Note: Descriptions are shown in the official language in which they were submitted.
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 ;. `
WO 95/15982 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.
21 75482
WO 95/15982 PCTIU594J14106
1
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
21 75482
Wo 95/15982 PC. ~uss4~14106
19
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
WO 95115982 23 PCT/US94114106
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
21 75482
WO 95/15982 25 PCTIUS94/14106
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
21 7548
WO 95/15982 PCTNS94114106
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~
WO 95/15982 36 PCT/IJS94/14106
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
38
-
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
WO 95/15982 2 1 7 5 4 8 2 PCI/US94/14106
" 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
42
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.
WO 95/1S982 5 1 2 1 7 5 4 8 2 PCI/US94/14106
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
54
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
H3 1 1 2
H4 3
F3 10 6
F4 10 6
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
GACDCAGTCT CCA 73
(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
GACMCAGWCT CCA 73
(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
33
(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
WO 95/15982 2 1 7 5 4 8 2 PCTIIJS94/14106
~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-
GGGAATTCAT GGAGYTTGGG CTGASCTGGS TTTT 34
(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
WO 95115982 67 2 1 7 5 4 8 2 PCT/US94/141/16
~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
1 5
( 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
ATGGCC GAG GTC CAG CTG CAG CAG TCT GGA CCT GAG CTG ATG ATG CCT 108
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Met Pro
1 s 10
GGG GCC TCA GTG AAG ATC TCC TGC AAG GCT ACT GGC TAC ACA TTG AGT 156
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser
15 20 25 30
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
35 40 45
TGG ATC 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 l~is Tyr Asn Glu
50 55 60
AAA TTC AAG GGC AAG GCC ACA TTC ACT GTA GAT ACA TCC TCC AAC ACA 300
Lys Phe l.ys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Asn Thr
65 70 75
GCC TAC ATG CAA CTC AGC AGC CTG ACA TCT GAG GAC TCT GCC GTC TAT 348
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser alu Asp Ser Ala Val Tyr
80 85 90
TAC TGT GCC AGA GGA GAC TAT GGT AAC TAC GGG GAC TAC TTT GAC TAC 39
Tyr Cyu Ala Arg Gly Asp Tyr Gly Asn Tyr Gly Asp Tyr Phe Asp Tyr
95 100 105 110
TGG GGC CAA GGC ACC ACT CTC ACA GTC TCC TCA GCC AAA ACG ACA CCC 444
Trp Gly Gln Gly Thr Thr I,eu Thr Val Ser Ser Ala Lys Thr Thr Pro
115 120 1as
CCA TCT GTC TAT CCA CTG GCC CCT GGA TCT GCT GCC CAA ACT AAC TCC 492
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
130 135 140
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
145 150 155
ACA GTG ACC TGG AAC TCT GGA TCC CTG TCC AGC GGT GTG CAC ACC TTC 5 8 8
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val lIis Thr Phe
160 165 170
