistant than vegetative bæterial cells or phage to chemical amd physical agents, and hence permit the use of a great variety of 20 affinity selection conditions. Also, Bæill~ls spores neither ætively metabolize nor alter the proteins on their surface. However, spores have the l;~ that the molecular mech anisms that trigger sporulation are less well worked out than is the formation of M13 or the export of protein to the outer membrane of El coli, though such a limitation is not a serious detractant from their use in the present invention Bacteria of tbe genus Bacillus form endospores that are extremely resistant to damage by heat, radiation, ~ ei~r.Atinn, and toxic chemicals (reviewed by Losick et al. (1986) Ann ~ev Genet 20:625-669). This l,l . .. -. ~ is attributed to extensive l~ t ~ cross-linking of the coat proteins. In certain ~ of the subject method, such as those which include relatively harsh affinity separation steps, Bacillus spores can be the preferred 30 display package. Endospores from the genus Bacillus are more stable than are, for example, exospores from Sl ~tu..~ . Moreover, Bacillus subtilis forms spores in 4 to 6 hours, whereas S~ tullly.,i,s species may require days or weeks to sporulate. In addition, genetic knowlçdge and 'A ~ " is much more developed ffir B. subtilis than for other spore-forming bacteria.
Viable spores that differ only slightly from wild-type are produced m B. subtilis even if any one of four coat proteins is missing (Donovan et al. (1987) J Mol Biol 196:1-10).
.

WO95/15982 29 2 1 754~2 PCT/IJS94/14106 Moreover, plasmid DNA is commonly included in spores, and plasmid encoded proteins have been observed on the surfæe of Bæillus spores (Debro et al. (1986) J Bacteriol 165:258-268). Thus, it can be possible during sporulation to express a gene encoding a chimeric coat protem comprising an antibody of the variegated gene library, without inter-5 fering materially with spore formation.
To illustrate, several polypeptide .. -, .......... , l ~ of B. subtilis spore coat (Donovan et al.
(1987) J Mol Biol 196:1-10) have been .1. A- ~ ;~ ;i The sequences of two complete coat proteins and - t, .lll u~l fragments of two others have been d ~ ' Fusion of theantibody sequence to cotC or cotD fragments is likely to cause the antibody to appear on the 10 spore surface. The genes of each of these spore coat proteins are preferred as neither cotC or cotD are post-l I ., 1 l- l f ;. .., -~Iy modified (see Lader et al. U.S. Patent No. 5,223,409).
IV. SPIP~tir~ Antihn~lirc to a T~r~P:t Anti~en Upon expression, the variegated antibody display is subjected to affinity enrichment 15 m order to select for antibodies which bind preselected amtigens. The term "affinity separation" or "affinity ~l.fi.,l~ lL" includes, but is not limited to (1) affinity utilizing immnhili7in~ antigens, (2) illllUUU10~ i . ' ' using soluble amtigens, (3) nuul~,~.,.,ll~,~ ætivated cell sorLing, (4) AL~ . and (5) plaque lifts. In each ~ ,ho,l; ,. . ~ the library of display packages are ultimately separated based on the 20 ability of the associated antibody to bmd an epitope on the amtigen of interest. See, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. T~ .,.,.l;.~-'l Publication No. WO 92118619; the Dower et al. T~ ;m. ~I Publication No. WO 91/17271;
the Winter et al. T"~ .,.,.1;...-~l Publication WO 92/20791; the Marklamd et al. Tll~.l,AI;"..=I
Publication No. WO 92/15679; the Breitling et al. T~` .,.,.1;..-.-l Publication WO 93/01288;
25 the McCafferty et al. T.. '~., .1;,~.. 1 Publication No. WO 92/01047; the Garrard et al.
TntPrr ~tinn~l Publication No. WO 92/09690; and the Læner et al. T, ~ Publication No. WO 90/02809. In most preferred ... 14~.1;....... ~ the display library v~ill be pre-enriched for antibodies specific for the rare epitope by first contæting the display library with a source of the ba~ Julld epitope, such as the toleragen, in order to further remove amtibodies which 30 bind the background epitopes. ~ J, the display pækage is contacted with the target antigen and antibodies of the display which are able to specifically bind the antigen are isolated.
With respect to affinity cLl ,, ,~ y~ it v~ill be generally understood by those skilled in the art that a great number of .,h. ~ techniques can be adapted for use in the present mvention, r~mgmg from column ~,1".. ~.~L;.,~I~I~y to batch elution, and including ELISA amd biopanning techniques. Typically the target antigen is ;.. ----.1,;1;,. ~I on an 0 95/15982 ~ ¦ 7 5 4 8 PCT/US941141~16 insoluble carrier, such as sepharose or pOl~a~lylall~i~ beads, or, al~llaiiv~ly, the wells of a microtitre plate. As described below, in instances where no purified source of the target antigen is readily available, such as the case with many cell-specific markers, the cells on which the antigen is displayed may serve as the insoluble matrix carrier.
iAhe population of display pækages is applied to the affinity matrix under conditions compatible with the binding of the amtibody to a target antigen. The population is then I by washing with a solute that does not greatly effect specific binding of antibodies to the tArget antigen, but which substantially disrupts amy non-specific binding of the display package to the antigen or matrix. A certain degree of control can be exerted over the binding ~ of the antibodies recovered from the display library by adjusting the conditions of the binding incubation and subsequent washing. IAhe ~Il,u~,la~ ., pH, ionic strength, divalent cation rnnrrntrAAtiA n and the volume and duration of the washing can select for antibodies within a particular range of affu~ity and specificity. Selection based on slow ;.", rate, which is usually predictive of high affinity, is a very prætdcal ~oute. This may be done either by continued incubation in the presence of a saturating amount of free hapten (if available), or by increasing the volume, number, and length of the washes. In eæh case, the rebinding of dissociated antibody-display package is prevented, amd with increasing time, antibody-display packages of higher and higher affinity are recovered. Moreover, additional III,A"III~,A-;"ll~ of the binding and washing procedures may be applied to fund antibodies with special ,1- A~ The affinities of some antibodies are dependent on ionic strength or cation ~.." ,1.-~;..l, This is â useful ~ ~ for antibodies to be used in affnity purification of various proteins when gentle conditions for removing the protein from the antibody are required. Specific examples are antibodies which depend on Ca~ for binding ætivity and which released their haptens in the presence of EGTA. (see, Hopp et al.
(1988) Biotechnology 6:1204-1210). Such antibodies may be identified in the 1,~.. "1,;"--.l antibody library by a double screening technique isolating fust those that bind hapten in the presence of Ca~, and by ~ llly identifying those in this group that fail to bind in the presence of EGTA.
After "washing" to remove non-specifically bound display packages, when desired,30 specifically boumd display packages can be eluted by either specific desorption (using excess antigen) or non-specific desorption (using pH, polarity reducing agents, or chaotropic agents).
In preferred ~.. . ,I-o~l ;, . .. -t~, the elution protocol does not kill the organism used as the display package such that the enriched population ,Df display pækages can be further amplified by r~,u.ud~l~,Lul~. The list of potential elumts includes salts (such as those in which one of the counter ions is Na+, NH4+, Rb+, S042-, H2PO4-, citrate, K+, Li+, Cs+, HSO4-, C032-, Ca2+, Sr2+, Cl-, po42-, HCO3-, Mg2+, Ba2+, Br, HPo42-, or æetate), acid, heat, and, when available, soluble forms of the target antigen (or analogs thereof). Because bæteria WO 9511S982 3~ 2 ~ 7 5 4 8 2 PCT/US94/14106 continue to metabolize during the affinity separation step and are generally more susceptible to damage by harsh conditions, the choice of buffer ...,.,1,...,~..,l~ (especially eluates) can be more restricted when the display package is a bacteria rather than for phage or spores.
Neutral solutes, such as ethanol, acetone, ether, or urea, are examples of other agents useful 5 for eluting the bound display packages.
In preferred rllll~u~l;l.. ~ affinity enriched display packages are iteratively amplified and subjected to further rounds of affinity separation until enrichment of the desired binding activity is detected. In certain n~ O~ , the specifically bound display packages, especially bæterial cells, need not be eluted per se, but rather, the matrix bound display 10 packages can be used directly to inoculate a suitable growth media for Al~
Where the display package is a phage particle, the fusion protein generated with the coat protein can interfere substantially with the subsequent A ~ of eluted phageparticles, ~uliculAuly in nll~llOIl;,l,. .,1~ wherein the cpIII protein is used as the display anchor.
Even though present in only one of the 5-6 tail fibers, some antibody constructs because of 15 their size andlor sequence, may cause severe defects in the infectivity of their carrier phage.
This causes a loss of phage from the population during reinfection and A",l,l;~.~;"., following each cycle of panning. In one . ..,I.o,l;" ~, the antibody can be derived on the surface of the display package so as to be susceptible to proteolytic cleavage which severs the covalent linkage of at least the antigen binding sites of the displayed antibody from the 20 remaining package. For instance, where the cplII coat protein of M13 is employed, such a strategy can be used to obtain infectious phage by treatment with an enzyme which cleaves between the antibody portion and cplll portion of a tail fiber fusion protein (e.g. such as the use of an cllt~,lvhill~c cleavage recognition sequence).
To further minimize problems associated with defective infectivity, DNA prepared25 from the eluted phage can be 1l. r~ i into host cells by Cli,~,llu~ ;ull or well known chemical means. The cells are cultivated for a period of time sufficient for marker expression, and selection is applied as typically done for DNA ,. .,~r"""-~;l." The colonies are amplified, and phage harvested for a subsequent round(s) of panning.
After isolation of display packages which encode antibodies having a desired binding 30 specificity for the il uU~lUlC~ " epitope, the nucleic acid encoding the V-genes for each of the purified display packages can be recloned in a suitable euharyotic or ~luh~Au~uLiC
expression vector and transfected into an appropriate host for production of large amounts of protein. Where, for example, the isolated V-gene lacks a portion of a constmt region and it is desirable that the missing portion be provided, simple molecular cloning techniques can be 35 used to add back the missing portions. The binding affinity of the antibody can be confirmed by weU known ;,- Illlll~ A,y techniques with the target epitope (see, for example, Harlow WO 9~115982 '2. l 7 5 4 8 ~ 32 PCTIUS94/14106 and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)).
V. F~lrfh-~r ~ ' (m Df~nfiht~ c ,~nfih~7~y CU~ arl~1 T~ "v Kifc Another aspect of the present invention concems chimeric antibodies, e.g., altered antibodies in which at least the antigen bulding portion of an; 1 ...., I .r~gl~ l ;1 l isolated by the method described above is cloned into another protein, preferably another antibody. Among C~ O1' of chimeric antibodies ~ by the present invention, further " of the subject antibodies can be used to complete tbe portion of the constant 10 region isolated from the V-gene library, as well as to facilitate "class switching" whereby all or a portion of the constant region of the alltibody isolated from the V-gene library is replaced with a different constant region, e.g., with the constant region(s) from a different IgG, such as IgGI, IgG2 or IgG3, or the constant region(s) from one of IgE, IgA, IgD or IgM. In similar fashion, single chain antibodies and other lc~....,l.;., ~ fragments can be generated from the 15 clonedgenes.
When antibodies produced in non-human subjects are used ~ ,.11y in humans, they are recognized to varying degrees as foreign and an immune response may be generated in the patient. Accordingly"c ' ' ~ - ;-... of the isolated amtibody gene, wherederived from a non-human V-gene library such as described in the Examples below, can be 20 used to "humanize" the antibody. The term "' ' antibody" is used to describe a molecule having an antigen binding site derived from an ~ from a non-human species, the remaining ,,' ~ ' derived portions of the molecule, as necessary tosubstantially reduce the ~ of the molecule in human subjects, being derived from a human i" - gl--l...l;., In a humanized antibody, the amtigen binding site may 25 include, for example, either complete variable domains fused to constant domains, or only the CDRs grafted to the appropriate framew~rk regions in hurnan variable domains. Such antibodies are the equivalents of the . ~ ....1... .1 antibodies described above, but may be less O when r ' .,d to humans, and therefore more likely to be tolerated upon injected in a patient.
In an illustrative Cl~ '- t, any of the H3-3, FB3-2 or F4-7 antibodies described in the Examples below can be prepared to include human constant regions for each of the heavy and light chains of these mouse-derived genes. For example, the portion of the antibody gene encoding the murine constimt region can be substituted with a gene encodmg a human constant region (see Robinson et al., T~ Patent Publication PCT/US8610226g;
Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; ~orrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) PNAS 84:3439-3443; Liu et al. (1987) J. ImmunoL 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst. 80:1553-1559).
The subject amtibodies can also be "llulll~li~i" by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions.
General reviews of "ll~lolli~d" chimeric antibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207; and by Oi et al. (1986) BioTec~zniyues 4:214. Those methods 10 include isolating, ", :~ ;"~ and expressing the nucleic acid sequences that encode all or part of an i~ ... gl~ variable region from at least one of a heavy or light chain.
Sources o~ such nucleic acids are well known to those skilled in the art. The cDNA encoding the chimeric amtibody, or fragment thereof, can then be cloned into an appropriate expression vector. Suitable "llulllalli~d" antibodies can be ' ~Iy produced by CDR Iciuld,~(see U.S. Patent 5,225,539 to Winter; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988)5cience239:1534;andBeidleretal.(1988)~1mmunol. 141:4053-4060).
The DNA sequence encoding the chimeric variable domain may be prepared by (~1,~." .... ,. 1..,1 ;~ir synthesis. This requires that at least the framework region sequence of the first antibody and at least the CDRs sequences of the subject antibody are known or can be 20 readily ~ trnnint d r. ~ these sequences, the synthesis of the DNA from nlit~.. .. It ~ and the preparation of suitdble vectors each involve the use of known teclmiques which can readily be carried out by a person skilled in the art in light of the teaching given herein.
Alternatively, the DNA sequence encoding the altered variable domain may be 25 prepared by primer directed ~ it site-directed ~ This technique in essence involves hybridizing an ol;~ ' ' codirlg for a desired mutation with a single strand of DNA containing the mutation and using the single strand as a template for extension of the ni ;c, .. . 1. ,,1;,1~ to produce a strand containing the mutation. This technique, in various forms, is described by: Zoller et al. (1982) Nuc Acids Res 10:6487-6500; Norriset al. (1983) Nuc Acids Res 11:5103-5112; Zoller et al. (1984) DNA 3:479-488; and Kramer et al. (1982) Nuc Acids Res 10:6475-6485. For various reasons, this technique in its simplest form does not always produce a high frequency of mutation. However, an improved technique for irltroducing both single and multiple mutations in an M13 based vector has been described by Carter et al. (1985) NucAcids Res l3:4431-4443. Using a long t li~. ", - It ~ lr, it has proved possible to introduce mamy changes ' '~/ (e.g., see Carter et al., supra) and thus single oli~u,~ v~id~s, each encoding a CDR, can be used to introduce the three CDRs from the subject antibody into the framework regions of a humam antibody (see also U.S. Patent wo 95/15982 ~ ~ 7 5 4 82 34 PCTIU594/14106 5,345,847 to Liu et al.). Not only is this technique less laborious than total gene synthesis, but it represents a particularly cor~veniem~ way of e~pressirlg a vanable domain of required specificity, as it can be simpler than tailoring an entire VH domain for insertion into an expression plasmid.
The ~ , u~ used for site-directed ~ may be prepared by ol;~ 1 ;rlr synthesis or may be isolated from DNA coding for the variable domain of the subject antibody by use of suitable restriction enzymes. Such long oli~nr lrlrnfirlre will generally be at least 30 residues long and n1ay be up to or over 80 residues in length.
In yet another ~..,1.~.11".~.,.l PCR techniques for generating fusion proteins can be 10 used to generate the chimeric antibody. PCR r~mrlifirr~rinn of gene fragments, both CDR and FR regions, can be carried out usmg anchor primers which give rise to . ....L,I~ .,,...l_ y overhangs between two consecutive CDR and FR fragments which can ~ lly be annealed to generate a chimeric V-gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
The antigen binding sites of the subject antibodies can also be used to generate a fusion protein which includes protein sequences from non-.~ .l,..l;.. molecules. For example, such chimeric antibodies cam include: proteins domains which render the protein cytotoxic or cytostatic, such as the addition of P. ' exotoxin or Diphtheria toxin domains (see, for example, Jung et al. (1994) Proteins 19:35-47; Seetharam et al. (1991) J
Biol Chem 266:17376-17381, and Nichols et al. (1993) JBiol Chem 268:5302-5308); DNA-binding poly,u~,u~idc~ for facilitating DNA transport (see, for example, U.S. patent 5,166,320); catalytic domains which provide am enzymatic activity associated with the ~ ~' ' ' , such as a ~ or peroxidase activity; and purification ,uuly~ id~
to simplify r~rifir:l1inn of the antibody, such as a glutathione-S-transferase poly,u~,~u~iJe for purification of the antibody with a ~lu~r~Ql;u~ derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)), or a pOly-(His)/~,..~lul.h~, cleavage site sequence to permit purification of the poly(His)-antibody by affinity ~ , ' y using a Ni2+ metal resin (e.g., see Hochuli et al.(l987)J. Chromatography411:177;andJanknechtetal.PNAS88:8972).
The present invention also makes available isolated forms of the subject antibodies which are isolated from, or otherwise ~ lly free of other ceUular and . .l.~. . ll.~l_.
proteins, especially antigenic proteins, or other . ,l.... rl'..l~. factors, v~ith which the antibodies normally bind. The term "substantially free of other cellular or r.~r.,,lllllqr proteins" (also referred to hereirl as " ,, proteirls") or " ' "!' pure or 35 purified ~ ,u~a~iul~" are defined as ~ "Ual.l~iUII:~ of the subject antibodies having less than 20% (by dry weight), g protein, and preferably having less than WO 9S/15982 2 1 7 5 4 8 2 PCTNSg4/14106 ~S% ~ Ig protein. Functional forms of the subject antibodies can be prepared, for the first time, as purified ul~lJalaLiull~ by using a cloned gene as described herein. By "purified", it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological Illa~
5 such as other proteins. The term "purified" as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological ~ o~ of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term "pure" as used herein preferably has the same numerical limits as 10 "purified" ' l~ above. "Isolated" and "purified" do not encompass either natural materials in their native state o} natural materials that have been separated into (e.g., in an acrylamide gel) but not obtained either as pure (e.g. Iacking .I.~
proteins, or ~LI~ reagents such as denaturing agents and polymers, e.g.
acrylamide or agarose) substances or solutions.
Yet another aspect of the present invention concerns ~ of the subject antibodies, ualli~,..l_ly 13 ~ - 1l l =~ 11.- Al ~JIqJala~iullS~ The antibodies of the present invention, or 1~ 1Y acceptable salts thereof, may be ~,ull~ ,l..ly formulated for Alllll' 1 ~11..1;1~.~ with a 777;ulogi~ally acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid pol~,;hyl~ , glycol and the like) or suitable 20 mixtures thereo The optimum c. of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicma7i chemists, and may depend on such as factors as intended route of ' age and body weight of patient. As used herein, "biologically æceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of 25 71, ,:~ ;.. of the ~ l preparation. The use of such media for ly active substances is known in the art. Except insofar as any 7,u1~7.,.~Liullàl media or agent is ;~ '''''I l;1,1~ with the activity of the antibody, e.g., its specificity and/or affinity, its use in the ~ ' ' preparation of the invention is ~ I Suitable vehicles and their r ~ inclusive of other proteins are described, for example, in the 30 book Re~nington's Pllu, ' Sciences (Remington's r~ Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit fonm71,AtiAnr". Basedontheabove,such1~lIA.",,,~..;.Al r ' include,athoughnot exclusively, solutions or freeze~7ried powders of the antibody in association with one or more IIII- II~ ; lly acceptable vehicles or diluents, and cont~ined in buffered media at a 35 suitable pH and isosmotic with ,ull~A;ùlù~i~,al fluids. For illustrative purposes on y and without being limited by the same, possible .~ ;-...s or 1`."", ,1 ;""~ which may be prepared in the form of solutions for the treatment of proliferative disorders with an anti-cancer cell antibody of the present invention are given in U.S. Patent No. 5,218,094. In the ~l 754~

case of freeæ-dried plctJ~ual;ul~, suppûrting excipients such as, but nût exclusively, mannitol or glycine may be used amd appropriate buffered solutions of the desired volume will be provided so as to obtr,in adequate isotorlic buffered solutions of the desired pH. Similar solutions may also be used for the ~ c of t_e antibodies in isotonic 5 solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable cr,nr~ntr:~tinne so as to obtain at all times isotonic ~ plc,uo~ iUIla of the desired pH, (for example, neutral pH).
Still another aspect of the present invention concerns assay kits that can be used for detecting an ;",1l "~ epitope(s) in a sample, for example. The assay kits generally 10 provide an antibody for the ill~ ullu~ a.-~., epitope, derivatized with a label group that can be ulhmately detected, as for example, by ~,~,L~ h ;- techniques (including FACS) or r~lir,pr~rhir techniques. For instance, the label can be any one of a number of ,,.,1ir,,~ u~ fluorescent cu~, ' enzymes, amd enzyme co-factors. To illustrate, the label g}oup can be a functional grûup selected from the group consisting of horseradish 15 peroxidase, al6aline 1~ ,al-- ~ ., luciferase, urease, fluorescein and analogs thereof, rhodar~ine and analogs thereof, allulullY~,o.,y, R~~ u~l~LLl;ll~ erythrosin, europiarn, luminol, luciferin, coumarin analogs, 125I, 13 II, 3H, 35S, 14C and 32p Assay kits provided according to ~the invention rnay include a selection of several different types ûf the subject antibodies. The antibodies may be in solution or in Iyophilized 20 form. In some clllb~ ' the antibodies may come pre-attached to a solid support, or they may be applied to the surface of the solid support when the kit is used. The labeling means may come pre-associated with the antibody, or may require ~ with one or more e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, priorto use. Many types of detectable labels are available and could make up one or more 25 ~ of a kit. Various detectable labels are known in the art, amd it is generally recogrlized that a suitable label group is one w_ich emits a detectable signal. Various label groups can be used, depending on the type of .r conducted. Useful labels includethose which are fluorescent, radioactive, . ' , ' ~ .lt, rh~-nni! , ,.. ~,:,,1 "";"~ ~,, .. 1 and free radical. Also, the label groups may include ~ulyl,~lid~ (e.g., enzymes or proteins), 30 polymers, pol~ - -- .1.... ;.1. ~ receptors, cofactors, and enzyme inhibitors. Kits of the invention may also include additional reagent. The additional reagent can include blocking reagents for reducmg nonspecific binding to the solid p~se surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of microtiter plates, I~ ,lua,ull."e ~, ûr the lik~, composed of pûlyvinyl chloride, pOlyalylcllc, or the like materials suitable for 35 ;,.,."i,l,:l;,;..~ proteins.

wo sS/I5982 37 2 1 7 ~ 4 8 2 PCT/IJS94,l4l06 Vl. F.Y~ ,nl~,v ~nlir~tilme of thl Subject ~rthml ant1 Antih~ irc DeriYed Th~ rewith The subject method of the present invention can be applied ~ ,vu:~ly to the production of antibodies useful in Au ;ri~ diagnostic, and therapeutic ,.~ In contrast to even the antibody display libraries which can be derived from immunized animals, 5 the antibody libraries which can be generated by the subject method provide a greater population of high affinity antibodies to the ;~lllllullvlc~,.,.~;ve epitope of interest, as well as establish a broader pool of display packages comprising antibodies specific for the hl~lll~.Jlc~ ;ve epitope. With respect to the illllllu..vlc~ ;ve epitope, the more effective access of the antibody repertoire provided by the display libraries of the present invention 10 allows more efficient enrichment to occur by, for exarnple, affinity selection means.
As described above, Cl,i,....l~, epitopes can be defined in terms of the toleragen and " used in the subtractive i ", ....; ,..l ;l .., step, and are therefore unique to the i", .n~.. with respect to the toleragen. Thus, where the desired antibody is to distinguish between various cells of common or similar origin or phenotype, the cell to be 15 specifically boumd by an amtibody of the present invention is used as an ;~ c . while the related cells from which it is to be ll;~l;..~;- ;~l. ~l are employed as the toleragen. Table I
provides exemplary systems of ,.., . ~c,.../toleragen sets which can be employed in the subject method to isolate antibodies which specifically l '. )~ unique to the ;",.. ,.... ,n~ ., The choice of toleragen and ;,.. ~,.. can provide antibodies specific to, for 20 example, tumor cell markers, fetal cell markers, and stem cell markers.
Likewise, the subject method can be used to generate antibodies which can .l:.. . ;",;, .t. between a variant form of a protein and other related forms of the protem by employing an ;........... ~,........... comprismg a variant protein, such as a mutant form of a protein or a particular isoform of a family of proteins, and a toleragen comprising the wild-type protein 25 or alternate isoforms of the variant protein. The difference in ~ (i.e. the cc.,~ivc epitopes) between the variant protein amd wild-type (or other isoforms) will typically consist of only a few differences in amino acid residues ~I.e. Iess than 15%, but preferably on the order of only one to three residues difference). For example, such ,,... 1.,.. ~:;.,,.~ of i.. ,-- -,.. ~, .. ~ and toleragens can be used in the present invention to derive 30 antibodies which can specifically bind variant forms of UIICU,UIU~ or tumor suppressor proteins, as well as of l. -..h~ , CLIJUlilJU~ ' E, LDL receptor, cardiac ,~-myosin, sodium or other ion channels, collagen, ~ , or il(lll~ iVll.

WO95/15982 ~ ~ 7 PCTII~S94/14106 -Table I
Targ~t Antigen Toleragen Immunogen Fetal nucleated red blood cells maternal erythroid cells fetal erythroid cells colon cancer rlormal colon cells colon carcjnoma cells ~e.g. epitheleal) ApoE4 ApoE ApoE4 Stem or embryonic nerve cell differentiated nerve cell embryonic nerve cdl cellcolrlmiKed stem cell I , stem cell meusutic tumor marker non-mesutic transformed cell metsutic transtormed ce p53 muunt wild-type p53 mutant p53 In an exemplary all,l.v.l;.,..,l of the present invention, the subject method isemployed to generate antibodies for a cell-type specific marker. As Example 2 illustrates, the S present method can be employed to produce amtibodies directed specifically to fetal cell-specific markers. For example, specific antibodies for markers of fetal nucleated red blood cells can be generated by the subject method employing maternal erythroid cells as a toleragen and fetal erythroid cells as an ~, When generated to distmguish between fetal cells and maternal cells, as by the specific recogtution of epitopes on cell surfæe 10 antigens such as of I ~ precursor cells, antibodies generated by the subject method can be used to separate fetal cells from maternal blood by, for instance, Il."....~
activated cell sorting (FACS). The isolated fetal cells, such as fetal nucleated ~lya~lv~,yl~s, represent a non-invasive source of fetal DNA for prenatal genetic screening and offf~r a powerful and safe alternative to more invasive procedures than, for example, :.
15 or chronic villus sampling.
In another ..,I.o.l;..l. ,l, the present invention I ' the generation of antibodies specific for a tumor cell-specific marker. As described in Example 1, the subject method can be employed a lva~ v.~,,ly to generate antibodies which are able to di~ between normal cells and their ~ r"~ 1 p~ . Such antibodies may 20 be suitable for both diagnostic and therapeutic uses. For example, antibodies can be selected in the present assay which detect cell-specific markers found on neoplastic or lly~ ic cells. Antibodies so obtained can be used to identify l" r~ cells and thereby used to diagnose cancers and tumors such as ad.,.lo~,a..,ihlu.lld~, papillomas, squamous and transitional cell carcinomas, anaplastic carcinomas, carcinoid tumors, "..~.,ll.~li..."~, 25 hepatomas, mPl~nnm~e and germ cell tumors. These antibodies mays also be used to selectively destroy l.. r.""" l cells, both in vivo and in vilro, such as through the " y activation of ~ at the cell surface of a l "" .~l; .., . ,r.l cell bound by the antibody, or by delivery of toxins, or by delivery of nucleic acid constructs for gene therapy.
For example, antibodies specific for colon cancer markers can be generated in the present invention by suing normal colon cells as a toleragen and cells derived from a colon S carcinoma as an ;"",....,n~,..., In similar fashion, the subject method can be engaged to produce antibodies that specifically inhibit metastasis of highly metastatic tumor cells. Such antibodies, designed to recognize unique epitopes on highly metastatic variants of tumor cells (i.e. whose expression is elevated relative to non-metastatic variants), can be used to interfere with the function of cell surface proteins containing these epitopes in the metastatic cascade.
In similar fashion, where specific antibodies for stem or embryonic nerve cell markers are desired, the immlm~fl~l~n~e-derived antibody repertoires used in the subject method can be generated using a diITt~ nerve cell as a toleragen and an embryonic nerve cell, such as a neural crest cell or ll ".. ~ progenitor cell, as an ~, For l~ cell specific antibodies, the ;l.. ~.. ~.. can comprise a 15 1.. ., ~ stem cell, and the toleragen can be a committed stem cell.
In yet a further r"ll~ the subject method can be applied to the generation of antibodies which can discern between variant proteins. Such antibodies can be used to distinguish various naturally oCcmring isoforms of a protein, as well as to detect mutations which may have arisen in a protein. In an illustrative ~ J~ I, antibodies can be20 produced by the present invention which can be used in i,., ~ h ~ 1 assays for detecting cell l~ . ~ ., ... ~ ;.. ~ arising due to mutation of an oncogene or anti-oncogene. For instance, the subject method can be used to generate antibodies which ~" between wild-typeras and a mutant form of ras. For example, useful antibodies for detecting ras-indueed 1,,.. ~.. -:;.. of a cell ean be generated by the subject method using a Ser-17~Asn variant of ras as arl ... - - ~,.. and wild-type ras as a toleragen Likewise, l; ~ .~ ly useful antibodies can be produced by the present invention which ~,~,ir.~,ally bind and ~' between wild-type and variant tumor suppressor protems. ~or example, i~ dil.g mutations of either the p53 or Rb tumor ~ can lead to escape from cell senescence and lead to ~ r~ ;..., The subject method can be 30 used to generate antibodies specific for a variant p53, the ability to distinguish between the wild-type and mutant forms arising through recoglution of a unique epitope created by mutation, such as Arg-273i'Cys, Tyr-163~Asn, Val-157~Phe, or Cys-238~Phe.
Appropriate ;~ uh~ ll sets would therefore inelude p53 mutants and wild-type p53.
The subjeet method can also be used to produce antibodies for detecting variant 1.. ,.. ~1.~1~,;., moleeules, and whieh ~ ly can be employed as diagnostic tools for ~ ~ 7 5 4 ~ ~ Pcr/US94/l4m6 detecting hPmo~lnl,;,..)ua~ sl such as sickle cell anemia and ~-thol~eePmi~ A large number of such ~ , most resulting from single-point mutations, have been observed as abnormal hPmnglnhine of embryonic, fetal, neonatal, and âdult disorders (see, for review, Huisman (1993) i3aillieres Clin ~aemato~ 6:1-30). Therefore, antibodies to unique epitopes 5 of l .~lhgl.~ll;l. variants can be of great use in detecting amd ~ rltit,~tin~ botb normal and abnormal hPmnnlnhin levels. .
Where the ~" is ~olipu~uli~l E4 (ApoE4) amd the toleragen comprises other ApoE isoforms, specific antibodies can be isolated by the subject metnod which can be used to measure ApoE4 levels in plasma or serum of a patient The presence of the ApoE4 10 variant has been linked to increased ~ y to Alzheimer's disease (Strittmatter et al.
(1993) PN~S 90:8098-8102) as well as significamt impact on variation of cholesterol lipid and lipoprotein levels in individuals (Rall et al. (1992) ~ Intern. Med 231:653-659; amd Weisgraber et al. (1990) J. Lipid Res. 31:1503-1511). In similar fashion, specific antibodies to other ApoE isoforms can be generated, including antibodies which can specifically bind 15 ApoE20rApoE5.
Other exemplary ~ o~ include the generation of specific antibodies for LDL
receptor variants which can be useful, for example, in predicting risk of diagnosing familial llyy~ Gl~ lul~luia~ specific amtibodies to cardiac ,~-myosin variants, wbich can be used to diagnose l~ v,ulu~ udiu~ll,yu~ ,y, specific antibodies to variant forms of sodium or ion 20 channels, such as which arise in congenital l.~ .lc periodic paralysis, antibodies to collagen variants, such as Cys-579 collagen, which can be imdicative of a ,u.edi,uoai~ factor in risk of familial ~ h~ , specific antibodies to a variant of yl; ,1;"~ e~, such as which arise in non-insulin-dependent diabetes mellitus, and antibodies specific for a mutant of IL<U~ such as which might arise in f.amilial amyloidotic pol,vl.~u.u,u~

VII. Antihnflipc ,CpPPifiP~lly Rp:lrtive With FPf~l ~PIl/(~"'`^Pr (`Pll F.~7it(1pPC
As described in detail below, the subject method has been applied ~lv ~ '~/ to the d~ ,lu~ lll of antibodies for cell-surface markers of fetal cells and l ,~ r.. ,.. 1 cells. In contrast to the use of cull~llliullal hybridor~a methods, or even prc ' phage display 30 libraries, practice of the subject method cfm yield a library of antibodies which are amenahle to very rapid ' This invention represents the first instance that antibodies specific for urlknownlurlisolated cell-surface antigens have been generated using a .
display library. Indeed, initial ~ ;nn using V-gene libraries derived from animals immunized with the c:~.,,,ive epitope, but not tolerized to 1 'c" u~.d epitopes (in 35 contrast to the present method), suggests that the subject antibodies are attainable only with wo 95/15982 4 1 2 1 7 5 ~ 8 2 PCT/US94/14106 great difficulty and expense, and perhaps not at all, by prior art i..""l,;,.~ l display techniques.
To illustrate, Figure SA reveals the rapid enrichment of specific antibodies from the ;," "l,,..l,,l..;,.d V-gene library. By c/~mr~ri~n Figure 5B ~l..,,.."~, .~ that phage 5 libraries prepared by prior art techniques (non-tolerized #1 and #2) do not show significant enrichment from one roumd of parming to the next (compare tolerized to non-tolerized #l and #2). Likewise, as set out in more detail below, despite several years of illV~,~Lio~Lillo l~b~;dulllds, even those generated usmg B-cells derived by i"".,.l.,~,l..l..;,~.;l", protocols, antibodies that l" between fetal and maternal blood cells with only the same . . r. " ", ~ as anti-CD71 antibodies were obtained.
The subject method, on the other hand, provides a library containing a rich source of high affinity antibodies which permit detection of specific antibodies by, for example, panning on live cells, FACS assays or cell based ELISA. To further illustrate, the appended examples describe that individual antibody display packages were enriched 5000 to

3,600,000 fold in only a single roumd of selection. DNA sequence amalyses of particular isolates depict a remarkable history of affinity maturation of both heavy and light chains, suggesting an ~m-~Yr-~t-~lly efficient access to the I O ' repertoire.
F~LI~.llllVlC, in addition to hastening antibody maturation, amd perhaps causing such enh_nced maturation, the instant method enables selection of amtibodies having both 20 .l;~. .;..,;" ~;- ~, specificity and high binding affinity for an c.,.,,,;vc epitope. Indeed, n ~nnrRri~--n of antibodies isolated by the subject method with antibodies available through the use of prior art techniques reveals that the cl~ ly-derived antibodies of the present invention tend to be orders of magnitude better with respect to each of specificity and affinity relative to antibodies available in the prior art.
The genes for three of the antibodies which ~l~ .l. ~,.~l .. '~ both desirable specificity and binding affinity have been sequenced. As described in Example 2, the F4-7 and H3-3 antibodies were originally isolated with a parming regimen including fetal liver cells. Further .;,,.;;nn of the H3-3 antibody confrimed that this antibody recognized fetal blood cells of early gestational age (e.g., <16 weeks), but also stained fetal cells of later gestational 30 ages, albeit less well. This probably reflects the use of fetal liver, which consists u~cdu~ ~lLly of the earliest blood cell precursors, for both ;.... ~ and ~ irhn~rnt However, it is ~'- -- ' below that the population of antibodies enriched from the library could bc biased to select antibodies specific for epitopes present on fetal blood cells of later gestational ages. One of the isolates, FB3-2, was .l.-, ~ l amd found to have an35 . ~ li, ;Iy low 1,...,~1~ ' staining level on adult blood cells (e.g., less than 0.1%). A
g~ude to the nucleic acid and amino acid sequences for eæh of these clones is provided in WO 95/15982 ~ ¦ 7 5 4 8 2 PCT/US94/14106 Table 2, and the overall structure of the variable region for each of the heavy and light chains are provided irl Figures 8A and 8B.
Table 2 P~ucleof ide and Amino Acid Sequences for Anti-~etal Antibodies Antibody H.C. nuclcotide H.C. a~Qino acid L.C. nucleotide L.C. amino acid Fs3-2 SEQ ID No. 50 SEQ ID No. 51 SEQ ID No. s2 SEQ ID No. 53 F4-7 SEQ ID No. 54 SEQ ID No. 55 SEQ ID No. 56 SEQ ID No. s7 H3-3 SEQ ID No. 58 SEQ ID~ No. 59 SEQ ID No. 60 SEQ ID No. 61 The antibodies isolated by the present method, derived from a V-gene library of ar ;, . ". l". ~l~ ,1. .; ~ .1 animal, are not apparently available by other prior art techniques and in fact displayed p r."",~ which greatly surpassed those obtained by previous 10 methods. Irl contrast to the antibodies achieved by the subject method, employing an identical ;. . l I . . " .- -If~ 1 step, but coupled instead with the use of hybridoma techniques, only a few antibodies which showed fetal cell selectivity were obtairled. The specificity for one of the best of these amtibodies, "anti-Em", is shown in Table 3. Fetal cell selective arltibodies isolated by other groups usirlg other hybridoma i ~ giP~ were also compared.
1~ As is understood in the art, anti-CD71 ant,ibodies are believed to be among the best of the fetal cell specific antibodies. However, as l ,..~ l in Table 3 (see also example 4), antibodies generated by the irlstant method perform with superior qualities relative to each of the arltibodies obtained by i" ~ (arlti-Em) and hybridoma (anti-CD71) techniques.
7'able 3 ~',-, .i.,,"~ofl.)~,''. ~rivedantibodieswitl:immunotoL,i~ rivedantibodies Antibody Amt Used Cell Type Stained % Positive Speciftcity Anti-Em 5.0 ,ug fetal liver 50.0% 2.5 fold 5.0 llg maternal PBMC 20.0%
H3-3 0.25 ,ug fetal liver 79.5% >125 fold F(ab')2 0.25 ~g maternal PBMC below b 'r k~lmrl FB3-2 0.25 ',lg fetal liver 85.4% 125 fold F(ab')2 0.025 ~Lg fetal liver 80.0%
0.25 ',lg materrlal PBMC 0.68%
anti-CD71 1.0 llg fetal liver 83.1% 7.7 fold (Beclcton- 1.0 !lg maternal PBMC 10.8%
Dickinson) WO 95/15982 43 2 1 7 5 4 8 2 PC~/US94~1410C
.
Each of the anti-Em and anti-CD71 antibodies are considered to be of excellent specificity with respect to anti-fetal cell antibodies deriYed by methods in the prior art. Yet, as Table 3 illustrates, the level background b*nding to maternal peripheral blood ",.",.." ~ l ~
5 cells (PBMC) is many times higher for these antibodies relative to the ~a~luuuld stairling of maternal cells using the subject antibodies. COI~C~ IILIY~ although the anti-Em, anti-CD71 antibodies and the like stain fetal cells very well, the* ba~ ;luulld stain*lg on maternal blood of greater than S percent provides subst~mtial room for ;IllplU.~,III~,..~ of antibodies useful for retriev*ng a very small population of fetal blood cells from maternal blood samples.
One estimate of fetal cell cnnr~ ntr~tinn~ in maternal blood provides I fetal cell *l 100,000 to I in one million (e.g., %0.001 to %0.0001) adult nucleated blood cells. The ~.. . r.,. ",~ , attributes on the antibodies derived by the subject method suggests that these antibodies are specific enough for use in purifying fetal cells from maternal samples. To further ~ . ` the improved L" r...,. ~ of antibodies isolated by the subject method, 15 relative to antibodies known in the art, 400 male fetal cells were spiked into I million maternal cells, and the mixture stained with nuvlcO~ .. conjugated H3-3 Fab. FACS sorting to recover stained cells, followed by in situ II,ybl;d;~ai;Ull to detect Y .1.,.,.,...~..,..~1 DNA, .1.. ,.. ~1,. '.. 1 a recovery of 75% of the male cells at alrnost 40% purity (300 male cells of 800 total cells recovered). The best results obtained for either of the anti-Em or anti-CD71 amtibodies described above âpproach the same percentage of recovery of fetal cells, but at orders of magnitude lower purity (e.g., a few humdred fetal cells amongst a ha~h~ of 100,000 maternal PBMC).
Another feature of the antibodies derived from the subject method, which feature also apparently exceeds the amtibodies of the prior art, pertains to the bind*ng affinity of these amtibodies for fetal cell-bound antigens. As described in Example 3, the affmity of the H3-3 and FB3-2 antibodies was determmed against human erythro-leukemic (HEL) cells ("HEL
scatchard assay"). In each instance, the association constant (Ka) exceeded 109. For instance, monomeric H3-3 and FB3-2 Fab' fragments displayed association constants of 6x1010M-I and 8x101M-I ~ . D*meric forms of the lc.,, ' antibodies had even greater binding affinities, with Kas of 5x10l2M-l for H3-3 and lx10l2M-1 for FB3-2 I C D~ l y .
As a result of the inventors' discovery, it is now possible to provide a .c,u.u~_;l,le and pr~dictable method for isolat*ng amtibodies ,.,. . -- l;vc with ;IIUIIUIIUIC~ ;VC
epitopes, which antibodies are .3~ - l by specificity and/or affinity for a CUllc.~pUIII~
35 antigen which exceed those presently attainable by either hybridoma or by phage display ,~,1. ~In~ Accord*ngly, in one aspect of the ~nvention, the subject method makes 48~
WO95/15982 11`15 44 PCT/US94/14106 available antibodies specific for illllll.lllJll,~ iVt: epitopes, in which antibodies are by association constants for the ill~ lVl~ ;V~: f pitopes which are greater than I o6M- I, preferably greater than I o8M- I, more preferably greater than about I o9M-I, and even more preferably greater than IOIM-I, IOIlM-l, or 1012M-I, e.g., Ka in the range of 1010M-1 to 1013M-I.
In another aspect of the invention, the 6ubject method ~c ~ ,..,.f.,; ~ ~ the isolation of antibodies which have a low level of b~L~y~ ' staining. The relative specifcity of these antibodies can be several fold, if not orders of maglutude, better than .,1 ' ' and hybridoma generated antibodies, particularly with respect to antibodies for cell surface 10 epitopes. For instance, the subject method can provide antibodies which have no substantial background binding to other related cel s, e.g., relative 'l~ greater than 10 fold binding to the target cells over b~ uulld binding to the related cells. As f'~
antibodies can be generated which do rlot substantially cross-react with other epitopes, preferably having specificities greater than 20 fold over background, more preferably 50, 75 or 100 fold over background, amd even more preferably more than 125 fold over l.d.,l~y.
For example, anti-fetal cell antibodies generated by the instant method" ~ f~ by the FB3-2 and H3-3 antibodies, were tested by n~.v.c~ activated cell sorting ("FACS
eff~ciency assay") and were each 1 ".~ to have relative ~ f ~ greater than 125 fold over background. In contrast, the anti-CD71 and amti-Fe antibodies were found to have 20 relative ~ . - of 7.7 and 2.5 fold over backgroumd, .~ ,lr. Furthermore, the specificity of feta, cell specific antibodies produced by the subject method cam a so be , 1 ,.. ~ .;,. .1 in terms of a bd~,L~yu~l~lJ stailung of maternal cells relative to antibodies of the prior art, such as arlti-CD71 antibodies. For instance, the subject antibodies preferably stain two times less non-fetal cells relative to an anti-CD71 antibody, more preferably at least five 25 times less, and even more preferably at least twenty times less than an anti-CD71 antibody.
Such ~ ".,c can be made using standard ;l ~ r~ " such as the FACS efficiency assay of Example 4. Exemplary anti-CD71 (e.g., anti-Transferrin receptor) antibodies include the 5E9 antibody (ATCC HB21), the L5.1 antibody (ATCC HB84) and the L01.1 antibody ~Beclcton DicLinson Catalog No. 347510).
With respect to the specific antibodies which have been sequenced, namely H3-3, F4-7 and FB3-2, it is ~ , J that, as described above, each antibody can be further engineered without departing from the purpose and intent of the present invention.
Accordingly, a chimeric FB3-2 antibody can be generated which includes the variable regions from the heavy chain (residues El-S121, SEQ ID No. 51) and light chain (residues Dl-K111, SEQ ID No. 53). Likewise, chimeric F4-7 antibodies cam be provided which include the heavy chain (residues El-S121, SEQ ID No 55) and light chain (residues Dl-KI 11, SEQ ID
No. 57) variable regions from the F4-7 antibody described below. F~lh~ ul~, chimeric H3-WO95/lS982 45 2~ 75~$2 PCT/I~S94/14106 3 antibodies are also ~" I ' i, as for example antibodies including the variable regions fromtheheavychain(residuesEI-S115,SEQlDNo.59)andlightchain(residuesDI-K111, SEQ ID No. 61 ) of the H3-3 antibody.
In similar fashion, chimeric antibodies cam be generated including heavy and light 5 chain variable regions, each represented by the general formula: FR(1)-CDR(I)-FR(2)-CDR(2)-FR(3)-CDR(3)-FR(4), wherein CDR(1), CDR(2) and CDR(3) represent y .l. . ."" "~ regions from the subject antibody, and FR(I), FR(2), FR(3) and FR(4) are framework regions from a second antibody. For example, chimeric FB3-2 amtibodies can be generated which include a heavy chain in which CDR(1) is SYWLE, 10 CDR(2) is EILFGSGSAHYNEKFKG and CDR(3) is GDYGNYGDYFDY, and a light chain in which CDR(I) is RASQSVSTSRYSYMH, CDR(2) is FASNLES and CDR(3) is HSWEIPYT. Likewise, a chimeric F4-7 antibody can be made mcluding a heavy chain in which CDR(1) is SSWLE, CDR(2) is EILFGSGSAHYNEKFKG and CDR(3) is GDYGNYGDYFDY, and a light chain in which CDR(1) is RVRQSVSTSSHSYMH, 15 CDR(2) is YASNLES and CDR(3) is ~i~W~ Yl. In similar fashion, chimeric H3-3 antibodies can be provided, which antibodies include a heavy chain having a CDR(I) of DYYMY, a CDR(2) of TISDDGTYTYYADSVKG and a CDR(3) of DPLYGS, and a light chain in which CDR(I) is RSSQSLVHSNGNTYLH, CDR(2) is KVSNRFS and CDR(3) is SQSTHVLT. In each mstance, the associated framework regions (FRI-FR4) can be derived 20 from an unrelated antibody, preferably a human antibody.
The present invention further pertains to methods of producing the subject .~.,,, hl,,_.,l antibodies. For example, a host cell transfected with nucleic acid vectors directing expression of nucleotide sequences encoding an antibody (or fragment) can be cultured under appropriate conditions to allow expression of the antibody to occur, and if 25 required, assembly of a heav,v/light chain dimer. The antibody may be secreted and isolated from a mixture of cells and medium containing the ~ , antibody. A cell culture includes host cells, media and other by-products. Suitable media for cell culture are well known in the art. The ~ antibody peptide can be isolated from cell culture medium, host cells, or both using techniques known in the a~t for purifymg antibodies, 30 including protein-A:sepharose and ion-exchange ~ y~ gel filtration ~,1." ~ itr-filtr:~ti ln and eh,~ ,;, In a preferred ...,I.o~l."~. ~1 the ,~....,.l..,. "l antibody is a fusion protein containing a domain which facilitates its ,.l.;l';.-:;..,.,suchasaGSTfusionproteinorapoly(His)fusionprotein, This invention also pertains to a host cell transfected to express a l~ . ", .l .;., . .l form of 35 the subject amtibody. The host cell may be any prok-ryotic or eukaryotic cell, and the choice can be based at least in part on the desirability of such post-translation ...---1;1~ as glycosylation. Thus, a nucleotide sequence derived from the cloning of an anti-fetal cell or WO 9S~15982 '2 l 7 5 4 8 ~ 46 PCI/US94114106 anti-oncogenic cell antibody by the subject method, encoding all or a selected portion of the variable region, can be used to produce a . ~ form of an antibody via microbial or eukaryotic cellular processes. Ligating the ~ 1 antibody gene into a gene construct, such as an expression vector, and 11 ~- - f ~ or i r ' _ into hosts, either eukaryotic 5 (yeast, avian, insect or ' ) or prokaryotic (bacterial cells), are standard procedures used in producmg otber well-known plotems, e.g., insulin, interferons, human grow~th hormone, IL-I, IL-2, as well as other l~c, ' antibodies. Similar procedures, or mnf~if / ,ltinne thereof, can be employed to prepare the subject amtibodies by microbial means or tissue-culture technology in accord with the subject invention.
Preferably, the cell line which is 1, -- r~""" 1 to produce the 1~ ,-- / amtibody is an i~ullvl41i~,d ,..,~ -, cell line, which is ad~ ,vu~ly of Iymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line. The cell line may also include a normal Iymphoid cell, such as a B-cell, which has been L~ by ~ r " ,. :,~,.. with a virus, such as the Epstein-Barr virus. Most prefelably, the illllllu~ l cell line is a myeloma cell 15 line or a derivative thereof.
The ~ antibody gene cam be produced by ligating nucleic acid encoding the subject antibody protein, or the heavy amd light cbains thereof, into vectors suitable for expression in either prokaryotic cells, eukaryotic cells, or botb. Expression vectors for production of 1~< . ,.~ - l forms of the subject antibody include plasmids and other vectors.
20 For instance, suitable vectors for the expression of an antibody include plasmids of the types:
pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in ~ k~uy~Lic cells, such as E. coli.
A number of vectors exist for the expression of 1.,~,. L' ' proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, 1~YES2, and YRP17 are cloning amd expression 25 vehicles useful in the hllludu~,lioll of genetic constructs into S. cerevisiae (see, for example, Broach et aL (1983) in E~ ' ; ' of Gene Expression, ed. M. Inouye Academic Press, p. 83, iu~lp~ ,l by reference herein). These vectors can replicate in ~.
coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication ,' of the yeast 2 micron plasmid. In addition, drug resistance markers such as 30 ampicillin can be used. In an illustrative r~ 1, an amtibody is produced Ir~ ly utilizing an expression vector generated by sub-cloning tbe coding sequences of the variable regions for each of the heavy and light chain genes of the H3-3 or FB3-2 antibodies.
The preferred ' expression vectors contain both prokaryotic sequences to facilitate the IJlupd~ali~ll of the vector in bacteria, and one or more eukaryotic 35 units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived WO 95/15982 47 2 ~ ~7 5 4 8 2 PCI/IIS94/14106 ectors are examples of " ,," "", ~ " expression vectors suitable for ~ of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both plul~yuLi~ amd eukaryotic cells. Alternatively, derivatives of viruses such as the bovme papillomavirus 5 (BPV-I), or Epstein-Barr virus (pHEBo, pREP-derived and p205) cam be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and ~ r " " ~ -- . of host organisms are well known in the art. For other suitable expression systems for both p -uhLu~uLi-, and eukaryotic cells, as well as general .,.. 1.;.,-- ~ procedures, see Molecular CloningA LaboratoryManual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17. In some instances, it may be desirable to express the .~ ~ . ." ,l .;. . ' antibody by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derivedvectors (such as pAcUWI), a~d pBlueBac-derived vectors (such as the l~-gal containing 15 pBlueBac III).
VIII. E~- "~l;ri- ~
The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of 20 certain aspects and n...hn.l ,....1~ of the present invention, and are not intended to limit the invention.
As described below, the subject method has been applied al~ L~,_uu~ly to the ~ ~ ~,Iu~ of antibodies for cell-surface markers of fetal and 1, rl ~ cells. In contrast to the use of ~UII~ '' 1 hybridoma methods, or even prc-:~ m~ni7f ~I phage display 25 libraries, the present invention can yield a remarkable library of antibodies which are amenable to very rapid ~nnrhn~nt In an exemplary . ~ ~ ' described in the Examples below, individual antibody display packages were enriched 5000 to 3,600,000 fold in only a single round of selection. DNA sequence amalyses of particular isolates gave a remarkable history of affinity maturation of both heavy and light chains, suggesting an ~ y30 efficient access to the I " ' repertoire.
1. M:-tPri~ls ~n~ lvlrthn~le Except where indicated otherwise, 1~ ~1-;.---.l DNA methods and lu~,lub;olu~;~,al techniques were carried out as described by Sambrooh, J. et al., Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

wo 95/15982 ~ 1 7 ~ 4 8~ 48 PCT/US94/14106 The materials amd methods described below were used to generate the antibody display librafies described in botb Example I and Example 2.
Bh~
DNA modifying enzymes were obtained from New England Biolabs ~Beverly, MA) 5 and used under conditions ~.. 1f ~I by the suppliers. Taq polymerase was obtained from Perkin Elme} (Norwalk, CT). A set of DNA fragments (l Kb ladder) obtained from Life Te~ rni~ , MD) was used as a standard for molecular weight of DNA
fragments by agarose gel el~,~.LIu~llull,a;~. DNA primers were custom synthesized by Genosys, Inc. ~fThe Woodlands, TX) or Cruachem, Inc. (Sterling, VA). D~v~ lvalll~ 5'[fCL
10 -(35S)thio]t, ~ was purchased from New England Nuclear (Boston, MA~. Polyclonal b;uLil~ anti-M13 antibody was obtained from 5 prime-3 prime (Boulder, CO).
Streptavidin-Alkaline ~ and Polyclonal goat anti-mouse kappa-alkaline ,n~ were from Fisher Biotech (Pittsburgh, PA).
~rrtPr;r~ Strf~ir~.~ an~l r~7/~t~/re ~. coli strains XL-I, SolR, and LE392 were obtained from Stratagene ~LaJolla, CA).
Lambda phage resistant XL-l was isolated ~vy standard methods and is described in this work.
~ coli was grown to stationary phase at 3al or 37C with shaking in Erlenmeyer flasks filled to one-tenth their nominal capacity with LB, SOB, 2X YT, NZY medium (Sambrook, 1989) or TB medium:0.1 M KH2PO4 buffer, buffer, pH 7.5 containing 12 g bacto-tryptone, 24 g 20 yeast extract, and 5.04 g glycerol per lite} (lphosphate buffer was autoclaved separately). For growth of bacteria on solid media, agar ~Difco, Detroit Ml) was added to a final .. 1 . Al 1 of 2% (wt/vol.). Glucose supplement was to 0.5% (~ bfnir.illin ~ and kanamycin were added when necessary to 50, 30, and 50 ug/ml, Il,a~
Tf~ectf~rsnn~b/ /~ ~c The E~ coli cloning vector, lambda SurfZapTM and helper phages ExAssistTM and VCS
M13 were obtained from Stratagene.
~~r""~.",,;, Whole blood from non-pregnant individuals was obtained from Interstate Blood Products ~fTennessee). Adult peripheral blood ' cells ("PBMC") were prepared by standard Ficoll-Hypaque gradient techniques. Fetal blood ~~~-~ ' cells were prepared from fetsl liver obtained from aborLuses at 12-20 weeks gestation, at which age the liver is the principal 1 r ' ~ organ. Cells were freed from ~ comnective tissue by passage through sterile Illiwva~ a in the presence of sterile Ca-Mg-free PBS.
The resulting cell suspension was diluted up to 20 ml m PBS and the blood .,,...... , 1 - cell wo 95115982 49 2 1 7 5 4 8 2 Pf-T/US94/14106 fraction obtained by standard Ficoll-Hypaque gradient ~ After recoYery from the Ficoll interface, both adult and fetal cells were w_shed twice in sterile Ca-Mg-free PBS
the ~ J~d in the PBS at 2xl 07 cells per ml.
~Aolerance ~. ,-,/;~,..~
S The use of c.y~ lr tolerance with intact, fixed, cells is a well known technique in the art. IAhe present procedure employed unfixed cells ~' '~/ afterisolation from whole blood, bone marrow or fetal liver, avoiding the issue of alteration of antigens by chemical amd/or physical processing of the cells. C~ was obtained from Sigma chemical and ~ 1 at 10 mg/ml sterile saline. The toleri7ation procedure used was essentially that of Matthew et al. ( 1987) Jlm~nunol Mefhods 100:73-82.
An alternating schedule of toleri_ation and ;~ was set up as follows: female Balb/c mice at 6 weeks of age were injected intra-peritoneally ("I.P.") with Ix107 adult PBMC in 500 ul PBS. Alhe adult PBMC injection was followed 10 minutes later by l.P.
injection of cy. ' r~ at 100 mg/kg. The cy- 1 ~.1.. .~l.l,A ..; 1 was repeated at 24 and 15 48 hours. After an additional 14 days, the toleri_ation was repeated.
Three weeks later, the mice were immuniæd with fetal " ,.. , .. 1. A blood cells by l.P. injection of IxlO7fetal cells in 500 ul PBS. After an additional 2 weeks, the mice were once again toleri7ed with adult PBMC as described for the first round of tol~ri7Ati/m Finally, three weeks later, the mice were again immunized with fetal blood ' cells by l.P.
imjection of Ix107 fetal cells m 500 ul PBS. IAhe fetal cell was repeated in 24 and 48 hours. After an additional 24 hours, the mice were sacrificed. The spleens were harvested amd ' 11~ fro_en in liquid nitrogen.
~cn~nf;f)n D f RNA and cDNA svnthesis Total RNA was isolated from spleens or from Hybridoma cell lines usmg standard methods (Cllo~ Li, U.S. Patent No. 4,843,155). RNA 1~ - were stored m RNAase free water (Sambrook, J. et aL, Molecular Cloning ,4 ~.aboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)) at -70C until use. A
Superscript pre ,' ~ kit from Life T l " was used to prepare first strand cDNA as 1~ .. , .. 1,"1 by the supplier.
30 rrn~mi~ 2 Qf DNA
Isolation of plasmid DNA from E co~i for DNA sequence or restriction analyses was by alkaline Iysis (Birnboim amd Doly, 1979). Bulk preparation of plasmid DNA was carrier out using ' ~ ' column .,LI. , .' y as described by the r Macherey-Nagel (Duren, Germany). All cultures for isolation of plasmid DNA from ~. coli clones W095/15982 21 7 ~48Z 50 PCT/US94/14106 containing antibody clones were grown overnight with shaking at 37C in 2xYT medium containing 0~5% glucose and 50 ug/ml carbenicillin.
p(~R, 7,, ~ " of antihoL~v ~,nn chnin L ~ hPnW ~ hnin L'~i~ re~ion~
A set of degenerate primers, showrl in Figures IA and IB, was designed to minimize 5 bias toward limited sets of PCR product~ from the repertoire of antibody coding regions encoded in spleenic mRNA, as well as to amplify >90% of the mouse kappa chain and heavy chain Fab encoding sequences. ~A".I.l,li.-l;.",~ of kappa chain or heavy chain coding sequences were ~ l using 5 separate primer pairs for each. The primers also contained restriction enzyme site to allow the ligation of the light and heavy chain PCR
10 products into a bacterial Fab expression cassette suitable for insertion in the Surf-Zap vector (Stratagene). PCR reactions were carried out in an Autornated BioSystems temp-cycler (Essex, MA) using the following protocol. Generally, 5-10 ug of total spleenic or hybridoma RNA was converted to cDNA using a Superscript first strand synthesis kit (BRL). 0.5-1 ug of first strand cDNA in 100 ul of buffer containing 10 mM Tris-HCI, pH8.3, 50 mM KCI, 1.5 mM MgC12, 0.01% gelatin with the appropriate primer pair (see Figures IA and IB) waS
incubated for 5 min. 98C, cooled to 60C, and 2 U of Taq polymerase was added. Products were then amplifed 35 cycles with the following four ~ ; profile: 72C for 120 seconds, 90 seconds at 54C, and 30 seconds at ~5C. After 35 cycles the samples were incubated an additional 10 min. at 72C. to erlsure complete product poly~ io.~. It is 20 important that individual reactions be adjusted to yield ~ 1-2 ug of 0.7 Kb PCR
product, with the minimal number of cycles (usually 30-38 cycles).
~4r~omh~vof~ 7hP~re~ ncn~otfo~ nnnn~hP~ychninp('~pr~
1-2 ug of each PCR product from fi~e separate reactions were combined to generate a kappa chain and separate heavy chain prod.uct pool. The pools were then purified by first 25 removing protein and debris with a PVDF spin filter (Millipore) followed by removal of low molecular weight l , using a 30,000 MW cut off spin filter as ' ' by the supplier (Millipore). A"pl. 'y 5 ug of each prodnct pool was digested in 300 ul of Sfil buffer with 50 units of Sfil for 2 h at 50C. Enzyme amd small end fragments generated by Sfil digestion were removed with the spin colunm procedure described above. Sfil digested 30 light chain products were ligated to Sfil digested heavy chain products (~ 'y 2 ug each) in a 50 ul volume overnight at 4C. The ligation mixture was then purified with spin columns as above amd digested with 50 units each of Notl and Spel restriction enzymes in 100 ul. The digestion products were resolved by agarose gel clc.,l~ D~ amd the 1.4 kb kappa chain heavy chain encoding dimer was purified using Gene Cleam II (Promega) as 35 l~ ..."....1.l..1 by the supplier.

A simpler more reliable method for uu~LIu~,Lioll of the Fab' expression cassette is 1 in Figure 2. Al~lv/dlll~tdly 10 ng of kappa and heavy chain product pools fromabove were amplified with primers designated in Figure 2 (and shown in Figures lA and lB) to give 3' kappa and 5' heavy chain sequence which when treated with T4 polymerase in the 5 presence of dTTP yielded an 8 base compatible overhang allowing highly efficient oriented ligation of kappa and heavy cl1ain sequences. PCR products were purified as described above. A,u~J~v~du~ .~ly 2 ug of each product was treated separately at 37C for I h in a 50 ul volume containing S units T4 ,uvl~lll.,laa~,, 5 mM dTTP. Products were purified as for PCR
products, and ~ 500 ng of each product was ligated at room ~IIly~ uc for 3 h 10 in a 25 ul volume of ligation buffer (Promega) containing 2 units of DNA ligase.
Fab encoding dimer from either method were amplified under standard conditions using a 5' kappa chain primer and 3' heavy chain primer shown in Figure 2 except that annealing was at 55C for I min., and the extension time was extended to 4 min. at 72C.
Generally 12-25 cycles under these conditions yielded .I,U,UlV~-iUl. ~t~ly 1-2 ug of 1.4 kb kappa-15 heavy chain dimer. This product was purified using spin columns as described above andthen digested in a200 ul volur~e containing 75 units each of Not I and Spe I rest~iction enzymes. Digestion products v~ere purified as described above except that a 100,000 MW
spin column (Amicon) was used to more efficiently remove primers from the digestion products. Purified 1.4 kb dimers were stored at 4C until use.
20 C. of variegntr~Fr~h clone ~ ' Ligation of Not l-Spe I digested 1.4 kb Fab encoding fragments was as follows: 0.2 ug of digested products was ligated to 2 ug of lambda surf-zap arms in 10 ul of Promega ligation buffer containing 3 units of T4 ligase overnight at 4C. Aliquots of the ligation mixture were then packaged into lambda heads using a Giga-pack Gold packagmg kit as 25 l,~.,...,,..,.1r(1 by the supplier (Stratagene). Packaging reactions were titered on E coli LE392 and pooled to yield a primary library. This primary library was then amplified in E.
coli LE392 using ~,ullv.,ll~iullrll methods. Generally 5x109 E. coli XLl cells were infected in 10 ml of 10 mM MgSO4 with 107 invitro packaged SURF-ZAP primary clones for 10 min. at 37C. The infected cdls were added to 100 ml of NZY top agarose at 50C. The mixture 30 was ~ lS~ plated onto two 20x20 cm plates containing NZY agar, allowed to solidify, and then mcubated for 8-16 h. The amplified library was harvested by rocking with an overlay of 25 ml of SM buffer of 2 h.
GPnPr~?tir)rl of Phr~e antibodv clone ~ ' A Phagemid clone bank was rescued from the primary lambda SURF-ZAP librar~v by 35 super infection with M13 exassist helper phage essentially as ~ 1 by Stratagen.
Generally 1011 E. coli XLI cells were infected with 1010 lambda clones from our amplified WO 95115982 Z ¦ 7 5 4 8 ~ 52 PCT/US94/14106 surf-zap library and 10~2 Exassist M13 phage. After growth for 3.5 h in LB or TB medium the cells were removed by f ~ . i r, Iy,r 1 ;l ~l ~ The exassist rescued library was treated for 70C for 20 min. and then stored at 4C.
Phage antibodies were generated by infection of E coli SOLR 1:1 with rescued 5 phagemid to generate a population of carbenicillin resistant antibody clone containing cells rl,ulc~ lg a 10-100 fold excess over the primary libraly size. Transduced cells were grown to early log phase in TB medium containing carbenicillin, and then infected with a ten fold excess of VCS M13 helper phage to cells. After I h at 37C, kanamycin was added and the culture was incubated at 30C with shaking until early stationary phase. Cells were removed 10 by . . .,1l ;rllV~ and phage antibodies were recovered from the supemat~mt by harvested by ~. ..I,ir,.~r:il,.., dissolved in I ml of TE buffer and then PEG ~u.,; ' a second time.
Phage antibodies were dissolved in I ml TE or PBS buffer and stored at 4C.
F,~-- ' of cell ~urfnrp bin~i~g phnh~ on whole cell.~
Cell specific phage antibodies were isolated by emichment on whole cells. Cells were prepared for enrichment by washing twice in blocking buffer (0.1% hydrolyzed casein, 3%
BSA, in Hanks Buffered Salt Solution). For the frrst round of emichment 101l phage antibodies in 200 ul of blocking buffer were added to 106 cells and incubated on ice for I h.
Non-specific phage antibodies were then removed by washing 8 times with cold blocking buffer. Cells were harvested after each wash by ~ 11 i r~v~: ;. at 3500 rpm in an Eppendorf micro centrifuge. Cell surface bound phage antibodies were then eluted nn 500 ul of 0.2 M
Glycine pH2.2 containing 3 M urea and 0.5% BSA. Debris was removed by ~ ltlirU~rLiUII
and the supematant was neutralized by addition of 50 ul of I M Tris pH 9.5. Urea and buffer VUIII,U ' were removed with three buffer changes using an amicon 100,000 MW cut off spin column. Emiched luvuul~lL;u1~ of phage amtibodies were titered on XLI cells, and then amplified by the following protocol. Eluted phage antibodies in 200 ul SM buffer were added to 5x109 XLI plating cells in I ml of 10 mM MgSO4 and incubated for 10 min. at room L~ a~. Infected cells were then used to inoculate 100 ml of TB broth in a 2 L
flask and incubated at 30C with shaking. After I h of incubation kanamycin and ~.~ub~li~,;lli11 were added to 50 ug/ml. Inc~lbation was contmued with shaking at 30C urltil early stationary phase (Increase in O.D.600 remained the same at two consecutive time points). Cells were removed by ~ .1 ir, V ;~ at 12000 rpm in a Dupont/Sorvall SS34 rotor for 10 min. Phage antibodies were then purified by PEG ,u1cc;~;~a~;ull. The stringency of washes m subsequent rourlds of enrichment were increased by reducmg the amount of phage antibodies loaded to 10 1 , increasing the number of washes (10-20 washes) and adding a 100 mM citrate buffer wash (pH 4.5 or pH3.5) before elution with Glycine-Urea.

WogS/15982 53 2 i 7 5 4 8 2 PCT/US94/14106 Prepar~tion Qf Inr'ivl~ J phaoe an~;h~7~'iP~ for ana~vse.~ of bin~ F s~ecifici~v E coli XLI was infected with dilutions of phage antibody pools and plated on LB
medium containing 0.5% glucose and 50 ug/ml carbenicllin. 20 mm culture tubes containing 2 ml of 2xYT medium with 50 ug/ml carbenicllin were inoculated with isolated colonies amd 5 grown overnight at 30C with sha,cing. The following morning I ml of culture was gently shaken at 37C for I h and then infected with 101l M13 VCS phage. A 250 ml flaskcontaining 25 ml of TB broth with carbencillin was inoculated with the VCS infected culture and shaken at 30C for 1 h, kanamycin was added to 50 ug/ml and incubation was continued until early stationary phase (O.D. 600 nm = 5-12). Cells were removed by .~ .;r~10 and phage antibodies were purified by PEG precipitation as previously described. The phage antibodies were dissolved in 200 ul TE (pH7.5) buffer.
SP~7/P77ri~g efAnfihod,v Isolates Individual isolates, such as the H3-3, FB3-2 and F4-7 clones, were isolated and each of the heavy and light chain inserts were sequenced using primers based on 3' and 5' flanking 15 sequence of the Surf-Zap vector using standard protocols (see Current Protocols in Molecular Biolog,v, eds. Ausubel et al. (John Wiley & Sons: 1992); and Molecular Cloning A
~aborator,v Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989)).
Flow cytometric ~rr~ of ~h~e antihodv hindin~ to whole ~Pn~
A flow cytometry protocol was devised for the testing of phage antibody binding to surfæe markers on whole cells. IX106 adult or fetal ."...,.... ~ l cells were dispensed into a 2 ml microtube and washed with blocking buffer as im the phage enrichment procedure. For initial assay, 2x101 phage were added to the washed cells and the volume brought to 100 ul with c~sein/BSA/HBSS. The phage were incubated with the cells for one hour at 4C. The phage-cells were washed three times with I ml blocking buffer. Biotinylated sheep amti-phage polyclonal antibody (5 Prime -2 Prime) was added to the phage-cells at 5-7.5 ul per sample, optimized for each lot of polyclonal antibody. Volume was once again brought up to 100 ul with blocking buffer. The anti-phage was incubated 90 minutes at 4C. Excess anti-phage was removed by washing three times with blocking buffer. Streptavidin-FlTC(Jackson Illll~ IUI~a~,~l,ll) was diluted 1:50 in Ca-Mg-free PBS and 250 ul added to each sample of phage-cells. After a 30 minute 4C incubation, the phage-cells were washed twice with blocking buffer and fixed by adding 400 ul o.5% r All samples were analyzed by flow cytometry. Flu~,lua.,.,u~e background was determined by using a non-display phage (VCS M13) as a negative control. Intensity of WO 95/15982 2 1 7 ~ ~ 8 2 PCTIUS94114106 FITC Iluv~ c~ above background on each cell was drrectly ~lul~vl~;ull~l to the number of specifically boumd phage.
Relative binding activity of each clone was determined by evaluation of two parameters: (I) scatte} pattern vs. intensity of nuul~ uc~ for ~ , .;" 'f ;"' I of relative cell 5 surface epitope number and uniformity of expression for each phage clone, with higher, more uniform, numbers being most desirable; (2~ titration of phage amd retention of fluorescent binding intensity - for !~ of relative phage antibody affinities.
In a variation of the above binding assay, soluble antibody ("Fab") was tested for activity. For the Fab fragments, the anti-~ v;v;ll-FITC was replaced by a goat 10 anti-lgG-FITC F(ab')2 polyclonal antibody (TAGO) that recogluzed the Ic chain of the Fab fragments. 30 ul of the goat anti-lgG-FlTC diluted 1:10 in 2.5% normal human serum was used per sample. The dilution in human serum ensured that any cross-reactivity of the polyclonal with human blood cell antigens would be minimized.
15 Il. F~n~ pl:Enrirhnnpntofrh~eanfih~ pc~n~nrprepllc A ...".1.;, '~.,;~1 phage display liibrary of IgGI amd kappa chain derived Fabs containing 6x107 primary clones was constructed from a mouse which had been tolerized v~ith adult human blood and immunized with fetal liver cells. In order to minimize clonal bias due to individual growth ..~ ,.. t` .;`1;. `, cultures containing antibody clones or pools of 20 clones were always in rich media (IB or 2xYT containing 1% glucose). In addition, cultures used to produce phage antibodies were harvested as close to peak growth as possible since binding activity was found to fall beyond the start of stationary phase of growth.
The human erythro-leukemic cell line (HEL) carries onco/fetal cell surface markers also found on fetal liver cells. This ~ and the ability to culture this cell made it a 25 reliable source of cells to develop methods for enrichment of cell line specific antibodies from the above phage library. The binding of phage antibody pools enriched on this cell line (HEL) are shown in Figure 3.
These results ~1. ."...,~:.,.~.1 an increase in binding from 2.5 x 10-7 to 1.25 x 10-3 after 3 rounds of pnrirl~rnPnf reflecting a 4 log increase m phage binding to the cell surface. We 30 tested the ~ of phage antibodies prepared from 16 ', ' isolates on HEL, Raji, and Adult blood cells by ni...., ~ . ~ activated eell sorting. Specific binding of phage antibody to the eell surfaee was deteeted by biotin-labeled anti-phage antibody followed by 11.. "1 .. - eonjugated :ILI.,~v ' Results from these assays shown in Figure 4 ' that at least 6 out of the 16 isolates (indieated by asterieks) from enrichment 3 ~095/15982 55 21 754~2 PCT/lJS94JI41~6 (i.e. phage isolated after three interative pannings) appeared specific for ~ found only on HEL cells.
In order to evaluate how dive}se the population of antibodies being enriching onwhole cells was, the DNA sequence encoding the regions including CDR3 from these six S phage antibody isolates was ~1 - ' Of the six EIEL specific isolates there was only a single duplicate. This result ~il ....,..~1l..~, 3 that the directed isolation of EIEL cell specific antibodies by enrichment on whole cells had been achieved.
III. FYP~IP 2: FnriPhmPnt of rh~P ~ntihr~ c nn fPtPI rPllc To maY~nize the chances of isolating fetal cell specific clones, the phage antibody library described in Example I was pre-absorbed on adult nucleated blood prior to each enrichment cycle on fetal liver cells in addition to ~ ", ;-1,.,....~ without pre-adsorption. The results of sequential rounds of pre-adsorption and enrichment on fetal liver cells are shown in Figure 5A. The increase in the percentage of phage antibodies binding to fetal liver cells 15 indicated enrichment for fetal ce~l binding phage antibodies.
Rernarkably, after only a single round of enrichment on fetal cells, it was observed that pure ~ of fetal cell binding phage antibodies had been isolated. This was ,. . 1 by ,1. ,..1..;,,.1;~.~, of a sampling of random isolates, from each stage of pnrj~hn~Pnt by DNA sequence analysis in ~,.,h;,.-li~." with an assay for binding of 20 individual isolates to fetal cells by FACS. The results of these ~ are shown in Figure 7. DNA sequencing delineated three classes of fetal cell binding antibodies based on the amino acid sequence of their heavy chains. E ach class included subtypes identified first, by amino acid changes in and around the CDR3 region which reflect affinity maturation, and second, by association of different kappa chains.
Table 4 shows the distribution of different phage antibody types at different stages of enrichment on HEL or Fetal cells with or without I.,r_.1~."l,1;~,.. on adult cells. It is likely that the three classes of phage antibodies recogluze three different epitopes based upon the difference in their staining profiles on fetal liver and adult cells.
After the fourth round of enrichment (in the enrichment series including ~
30 on adult cells) only phab type 5 was eluted from fetal liver cells. The selection for this type umder the most stringent wash conditions suggests that it is the highest aff~nity of heavy and light chains.

WO95115982 ~7548~ 56 PCT/US94/14106 Table 4 Ph~ge Ant body Type Phab 1 2 3 4 5 6 7 ua Pool Primary 1 7 Hd3 5 Hd4 16 Fdl 8 Fd2 6 Fd3 12 1 20 Fd4 1 6 a Unknown crPrifir tiPC
Mock enrichment 1, ;,1 ,1~ in ~hich tbree different phage antibodies spiked I in I o6 non-specific control Ml 3 phage were enriched on fetal liver cells (Table 5), fi - - ' a dramatic 1 05-l o6 fold single round enrichrnent of these phage antibodies on fetal liver cells.
This order of magnitude of enrichment is at the upper limits of those reported for enrichment of ~.,.,.1~ .. ~ ..;-lly-derived antibodies using purified antigen, which is of import when it is 10 considered that the epitopes targeted in the present example are highly complex cell-surface antigens and have not been purified in any way.
Table 5 ~nrichmer~t of phege antibodies on fetel liver cells.
Starting Fmal Phab %Bindingb RatioC Ratiod r",i,l.. ~ ~,, H3-31.6 1 in 4,500,000 1.6:1 3,600,000 fold Fd3-1 1.1 1 in 150,000 1.1:1 165,000 fold F4-70.5 1 in 100,000 3.0:1 300,000fold b Number of phabs eluted from cells after 10 washes, divided by the total phage loaded.
c Ratio of specific to non-specific phab in starting population.
d Ratio of specific to IIO., ~,~,;rl., phab after elution from fetal cells.
e Final ratio divided by starting ratio.

WO 95115982 2 1 7 5 ~ ~ 2 - PCTtUS94tl4106 The vast number of cell surface binding isolates seen on a consistent cell source (HEL) reflects the efficiency and diversity of the library constructed. The enmhinqfinn of tolerance ; " " . ., 1 ~ " along with the efficiency of the prescnt methods for library construction and ,qmrlifi~qfi-m have yielded a remarkable library of phage antibodies which S can be very rapidly emiched on whole cells. Such results are important for identifying phabs _ighly specific for a particular target cell-fype from different individuals. This point is particularly ~ .l ,A.; ,. .1 by the fact that most of the phabs (even with tolerization ;,.,....-..,-~;--~) isolated without prPqrlenrrtinn also recognize adult cells. In addition, enrichment at each stage with fetal liver cells from an ;...1. IJ' .,.I. .II fetus eliminates those 10 antibodies which recogniæ individual specific markers. This added stringency in the emichment for pan-fetal specific antibodies emphasizes the power of the present approach, which yielded 13 different versions of three classes of pan-fetal specific antibodies. In contrast, using the identical toleri~ation approach with uu~ Liullal hybridoma methods, only two IgGs of the same type were obtained.

IV. FYq~lp 3: Affinif~y ~ of l:Tl-3 ~qn~1 Fg3-2 Antihn(liPe Affinity of purified antibodies was determined by Scatchard analysis. Varying amounts of antibody in significant excess were incubated for 16 hours at 4C with a constant number of HEL cells. After extensive washes, bound antibody was eluted from cells at pH 2, 20 and quatitated in an ELISA. For Scatchard analysis, free amtibody was assumed to be equivalent to the total added. The Ka for each antibody was obtained from the negative slope of the line from the plot of bound versus bound/frPe antibody. All points were done in triplicate; the correlation coefficient for all reported sIopes was greater than 90%.
25 V. FYqrr~"~le 4: SnPrifirif,y of H~-3 ,qnll FB~-2 AntihmliPc In order to compare hybridoma-derived amtibodies, such as anti-CD71 and anti-EM,with the subject antibodies, reactivity of these antibodies with fetal and maternal cells was tested by analytical flow cytometry (FACS effciency assay). Briefly, lx106 cells per sample were stained with indicated amounts of FITC-conjugated pure ~mtibody. 10,000 cells were 30 analyzed for each sample. The results, provided in Table 3 above, are reported as "%
positive", indicating the percentage of cells that were found to stain above background nUul.,~ ,e as established by an isotype-matched negative control antibody.
The highly specific " of the H3-3 antibody for fetal as opposed to adult 1.. -- ~`'1-"~;' :;r cells was further ' ' by FACS and subsequent fluorescent in situ 35 l~yblidi~iiull (FISH) analysis of sorted cells. Briefly, 400 fetal liver cells, .1. ...~ to wo gs/lsg82 ~ 2 58 PCTIUS94/14106 be male by Y-PCR, were spiked into one million adult PBMC. The spiked sample was then stained with Hoescht-DNA dye and 11l8 of FITC-conjugated H3-3 antibody. The nucleated cells (those positive for Hoescht) were sorted for H3-3-positives, fixed to slides and analyæd for the presence of male cells by FISH. Male (Y) probe was detected with Cy3; female (X) S probe by FITC. The results are ~.,..",.,.. ;, ~I as follows: starting purity of fetal cells = 0.04%;
backgroundstainingofadultcells=0.05%;fetal(male)cellsrecovered=301;purityofH3-3 sorted fetal cells = 36.4%.
All of the above-cited references and ~ are hereby ;II~,Ulp~ ' ~ by I 0 reference.
Fq~ ~t~
Those skilled in the art will recognize, or be able to ascertain using no more than routine ~ l, numerous equivalents to the specific method and reagents described herein. Such equivalents are considered to be within the scope of this invention and are 15 covered by the follûwing claims.

WO 95/15982 59 2 1 7 :~ 4 8 2 PCT/US94114106 SEQ~ENCE LISTING
(l) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Genzyme Corporation (B) STREET: One Rendall Slluare (C) CITY: Cambridge 0 (D) STATE: MA
( E ) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02139 (G) TELEPHONE: (508) 872-8400 (H) TELEFAX: (508) 872-5415 (ii) TITLE OF INVENTION: ProceE~s for n~n~ ;n~ Specific An~;hr~
(iii) NUMBER OF SEQUENCES: 61 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC, _ ;hl ~
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text) (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE ~DZ~ l~b:
1A) LENGTH: 73 ba~e pairs (B) TYPE: nucleic acid (C) STI~ : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
A;~ GCAGGTCTCC TCCTCTTAGC ~r.rDr~ r2~ GCAATGGCCG ACATTSTGAT 60 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQ~ENCE rll7~ rul.'~ b:
(A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (c) .~ "~ C single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: Other nucleic acid (xi) SEQI~ENCE IJ~:b~lCl~llVN: SEQ ID NO:2:

WO95/15982 ~ ~ 7 ~ 60 PCrlUS94/14106 ATATGCGGCC GCAGGTCTCC TrrTrTTArr Ar.r~r~7~rrA GCAA~ C~ A'rA'rt.:('AC;A'r iU
C~--ArArArT HCA 73 ~2~ INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE r~D~
(A) I.ENGTH 73 ba~e pairs (B) TYPE nucleic acid 0 (C) sT~pr - ~n - q single (D) TOPOLOGY linear (ii) MOI.ECULE TYPE Other nucleic acid (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AIA~ jLU GCAGGTCTCC TCCTCTTAGC ~rrAr1~rrA GCAATGGCCG 1~ rll 60 r~rrr~ArT CcA 73 (2) INFOR~ATION FOR SEQ ID NO:4:
(i) SEQUENCE r~rTR~TCTICS
(A) LENGTEI 73 ba~e pairs (~3) TYPE nucleic acid (C) ST~?A -: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE Other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID ~:4:
ATATGCGGCC GCAGGTCTCC TCCTCTTAGC ArCArz~ rA GCAATGGCCG ACATTGTGMT 60 (2) INFORMATION FOK SEQ ID NO:5 ( i ) S EQUENOE r~T'` ~ ~ X l l (A) ~ENGTH 73 base pair~
(B) TYPE: nucleic acid ~C) ."~./''''I~.IIN~:X`i: ~ingle (D) TOPO~OGY: linear (ii) MOLECULE TYPE Other nucleic acid (Xi) SEQUENCE ~;~K1~11UN: SEQ ID NO:5:
A~ GCAGGTCTCC TCCTCTTAGC Aar~r~r'rr~ arp7~TGarrr AAATTGTTCT 60 CACCCAGTCT CCA ~ - - ' 73 WO 951l5982 ~ 1 7 S 4 8 2 PCr~'[rS941141~6 2 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE rTT7~oDrTrRT~cTIcs:
(A) LENGTH: 107 base pairs S (B) TYPE: nucleic acid (C) sT~DNn~!nN~!~q~c: single (D) TOPOLOGY: linear (ii) MOLECUL13 TYPE: Other nucleic acid (xi) SEQUENCE 1~;~KlrllUN: SEQ ID NO:6:
5CATGGCCGGT 1~ r.TD~T7`D~D~ TCCAGCGGCT rrrrTDr.rrD ATAGGTATTT 60 CATTATGACT lil~L~ll~i~ T~TTAACACT CATTCCTGTT GAAGCTC 107 (2) LN~ UN FOR SEQ ID NO:7:
(i) SEQUENOE r (A) LENGTH: 33 base pair~
(B) TYPE: nucleic acid (C) :~ : ~ingle 25 (D) TOPOLOGY: linear (ii) MOLECIJLE TYPE: Other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N-0:7:
~ l ~u~ l cuu~ CAT~TGCGGC CGCAGGTCTC CTC 3 3 35 (2) INFûRMATION FOR SEQ ID NO:S:
(i) SEQUENOE rT DoD~
(A) LENGT~: 33 base pairs (B) TYPE: nucleic acid 40 (c) ST7` : single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE J~:~I~llUN: SEQ ID NO:8:
Ll~LilJ~ CC~CATGGCC Wl ~uu~:, CGA

(2) IN-FORMATION FOR SEQ ID NNO:9:
(i) SEQI~ENOE rTT~T'DrTr:~TqTICS
(A) LENGTE~: 33 ba~e pair~
55 (B) TYPE: nucleic acid (C) ~ ingle (D) TOPOLOGY: linear ~7548~
WO 95/15982 62 PCTIITS94/l4l06 (ii) MOLECULE TYPE: Other nUC1eiC aCid (xi) SEQUENCB ~ K1~11UN: SEQ ID NO:9:
L1.~ CATCGCGGCC r~71rrrr.rr~ TGG . 33 (2) 1NL~ UN FOK SEQ ID NO:10:

( i ) SEQUENCE rFn~ ~ ~ rT~ T .CTI CS:
(A) LENGTH: 33 ba~;e Pair9 (B) TYPE: nUC1eiC aCid _ - -(C) ;I~ S ~ing1e (D) TOPOLOGY: 1inear (ii) MOLECULE TYPE: Other nUC1eiC aCid (Xi) SEQUENCE DESCKIPTION: SEQ ID NO:10:
~1~11~;~ CC~AGGCTTA rTPrT~r~7`T CCC 33 25 (2) INFOKMATION FOR SEQ ID NO:11:
(i) SEQUENCE rlT7 ~ D
(A) LENGTH: 22 ba8e Pair8 (B) TYPE: nUC1eiC aCid (C) DIr/~ 1)N~ ing1e (D) TOPOLOGY: 1inear (ii) MOLECULE TYPE: Other nUC1eiC aCid (Xi) SEQUENCE LIC~DLK1~ N: SEQ ID NO:11:
r.rrr,rrr~7~r CGGCCATGGC CG 22 (2) INFOKMATION FOK SEQ ID NO:12:
( i ) SEQ~ENCE ~ D:
(A) LENGTH: 6B ba~;e Pair8 (B) TYPE: nUC1eiC aCid (C) sT~ n~:m~cq: 8ing1e (D) TOPOLOGY: 1inear (ii) MOLECULE TYPB: Other nUC1eiC aCid (Xi) SEQUENCE IJ~D~K1~11UN: SEQ ID NO:12:
r,r~r:rrrrr~r T~T7~nr~T CCAaCGGCTG rrr~T~rrr~7~ TAGGTATTTC ATTATGACTG 60 TCTCCTTa 68 ~2) INFORMATION FOR SEQ ID NO:13:
(i~ SEQUENCE t~TDDDrT~DTcTIcs (A) LENGTH: 45 ba~ie pairs (B) TYPE: nucleic acid (C) STD~ n~--q~ ingle (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: Other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
15 lC~U~ C~ ACCGGCCATG GCCGAGGTCC arr~TKrP~ D GTCWG 45 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE t'MDDD~'Tl;DTqTICS:
(A) LENGTH: 45 ba~e pairs (B) TYPE: nucleic acid (C) sTDDNnDnN~cc: single ( D ) TOPOLOGY: 1 inear 25(ii) MOLECULE TYPE: Other nucleic acid (xi) SEQIJENCE J~ lU~: SEQ ID NO:14:
'lC~iU~i:~ ACCGGCCATG GCCGAGGTGA Wla`:l~lil~ RTCTG 45 (2) INFOD~IATION FOR SEQ ID NO:15:
35(i) SEQUENCE ~7`Da~'T~DT':TICS:
(A) LENGT~: 45 base pairs (B) TYPE: nucleic acid (C) sTDrr~n~: 3ingle (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: Other nucleic acid 45(xi) SEQUENCE L/~U~l~llU~: fiEQ ID NO:15:
l~iUVi~ ~ ACCGGCCATG GCCCAGGTYC AGCTGMl~GCA GTCTG 4s (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE ~ `DD~ ~T~ll~
(A) LENGTH: 45 baae pairs (B) TYPE: nucleic acid (C) 5TDP : single 55 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid WO9~/15982 ~7 54~ 64 PCT/US94/14106 ~xi) SEQUENCE L).t:S~lrLlVs~: SEQ ID NO:16:
SlU~ ACCGGCCATG GCCGAGGTYC DrrT.qrDr~rD GTCTG 4s (2~ INFORMD~TION FOR SEQ ID NO:17:
(i) SEQUENCE rT~ DrTR~TqTIcs 0 (A~ LENGTH: 45 base pair (B) TYPE: nucleic acid (C) qT~Dl~m~nNRq~q: ~ingle (D) TOPOLOGY: linear 5(ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE ~ KlrLlU~: SEQ ID NO:17:
U~ ~ ACCGGCCATG GCCGAGGTGA Dr~rTTn~TqrD GTCTG 45 (2) INFORMDTION FOR SEQ ID NO:18:
25(i) SEQUENCE ~lA~Dr~rR~rgTIcs:
(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) o ~: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid 35(xi) SEQIJENCE ~ lrllUI`I: SEQ ID NO:lB:
~ ACCGGCCATG GCCCAGGTGC ArTK7~ GTCAG 45 (2) INFORMDTION FOR S13Q ID NO:19:
(i) SEQUENCE rHDRD~ S:
(A) LENGTH: 41 basc pairg (B) TYPE: nucleic acid (C) ST~D ~: single 45 (D) TOPOLQGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE L/~ Kl~llU~Y: SEQ ID NO:19:
Ul~rrD7~r~TDr~TD CAATCCCTGG GCACAATTTT C 41 SS (2) INFORWATION FOR SEQ ID NO:20:
(i) SEQUENCE rT~7~D,,-~l '`llW:
(A) LRNGTH: 42 base pairs WO 95115982 ~ ~ 7 5 4 8 2 PCT~lTS94/1410C
(B~ TYPE: nucleic acid (C~ ST~DNnT:nNrqq: single (D~ TOPOLOGY: linear (ii~ MOLBCULE TYPE: Other nucleic acid (xi~ SEQUENCE lJ~ Kl}'ll~N: SEQ ID NO:20:
8~ CCAGATATCA CTAGTGGGCC C~i~l~i~l~ AA 42 (2~ INFORMATION FOR SEQ ID NO:21:
5 ( i~ SEQUENOE rN7~T~D,; r.. , ~
(A~ LENGTH: 39 ba~e pairs (3~ TYPE: nucleic acid (C) ~ I)N~ .C. single (D) TOPOLOGY: linear (ii) MOLECULE TYPB: Other nucleic acid (xi) SEQUENCE ~J~;~K1~11~1N: SEQ ID NO:21:
-- rrDDrTDrTD r~~rrTrr~r~r~ GGGTACTGG 39 (2~ INFORMATION FOR SEQ ID NO:22:
(i~ SEQ~BNCE rp~Dl, ~
(A~ LENGTH: 42 ba3e p~irs (B) TYPB: nucleic acid (C) sT~r : 13ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:22:
~ . G~,, cw~ CCATCTGCAC TAGTTGGAAT wGCACATGC AG 42 (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUBNCE rTT7~D , (A) LBNGTH: 35 ba~e pair~
(B) TYPB: nucleic acid (C) ST~ rn`~qq: ~:ingle (D) TOPOLOGY: linear (ii) MOLECULB TYPE: Other nucleic acid (xi) SEQIJENCE ll~:~Kl~ll(JN: SEQ ID NO:23:

s4a~
W095115982 '1~1 66 PCTIUS94/14106 GGGAATTCAT GGACTGGACC TGGAGGRTCY TCT~CC 3 s ~2) INFORMATION POR SEQ ID NO:24:
(i) SEQUENCE r~H~D~ l~S:
(A) LENGTH: 34 ~ase pairs (B) TYPE: nucleic acid (C) STR~ ~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPR: Other nucleic acid ~;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24-(2) INFORMATION FOR Sl~Q ID NO:25:
(i) SEQUENCE rT7~v~T=RTqTIcs:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STR~ Rrl'lE.qS: single 25 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE L:~ lJrl: SEQ ID NO:25:
GGGAATTCAT GRAMMWACTE~ L-~Wa~wr~C TYCTG 35 35(2) INFORMATION FOR 8EQ ID NO:26:
(i) 9EQUENCE ~ 'T=RTqTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid 40 (c) ~ r~ q: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GGG~ATTCAT GGACATGRRR ~JY~(2tlVliY(iL CASCTT 36 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE t~T~R~f'T=RTqTICS:
(A) LENGTH: 35 base pairs 55 (B) TYPE: nucleic acid (C) STR~D=r .q: single (D) TOPOLOGY: liTLear ~ii) MOLECULE TYPE: Other nucleic acid ~xi) SEQUENOE DESCRIPTION: SEQ ID NO:27:
GGGA~TTCAT ~ w~r ~ ,lU~ ~ TS-wYC 3 (2) INFORMATION FOR 8EQ ID NO:28:
(i) SEQ~ENCE rT~ rTR~T~TICS:
(A) LENGTE~: 2a base pair~
(B) TYPE: nucleic acid (C) ~ : Lingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE lJl:;:~UKll:'llUI\I: SEQ ID NO:28:
CCAAGCTTAG ~rr~ n AaAGGGTT 28 25 (2) INFORMATION FOR SEQ ID NO:29:
( i ) SEQUENCE rua~ w l l~:j:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (c) 8~i~Pr~ nNT;.cc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPB: Other nucleic Pcid (xi) SEQUENCE U~ Kl.''lUl!J: SEQ ID NO:29:
CCAAGCTTGG P''''n'"''l'Gr CAGGGGG 27 (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE ,run~
(A) LENGTEI: 27 base pairs (B) TYPE: nucleic acid (C) ~ : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENCE J~l~.LlU~Y: SEQ ID NO:30:
ccaAGcTTGA AGCTCCTCAG AGGAGGG 27 (2) INFORNATION FOR SEQ ID NO:31:

WO 95115982~ ~ 7 5 4 8 ~ 68 PCT/US94~14106 (i) SEQUENCE ~ D~ ;ll~b: -~A) LENGTH: 27 base pairs (S) TYPE: nucleic aci~l (C) ST~p~)RTlN~q.c: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (xi) SEQUENC~ DESCRIPTION: SEQ ID NO:31:
CC~AGCTTTC ATCAG~TGCIC GGGAAGA 2 7 5 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE rT~ rT~TcTIcs:
(A) LENGTH: 6 amino acids (3) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE lJ~;SL~ : SEQ ID NO:32:
Asp Pro Leu Tyr Gly Ser (2) lNrl -' FOR SEQ ID NO:33:
(i) 8EQUENCE rN7~
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQIJENCE lll;:~LlCl~ N: SEQ ID NO: 33:
Ser Gln ser Thr His Val Leu Thr (2) lNl~ --TtlN FOR SEQ ID NO:34:
~i) SEQUENCE rTJ~rT~TqTICS:
(A) LENGTH: 6 amino acids (3) TYPE: amino acid ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide WO 9511598~ 7 5 ~ 8 2 PCT/US94/14106 ~v) FRAGMENT TYPE: internal (xi) SEQl~ENCE DESCRIPTION: SEQ ID NO:34:
Ala Leu Lys Val His Met ( 2 ) INFORMATION FOR SEQ ID NO: 3 5:
(i) SEQ~ENCE ~v~
(A) LENGTH: 6 amino acidL
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FPAGMENT TYPE: internal (xi) SEQI~ENCE DESCRIPTION: SEQ ID NO:35:
Asp Pro Leu Tyr Gly Asn 12) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE ~ rTFVTcTICS:
(A~ LENGT~I: 9 amino acids (P ) TYPE: amino aaid ( D ) TOPOLOGY: l inear (ii) MOLECHLE TYPE: peptide (v) FRAGMENT TYPE: internal~

(xi) SEQIJENOE DESCRIPTION: SEQ ID NO:36:
Gln Gln Trp Ser Ser Asn Pro Pro Thr l 5 (2~ INFO.~MATION FOR SEQ ID NO:37:
(i) SEQUENCE ~'`~''~FVT~TICS:
(A) LENGTH: 8 amino aoids (S) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal WO95/15982 ~1 548~ 70 PCI/US94/14106 ~xi~ SEQUENCE l)K::i(.:Kl~llUN: SEQ ID NO:37:
ser Gln Ser His His Val I.eu Thr ( 2 ) INFODMATION FOR SEQ ID NO: 3 8:
(i) SEQUENCE ICTTDDD-'~RDTqTICS:
0 (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: li~ear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: int~rral ~0 (Xi) SEQUENCE Jl~b~ luN: SEQ ID NO:38:
Asp Pro Leu Tyr Gly Asp (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE ~l~DD~-TRRrcTIcs (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FR~GMENT TYPE: interr,al (xi) SEQUENCE 11~L.7L'Kll:'llUN: SEQ ID NO:39:
Gly Asp Tyr Gly Asn Tyr Gly A6p Tyr Phe Asp Tyr (2) llNrL`Kl~lUrl FOR SEQ ID NO:40:
(i) SEQIJENCE ~7~DD~
(A) LENGTH: 9 amino acids (B) TYP3: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: illterIIal (xi) SEQUENCE Jl!;b~Kl.t'llUN: SEQ ID NO:40:
Gl~ His Ser Trp Glu Ile Pro Tyr Thr WO9511S982 71 ~ 1 7 ~ 4 8 2 PCT~U594/14106 (2) INFORM~TION FO~ SEQ ID NO:41:
(i) SEQUENCE rH2~VD~ hl~
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 0 (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE L~;~l~llVN: SEQ ID NO:41:
Gln A6p Ser Trp Glu Ile Pro Tvr Thr (2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE rH~V~rTF~TCTICS:
(A) LENGTB: 9 amino acids 25 (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLEC~LE TYPE: pept ide 30 (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Gln Gln Ser Asn Glu Asp Pro Tyr Thr (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQIJENCE CEIARACTERISTICS:
(A) LENGTB: 9 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECCLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE l~ i~lrllUN: SEQ ID NO:43:
Gln Gln Ser Asn Glu Asp Pro Phe Thr (2) lN~ -rr~ FOR SEQ ID NO:44:

wO95/1598~'\754a~ 72' PCI/IJS94/14106 (i) SEQUENCE r~D~r~rRDr.CTIcs:
(A) LENGT~I: 12 amino acids (B)_ TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO-44:
Gly Asp Tyr Gly Lys Tyr Gly A~ip Iyr Phe Asp E~i~
1 5 l0 ~2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE rTJnDDr~R~TqTIcs:
20 (A) LENaTH: lZ amino acidS
(S) TYPE: amino acid (D) ToPOLor,Y: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: i~terral (xi) SEQUENCE J~;S~ : SEQ ID NO:45:
Gly Val Tyr Gly LYD Tyr Gly A13p Tyr Phe A~p Uis 35 (2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE rT~nDDrrRDT.~TIcs:
(A) LENGTH: 9 amino acid~
(B) TYPE: amino acid 40 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: intenlal (xi) SEQUENCE L~ lUD~: SEQ ID NO:46:
50 Glr ~is Ser Trp Glu Ile Pro Phe Thr (2) INFORMATION FOR Sli:Q ID NO:47:
55 (i) SEQUENCE rTT~r~F~TcTIcs:
(A) LENGTEI: 4 amino acid~
(PD) TYPE: amino acid ~D) TOPOLOGY: linear WO 95115982 ~ 1 7 S 4 8 2 PCT~US94/14106 (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal s (xi) SEQUENCE ll~Kl~llU3\1: SEQ ID NO:47:
0 Cys Gly Gly Arg 15 (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE ruD~DrTr~TcTIcs (A) LENGTH: 14 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMBNT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Glu Gly Tyr Gly Pro Thr Gly Tyr Tyr Ser Ala Met Asp Tyr 30 1 s lo (2) INFORMATION FOR SEQ ID NO:49:
(i) SEQbENOE ru~.DDrTR7rATIcs 35 (A) LENGTH: 8 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: l inear (ii~ MOLEC~LE TYPE: peptide (v) FRAGMENT mB: interral (xi) SEQUENOE li~;:i~Kl!'llUN: SEQ ID NO:49:
Gln Gln Gly Tyr Ser Tyr Leu Thr ( 2 ) INFORMATION FOR SEQ ID NO ~ 5 0:
(i) SEQl~ENCE rF-.~DrTR~TATICS:
(A) LENGTH: 735 base pairs (B) TYPE: nucleic acid (C) ~ oth (D) TOPOLOGY: linear W0 95/15982 ~ 4 ~ PCT/IJS94/14106 (ii) MOLECCLE TYPE: cDNA
( ix ) FEATURE:
( A) NAME/}~EY: CDS
(B) LOCATIO~: 67. .735 (xi) SEQ~IENCE lJ~ LK~1~5~r~: SEQ ID NO:50:
ATGAAATACC TATTGCCTAC r~.~r~rrr~oT GGATTGTTAT TACTCGCGGC rr~rrrror 60 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Met Pro 1 s 10 Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser AGT TAC TGG CTA GAG TGG GTG AaA CAG AGC CCT GGA CAT GGC CTT GAA 204 Ser Tyr Trp Leu Glu Trp Val Lys Gln Ser Pro Gly ~is Gly Leu Glu Trp Ile Gly Glu Ile Leu Phe Gly Ser Gly Ser Ala l~is Tyr Asn Glu Lys Phe l.ys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser alu Asp Ser Ala Val Tyr Tyr Cyu Ala Arg Gly Asp Tyr Gly Asn Tyr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr I,eu Thr Val Ser Ser Ala Lys Thr Thr Pro 115 120 1as Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser ATG GTG ACC CTG GGA TGC CTG GTC AAa GGC TAT TTC CCT GAG CCA GTG 54 o Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val lIis Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser ser Ser Val Thr WO95/1~5982 ~ 7~i~T~ PCT/US94/14106 ~s 180 185 lYU

Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cy6 Asn Val Ala His Pro Ala Ser Ser Thr Lyu Val Asp Lys Lys Ile Val Pro Arg Aup Cys (2) INFORMATION FOR S=!Q ID NO:51:
(i) S~QULNCB ~TJ7`~2~TTz~TcTIcs:
(A) LENGT~I: 223 amino acids (}3) TYPE: amino acid ( D ) TOPOLOGY: l inear (ii) MOLI;:CI~L13 TYPL: protein (xi) SEQ~ENC13 111~ Kl~ll~N: S~Q ID NO:51:
lu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Uet Met Pro Gly Ala 30 Ser Val Lys Ile Ser Cys Lya ~la Thr 31y Tyr Thr Leu Ser Ser Tyr Trp Leu Glu Trp Val Lys Gln Ser Pro Gly ~lis Gly Leu Glu Trp Ile Gly Glu Ile Leu Phe Gly Ser Gly Ser Ala P;in Tyr Aun Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Aup Ser Ala Val Tyr Tyr Cys Ala Arg Gly Asp Tyr Gly Asn Tyr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Aun Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val E~is Thr Phe Pro Ala WO 95/15982 ~ 7 5 4 8~ PCT/US94/14106 al Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala Eis Pro l9S 200 205 Ala Ser Ser Thr Lys Val Asp LYB Lys Ile Val Pro Arg Asp Cys (2) INFORMATION FOF~ SEQ ID NO:52:
(i) S~QUENCE t~T~f'T~TCTICS:
(A) LENGTE: 399 base pair3 (B) TYPE: nucleic acid (C) STI~ oth (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/~EY: CDS
(B) LOCATION: 67..399 (xi) SEQUENCE L~ lO~: SEQ rD NO:s2:
ATGAaATACc TATTGCCTAC ~ W~,L~ TCTTAGCaGC ~r~rr~r~r~ : 60 ATGGCC GAC ATT GTG ATG ACC CAG TCT CCT GCT TCC TTA GCT GTA TCT loa Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cy8 Arg Ala Ser Gln Ser Val Ser ~CA TCT AGA TAT AGT TAT ATG CAC TGG TAC CAA CAG A~A CCA GGA CAG 204 Thr Ser Arg Tyr Ser Tyr Met Eis Trp Tyr Gln Gln Lys Pro Gly Gl CCA GCC AAA CTC CTC ATC AAG TTT GC~TCC A~C CTA GAA TCT GGG GTC 252 Pro Ala Lys Leu Leu Ile Lys Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phc Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Eis Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln Eis 5~ AGT TGG GAG ATT CCG TAC ACG TTC GGA GGG GGG ACC AaG CTG GAA ATA 396 Ser Trp Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile WO95llSg82 77 21 7~482 PCT/US94/14l06 ~Y~
s (2) INFORMATION FOR SEQ ID NO:53:
(i) SEOUENOE r~rT~TCTICS:
(A) LENGTEI: 111 amino acid3 0 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser Arg Tyr Ser Tyr Met ~ Trp Tyr Gln Gln Ly~i Pro Gly Gln Pro Ala Lys Leu Leu Ile Ly~ Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His ro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cy~ Gln ~Iis Ser Trp 35 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 2) INFORMATION FOR SEQ ID NO:54:
40 ( i ) SEQ~CE r~ b:
(A) LENGTEI: 735 base pairs (B) TYPE: nucleic acid (C) sTl7~Nn~nN~cq: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( ix ) FEAT~RE:
50 (A) NAME/~EY: CDS
(B) LOCATION: 67 . . 735 (xi) SEQUENCE Ll:;b~ J: SEQ ID NO:54:
ATGAI~ATACC TATTGCCTAC r~r~r~r~rrr~rT GGATTGTTAT TACTCGCGGC rrD~rrr~rr 60 WO95/15982 ?~15~ 78 PCT/US9q/14106 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Met Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser AGT TCC TGG CTA GAG TGG GTG A~A CaG AGC CCT GGA CAT GGC CTT GA~ 204 Ser Ser Trp Leu Glu Trp Val Lys Gln Ser Pro Gly Pis Gly Leu Glu TGG ATT GGA GAG ATT TTA TTT GGA AGT GGT AGT GCT cac TAC AAT GAG 252 Trp Ile Gly Glu Ile Leu Phe Gly Ser Gly Ser Ala EIis Tyr Asn Glu 50 5!~ 60 A~A TTC A~G GGC AP~G GCC ~ TTC ACT GTA GAT ACA TCC TCC A2 C ACA 3 0 0 Lys Phe Lys Gly Lys Ala Thr Phe Thl- Val ~sp Thr Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Asp Tyr Gly Asn Tyr Gly ASp Tyr Phe Asp Tyr Trp Gly Gln Gly Gln Ala Leu Thr Val Phe Ser Ala Lys Thr Thr Pro TCA TCT GTC TAT CCA CTG GCT GCT GGA TCT GCT GCC CAA ACT A~C TCC 492 Ser Ser Val Tyr Pro Leu Ala Ala Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val ~Iis Thr Phe CCA GCT GTC CTG caG TCT GAC CTC TAC ACT CTG AGC AGA TCA GTG ACT 636 Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Arg Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala CAC CC9 GCC AGC AGC ACC AaG GTG GAC AaG A~A ATT GTG CCC AGG GAT 732 E~is Pro Ala Ser ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys -WO 95/15982 79 ~ ~ 7 S 4 ~ 2 PCT~US94~14106 .
(2) INFORMATION FOR SEQ ID NO:55:
S (i) SEQUENCE t'T~DVDf'TE~TCTICS:
(A) LENGTH: 223 amino acidR
(B) TYPE: amino acid (D) TOPOLOGY: linear 0 (ii) MOLECULE TYPE: proteir, (xi) SEQUENCE L~ lO~: SEQ ID NO:55:
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Met Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser Ser Ser Trp Leu Glu Trp Val Lys Gln Ser Pro Gly His Gly Leu Glu T Ile 35 40 45 rp Gly Glu Ile Leu Phe Gly Ser Gly Ser Ala His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 8s go 9s Ala Arg Gly Asp Tyr Gly Asn Tyr Gly Asp Tyr Phe Asp Tyr Trp Gl Gln Gly Gln Ala Leu Thr Val Phe Ser Ala Lys Thr Thr Pro Ser Ser Val Tyr Pro Leu Ala Ala Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cy9 Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Arg Ser Val Thr Val Pro la0 185 190 Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cy6 Asn Val Ala His Pro 195 200 20s Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys (2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENOE ~7~VD(, ~

754~
WO 95/15982 ~ ~ PCT/US94114106 (A) LENGTH: 723 base pairs ~B) TYPE: nucleic acid (C) ~ ~ " "c"~ ... both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
ix ) FKATGRE:
0 (A) NAME/KEY: CDS
(B) LOCATION: 67..720 (xi) SEQUENOE L1~i~Kl~llUN: SEQ ID NO:56:
ATGADATACC 'l'Dl-rr'rr'T'Ar ~ J~'I'r'.'L: ~ ~ TCTTAGCAGC l~r~ r~Dr.rz~ 60 A5p Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser 1 s lo Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Val Arg Gln Ser Val Ser ACA TCT AGC CAT AGT TAT ATG CAC TGG TAC CAA CAG AaA CCA GGA CAG 2 04 Thr Ser Ser His Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln CCA CCC ADA CTC CTC ATC AAG TAT GCA TCC D,AC CTA GAA TCT GGG GTC Z52 Pro Pro Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val CCT GCC AGG TTC AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC A;~C 3 0 0 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His 80 85 go Ser Trp Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile A~A CGG GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA CCA TCC AGT 444 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn TTC TAC CCC A~A GAC ATC AAT GTC AP~G TGG AAG ATT GAT GGC AGT GAA s40 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 21 7~48~
WO gS/15982 PCT/IJ594/14106 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp AGC ACC TAC AGC AGG AGC AGC ACC CTC ACG TTG ACC AaG GAC GAG TAT 636 5Ser Thr Tyr Ser Arg Ser Ser Thr Leu Thr Leu Thr Lys A6p Glu Tyr 175 150 ~ 185 190 Glu Arg Bis Asn Ser Tyr Thr Cyu Glu Ala Thr His Lys Thr Ser Thr TCA CCC ATT GTC AAG AGC TTC AAC AGG AAT GAG TGT TA~ 723 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys (2) INFORMATION FOR SEQ ID NO:57:
(i) SEQ~JENOE ~T~ TcTIcs (A) LENGT~: 218 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECIJLE TYPE: protein (xi) SEQI~ENOE DESCRIPTION: SEQ ID NO:57:
Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Val Arg Gln Ser Val Ser Thr Ser 20 2s 30 Ser ~Iis Ser Tyr Met l~is Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile ~Iis Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln ~is Ser Trp Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln llS 120 125 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 55 Pro Lys Asp Ile Asn Val Lys Trp Ly~ Ile Asp Gly Ser Glu Arg Gln A3n Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr W09S/15982 ~1548~ 82 PCI~/IJS9V14106 yr Ser Arg Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg i~ Asn Ser Tyr Thr Cys Glu Ala Thr Uis Lys Thr 5er Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys (2) INFORMATION FOR SEQ ID 1~0:58:
(i) SEQUBNCE ~TD~DrT~TCTICS
(A) LENGTEI: 717 ~ase pairs (B) TYPE: nucleic acid (C) sTl~Nn~nNRq~ 0th (D) TOPOLOGY: linear 20 (ii) MOLECULE TYPE: rDNA
( ix ) FEATURE:
(A) NA~E/KEY: CDS
(B) LOCATION: 67. .717 (xi) SEQUENCE l~ ,'~lrLlL1N: SEQ ID NO:58:
30GTGADATACC TATTGCCTAC nr.rDr-crr~rT GGATTGTTAT TACTCGCGGC -r7~Drrrr,rr 60 ATGGCC GAG GTG AAG CTT ATG GAG TCT GGG GGA GAC TTA GTG Al~G CCT 108 Glu Val Lys Leu Met Glu Ser Gly Gly Asp Leu Val Lys Pro lo Gly Gly Ser Leu Lys Leu Ser Cya Ala Ala Ser Gly Phe Thr Phe Ser 15 20 2s 30 40GAC TAT TAC ATG TAT TGG GTT CGC CAG ACT CCG GAD. AAG AGG CTG GDG 204 Asp Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu 35 40 4s TGG GTC GCA ACC ATT AGT GAT GAT GGT ACT TAC ACC
45Trp Val Ala Thr Ile Ser Asp Asp Gly Thr Tyr Thr Tyr Tyr Ala As 252 so ss 60 AGT GTG AAG GGG CGA TTC ACC ATC TCC AGA GAC AAT GCC D,AG AAC AAC 300 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys A~n Asn 5065 70 7s Leu Tyr Leu Gln.Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr TAC TGT GCA AGA GAT CCC CTT TAT GGC AGC TGG GGC CD,A GGC ACC ACT 396 Tyr Cy~i Ala Arg Asp Pro Leu Tyr Gly Ser Trp Gly Gln Gly Thr Thr WO 95/15982 ~ ~ 7 S 4 8 2 PCT/I~S94/l.llOC

CTC AC~ GTC TCC TCA GCC A8A ACG ACA CCC CC~ TCT. GTC TAT CCA CTG 444 Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu GCC CCT GGA TCT GCT GCC CAA ACT AAC TCC ATG GTG ~CC CTG GGA TGC 492 Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys 0 CTG GTC AAG GGC TAT TTC CCT GAG CCA GTG ACA GTG ACC TGG AAC TCT s40 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val Fris Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Ser Ser Glu Thr Val Thr Cys Asn Val Ala Bis Pro Ala Ser Ser Thr AAG GTG GAC AAG A~A ATT GTG CCC AGG GAT TGT 717 Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys (2) INFORMATION FOR SEQ ID NO:59:
( i ~ SEQUENCE ~7~
(A) LENGTE~: 217 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESC~IPTION: SEQ ID. NO:59:
Glu Val LYG Leu Met Glu Ser Gly Gly Asp Leu Val Ly~ Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Asp Asp Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu l~sp Thr Ala Met Tyr Tyr Cys WO 95/15982 ~ 84 PCTNS94/14106 la Arg Asp Pro Leu Tyr Gly Ser Trp Gly Gln Gly Thr Thr Leu Thr S Val Ser æer Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly ser Ala Ala Gln Thr Aqn Ser Met Val Thr Leu Gly Cyli Leu Val Ly~ Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val Eli~ Thr Phe Pro Ala Val Leu Gln Ser Asp Leu yr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Ser Ser 20 Glu Thr Val Thr Cys Asn Val Ala ~is Pro Ala Ser Ser Thr Ly9 Val Asp Lys Lys Ile Val Pro Arg ~sp Cys (2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE r'~D~DrTR~r.STICS:
(A) LENGTH: 723 l~ase palr~
(B) TYPE: nucleic acid (C) ST~ Tr)Rn~1Rqc: ~oth (D) TOPOLOGY: linear (ii) MOLECtJLE TYPE: cDNA

(ix) FRATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 67..720 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
ATGA~ATACC TATTGCCTAC ~ l'D ~ rJ~I~IC~ TCTTAGCAGC Dn~ rDr:n~ 60 Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Gly Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val 15 . 20 25 30 CAC AGT AAT GGA A~C ACC TAT TTA CAT TGG TAC CTG CAG AAG CCA GGC 204 E~is Ser asn Gly ADn Thr Tyr Leu ~is Trp Tyr Leu Gln Ly~ Pro Gly WO9~;/15982 85 21 75482 PCT/IJS94/14106 ln Ser Pro Ly6 Leu Leu Ile Tyr Ly6 Val Ser A6n Arg Phe Ser Gly Val Pro A6p Arg Phe Ser Gly Ser Gly Ser Gly Thr A6p Phe Thr Leu Ly6 Ile Ser Arg Val Glu Ala Glu A6p Leu Gly Val Tyr Phe Cys Ser ao 85 90 Gln Ser Thr ~li6 Val Leu Thr Phe Gly Ala Gly Thr LyY Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Vai Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Gly Phe Leu A6n A6n TTC TAC CCC AaA GAC ATC AAT GTC AAG TGG AAG ATT GAT GGC AGT GAA s40 Phe Tyr Pro Ly6 A6p Ile Asn Val Ly6 Trp Ly6 Ile A6p Gly Ser Glu CGA CAA AAT GGC GTC CTG A~C AGT TGG ACT GAT CAG GAC AGC AAA GAC 588 Arg Gln A6n Gly Val Leu A6n Ser Trp Thr A6p Gln A6p Ser Ly6 A6p AGC ACC TAC AGC AGG AGC AGC ACC CTC ACG TTG ACC A~G GAC GAG TAT 636 Ser Thr Tyr Ser Arg 8er Ser Thr Leu Thr Leu Thr Ly6 A6p Glu Tyr Glu Arg ~is A~in Ser Tyr Thr Cy6 Glu Ala Thr ~i6 Ly6 Thr Ser Thr Ser Pro Ile Val Lys Ser Phe A6n Arg A6n Glu Cys ~2) lN~ --Ttw FOR SEQ ID NO:61:
(i) SEQIJENCB rTJ7~ ~E~TcTIc8 (A) LENGTEI: 218 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECl lLE TYPE: protein (xi) SEQI~ENCE l~ Kli~ N: SEQ ID NO:61:
A6p Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly ~154~ 86 Gly Gln Ala Ser Ile Ser Cy~ Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Hi~ Trp Tyr Leu Gln Ly~ Pro Gly Gln Ser 3s 40 45 Pro Ly~ Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 0 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr L.eu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cy~ Ser Gln Ser Thr His Val Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala ABP Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Gly Phe Leu Asn Asn Phe Tyr ~5 Pro Lys Asp Ile Asn Val Ly~ Trp Lys Ile aSp Gly Ser Glu Arg Gln sn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Ly~ Asp Ser Thr 30yr Ser Arg Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 180 185 l90 is Asn Ser Tyr Thr Cys Glu Ala Thr His Ly~ Thr Ser Thr Ser Pro 195 200 20s le Val Lys Ser Phe Asn Arg Asn Glu Cys

Claims (85)

CLAIMS:

1. A method for generating a specific antibody for an immunorecessive epitope, and nucleic acid encoding said antibody, comprising the steps of generating an immunotolerance-derived antibody repertoire for an immunorecessive epitope;
generating an antibody display library comprising a variegated V-gene library expressed by a population of display packages, said V-gene library cloned from said antibody repertoire, and selecting display pakages of said antibody display library which have a desired binding specificity for said immunorecessive epitope.

2. A method for generating a specific antibody for immunorecessive epitope, and nucleic acid encoding said antibody, comprising the steps of generating a variegated display library of antibody variable regions, said antibody variable regions cloned from an immunotolerance-derived antibody repertoire, andselecting antibody variable regions of said display library which have a desiredbinding specificity for an immunorecessive epitope.

3. The method of claim 2, wherein said display library is a phage display library.

4. The method of claim 2, wherein said display library is a bacterial cell-surface display library or a spore display library.

5. The method of claim 2, wherein said antibody variable region is a heavy chain, a light chain, a heavy-light chain pair, a VH, a VL, an Fab, an Fd, am Fv, or an scFv.

6. The method of claim 2, wherein said immunorecessive epitope is a cell-type specific marker.

7. The method of claim 6, wherein said immunorecessive epitope is a cancer cell marker.

8. The method of claim 6, wherein said immunorecessive epitope is a fetal cell marker.

9. The method of claim 6, wherein said immunorecessive epitope is a stem cell marker.

10. The method of claim 2, wherein said immunorecessive epitope comprises at least one amino acid residue in a variant protein that is different from a related or parent protein.

11. The method of claim 2, wherein said immunotolerance-derived antibody repertoire is generated by chemical immunosuppression.

12. The method of claim 11, wherein said immunotolerance-derived antibody repertoire is generated by cyclophosphamide-induced immunosuppression.

13. The method of claim 2, wherein said immunotolerance-derived antibody repertoire is generated by neonatal tolerization.

14. The method of claim 2, wherein said antibody variable regions are selected from said display library by a differential binding means comprising affinity separation of antibody variable regions which specifically bind said epitope from antibody variable regions which do not specifically bind.

15. The method of claim 14, wherein said differential binding means comprises panning said display library on a cell surface comprising said epitope.

16. A method for generating a specific antibody to an immunorecessive epitope, and genes encoding said antibody, comprising:
(a) transforming suitable host cells with a library of replicable phage vectors encoding a library of phage particles displaying a fusion antibody/coat protein, said fusion protein comprising a phage coat protein portion and an antibody variable region portion, said antibody variable region portion being obtained from an immunotolerance-derived variegated V-gene library;
(b) culturing said transformed host cells such that said phage particles are formed and said fusion protein are expressed; and (c) selecting any of said phage particles having an antibody variable region portion which binds to a an immunorecessive epitope.

17. The method of claim 16, wherein said transformed host cells further comprise a second antibody gene encoding a second variable region which is expressed in said transformed host cells, and which associates with said antibody variable regionportion of said fusion protein to form a heterodimeric Fv.

18. The method of claim 17, wherein said second antibody gene is obtained from said V-gene library.

19. The method of claim 17, wherein said second variable region is a polypeptide chain apart from said fusion protein and further comprises a secretion signal sequence that enables said second variable region to be secreted from said transformed host cells.

20. The method of claim 17, wherein said phage vector further comprises said second antibody gene.

21. The method of claim 20, wherein said fusion protein further comprises said second variable region covalently linked to said antibody variable region portion to form a single polypeptide chain antibody.

22. The method of claim 16, wherein said phage particle is selected from a groupconsisting of M13, fl, fd, If1, Ike, Xf, Pf1, Pf3, .lambda., T4, T7, P2, P4, ?X-174, MS2 and f2.

23. The method of claim 16, wherein said phage particle is a filamentous bacteriophage specific for Escherichia coli and said phage coat protein is coat protein III.

24. The method of claim 23, wherein said filamentous bacteriophage is selected from a group consisting of M13, fd, and f1.

25. The method of claim 16, wherein said transformed host cells are cultured with a helper phage suitable for inducing formation of said phage particles.

26. The method of claim 16, wherein said phage particles are selected by a differential binding means comprising contacting said phage particles with said immunorecessive epitope and separating phage particles which specifically bind said epitope fromphage particles which do not specifically bind said epitope.

27. The method of claim 26, wherein said differential binding means comprises an affinity chromatographic means in which said immunorecessive epitope is provided as a component of an insoluble matrix.

28. The method of claim 27, wherein said insoluble matrix comprises said immunorecessive epitope attached to a polymeric support.

29. The method of claim 27, wherein said insoluble matrix comprises a immunorecessive cell displaying said target epitope.

30. The method of claim 26, wherein said differential binding means comprises immunoprecipitating said phage particles with a multivalent form of said immunorecessive epitope, and subsequently removing non-specifically bound phage particles from said precipitate.

31. The method of claim 16, wherein said immunorecessive epitope is a cell-type specific marker.

32. The method of claim 31, wherein said immunorecessive epitope is a cancer cell marker.

33. The method of claim 31, wherein said immunorecessive epitope is a fetal cell marker.

34. The method of claim 31, wherein said immunorecessive epitope is a stem cell marker.

35. The method of claim 16, wherein said immunorecessive epitope comprises at least one amino acid residue in a variant protein that is different from a related or parent protein.

36. A method for generating a specific antibody for an immunorecessive epitope, and genes encoding said antibody, comprising:
(a) generating an immunotolerance-derived population of antibody-producing cells enriched for cells producing antibodies to an immunorecessive epitope;
(b) generating a variegated V-gene library encoding at least a variable region of immunoglobulin chains expressed by said enriched population of antibody-producing cells;
(c) generating a library of replicable phage vectors encoding a library of phageparticles displaying a fusion coat protein, each of said phage vectors comprising a chimeric coat protein gene encoding said fusion coat protein, said chimeric gene including (i) a first antibody gene encoding a variable region derived from said V-gene library, and (ii) a second gene encoding at least a portion of a phage coat protein, such that said library of phage vectors encodes a plurality of cloned variable regions;
(d) transforming suitable host cells with said library of replicable phage vectors;

(e) culturing said transformed host cells such that said phage particles are formed and said fusion coat protein are expressed; and (f) selecting any of said phage vectors corresponding to phage particles which display a variable region which binds to said immunorecessive epitope.

37. The method of claim 36, wherein said immunotolerance-derived population of antibody-producing cells are generated by chemical immunosuppressionof antibody production to immunodominant epitopes normally associated with said immunorecessive epitope in an immunogen.

38. The method of claim 36, wherein said immunotolerance-derived population of antibody-producing cells are generated by neonatal tolerization to suppress production of antibodies directed to immunodominant epitopes normally associated with saidimmunorecessive epitope in an immunogen.

39. A method for generating a specific antibody for a fetal cell-specific antigen, and nucleic acid encoding said antibody, comprising the steps of generating a variegated display library of antibody variable regions, said antibody variable regions cloned from an immunotolerance-derived antibody repertoire enriched for antibodies to a fetal cell-specific antigen, and selecting antibody variable regions of said display library which have a desiredbinding specificity for said fetal cell-specific antigen.

40. The method of claim 39, wherein said antibody variable regions of said display library are separated by a step comprising panning said display library on a fetal cell comprising said fetal cell-specific antigen.

41. The method of claim 39, wherein said fetal-cell specific antigen is a marker for fetal nucleated red blood cells.

42. An antibody that specifically binds an onco/fetal antigen said antibody having a heavy chain variable region comprising a CDR3 amino acid sequence selected from the group consisting of DPLYGS, DPLYGN, DPLYGD, GDYGDYGDYFDY, GDYGNYGDYFDY, GDYGKYGDYFDH, GVYGKYGDYFDH, and EGYGPTGYYSAMDY.

43. The antibody of claim 42, further comprising a light chain variable region comprising a CDR3 amino acid sequence selected from the group consisting of SQSTHVLT, ALKVHM, HSWEIPYT, QQWSSNPPT, SQSHHVLT, QHSWEIPYT, QDSWEIPYT, QQSNEDPYT, QQSNEDPFT, QQWSSNPPT, QHSWEIPFT, and GQGYSYLT.

44. An antibody isolated by the method of claim 1.

45. An antibody isolated by the method of claim 16.

46. An antibody isolated by the method of claim 36.

47. An antibody isolated by the method of claim 39.

48. An antibody display library enriched for specific antibodies to an immunorecessive epitope comprising a variegated V-gene library expressed by a population of display packages and enriched for specific antibodies by differential binding with an immunorecessive epitope, said V-gene library cloned from an immunotolerance-derived antibody repertoire.

49. The antibody display library of claim 48, wherein said display package is a phage particle.

50. The antibody display library of claim 48, wherein said immunotolerance-derived antibody repertoire is generated with a set of immunogen and toleragen in which said immunorecessive epitope comprises a cell-type specific marker.

51. The antibody display library of claim 50, wherein said cell-type specific marker is a fetal nucleated red blood cell marker, and said toleragen comprises a maternal erythroid cell and said immunogen comprises a fetal erythroid cell.

52. The antibody display library of claim 50, wherein said cell-type specific marker is a tumor cell marker.

53. The antibody display library of claim 52, wherein said tumor cell marker is a colon cancer marker, and said toleragen comprises a normal colon cell and said immunogen comprises a colon carcinoma cell.

54. The antibody display library of claim 52, wherein said tumor cell marker is a metastatic tumor cell marker, and said toleragen comprises a non-metastatic tumor cell and said immunogen comprises a metastatic tumor cell.

55. The antibody display library of claim 50, wherein said cell-type specific marker is a precursor nerve cell marker, and said toleragen comprises a differentiated nerve cell and said immunogen comprises an embryonic nerve cell.

56. The antibody display library of claim 50, wherein said cell-type specific marker is a hematopoeitic cell marker, and said toleragen comprises a committed stem cell and said immunogen comprises a hematopoietic stem cell.

57. The antibody display library of claim 48, wherein said immunotolerance-derived antibody repertoire is generated with a set of immunogen and toleragen in which said immunorecessive epitope comprises a determinant unique to a variant form of a protein.

58. The antibody display library of claim 57, wherein said variant protein is Apolipoprotein E4, and said toleragen comprises a Apolipoprotein E and said immunogen comprises Apolipoprotein E4.

59. The antibody display library of claim 57, wherein said variant protein is a p53 mutant having one or more amino residues different from wild-type p53, and said toleragen comprises a wild-type p53 and said immunogen comprises said p53 mutant.

60. The antibody display library of claim 57, wherein said variant protein is a ras mutant having one or more amino residues different from wild-type ras, and said toleragen comprises a wild-type ras and said immunogen comprises said ras mutant.

61. A variegated population of antibodies cloned from the antibody display library of claim 48.

62. An isolated antibody of the antibody display library of claim 48.

63. A method for generating an antibody having a binding association constant for an immunorecessive epitope of greater than 1x108M-1, and nucleic acid encoding saidantibody, comprising the steps of generating an immunotolerance-derived antibody repertoire for an immunorecessive epitope;
generating an antibody display library comprising a variegated V-gene library expressed by a population of display packages, said V-gene library cloned from said antibody repertoire, and selecting display packages of said antibody display library which have a bindingassociation constant for said immunorecessive epitope of greater than 1x108M-1.

64. A method for generating an antibody selective for an immunorecessive epitope, and nucleic acid encoding said antibody, comprising the steps of generating an immunotolerance-derived antibody repertoire for an immunorecessive epitope;
generating an antibody display library comprising a variegated V-gene library expressed by a population of display packages, said V-gene library cloned from said antibody repertoire, and selecting display packages of said antibody display library which have a bindingassociation constant for said immunorecessive epitope of greater than 1x108M-1 and a relative specificity of at least 10 fold over binding to background antigens.

65. A method for generating an antibody which selectively binds an immunorecessive epitope unique to a first cell phenotype of a related population of cells, and nucleic acid encoding said antibody, comprising the steps of generating an immunotolerance-derived antibody repertoire for an immunorecessive epitope on said first cell phenotype;
generating an antibody display library comprising a variegated V-gene library expressed by a population of display packages, said V-gene library cloned from said antibody repertoire;
generating an enriched display library by one or more of the steps of (i) removing from said antibody display library those display packages with substantial background binding to cells of said related cell population other than said first cell phenotype, and (ii) removing from said antibody display library those display packages which bind to said first cell phenotype in an individually selective manner, said enriched display library comprising remaining display packages of said antibody display library; and selecting display packages of said enriched display library which have a desiredbinding affinity for said first cell phenotype.

66. The method of claim 65, wherein said immunorecessive epitope is a fetal cell marker.

67. The method of claim 65, wherein said immunorecessive epitope is a cancer cell marker.

68. The method of claim 65, wherein said immunorecessive epitope is a stem cell marker.

69. The method of claim 65, wherein said display packages of said enriched display library are selected by panning said display packages will cells of said first cell phenotype.

70. An antibody immunoreactive with a fetal cell surface antigen, said antibody having a binding association constant for said antigen in excess of 1x108 M-1 and having no substantial background binding to maternal cells.

71. An antibody specifically immunoreactive with a fetal cell surface antigen and characterized by a specificity of at least 10 fold over background binding to maternal antibodies.

72. An antibody specifically immunoreactive with a fetal cell surface antigen, said antibody having a background binding to maternal cells of at least 2 fold less than an anti-CD71 antibody selected from the group consisting of a 5E9 antibody, an L5.1antibody, and an L01.1 antibody.

73. An antibody that binds an onco/fetal antigen, which antibody includes an antigen binding site comprising one or both of a first variable region and a second variable region, each of said first and second variable regions including complementaritydetermining regions of an H3-3 antibody, an FB3-2 antibody or an F4-7 antibody.

74. The antibody of claim 73, wherein each of the first and second variable regions are represented by the general formula FR(1)-CDR(1)-FR(2)-CDR(2)-FR(3)-CDR(3)-FR(4) wherein FR(1)-FR(4) represent polypeptides from antibody framework regions, and CDR(1)-CDR(3) represent polypeptides from complementarity determining regions of an H3-3 antibody, an FB3-2 antibody or an F4-7 antibody.

75. The antibody of claim 74, wherein each of the CDR(1), CDR(2), and CDR(3) for a single variable region have amino acid sequences selected from the group consisting:
CDR(1) = SYWLE, CDR(2) = EILFGSGSAHYNENKG, CDR(3) = GDYGNYGDYFDY;
CDR(1) = RASQSVSTSRYSYMH, CDR(2) = FASNLES, CDR(3) = HSWEIPYT;
CDR(1) = SSWLE, CDR(2) = EILFGSGSAHNYNEKFRG, CDR(3) = GDYGNYGDYFDY;
CDR(1) = RVRQSVSTSSHSYMH, CDR(2) = YASNLES, CDR(3) = HSWEIPYT;
CDR(1) = DYYMY, CDR(2) = TISDDGTYTYYADSVKG, CDR(3) = DPLYGS; and CDR(1) = RSSQSLVHSNGNTYLH, CDR(2) = KVSNRFS, CDR(3) = SQSTHVLT.

76. The antibody of claim 73, wherein the variable regions are selected from group consisting of E1-S121 of SEQ ID No. 51, D1-K111 of SEQ ID No. 53, E1-S121 of SEQ ID No. 55, D1-K111 of SEQ ID No. 57, E1-S115 of SEQ ID No. 59, and D1-K111 of SEQ ID No. 61.

77. The antibody of claim 73, which antibody further comprises framework region polypeptides from a human antibody.

78. The antibody of claim 73, which antibody further comprises a constant region polypeptide from a human antibody.

79. An antibody display library enriched for antibodies having binding constants for a cell surface antigen greater than 108M-1, which antibody library comprises a variegated V-gene library expressed by a population of display packages and enriched for specific antibodies by differential binding with an immunorecessive epitope of said cell surface antigen, said V-gene library cloned from an immunotolerance-derived antibody repertoire.

80. The antibody display library of claim 79 wherein said cell surface antigen is a fetal nucleated red blood cell marker.

81. The antibody display library of claim 79, wherein said cell surface antigen is a tumor cell marker.

82. A library of isolated nucleic acids encoding antigen binding sites immunoreactive with an immunorecessive epitope, comprising a variegated V-gene library encoding at least a variable region of immunoglobulin chains expressed by antibody-producing cells of an animal, which antibody-producing cells are enriched by immunotolerization for cells producing antibodies to the immunorecessive epitope.

83. The gene library of claim 82, wherein said V-gene library is expressed by a population of display packages.

84. The gene library of claim 83, wherein said display package is a phage particle.

85. The gene library of claim 82, wherein said immunorecessive epitope comprises an onco/fetal cell surface marker.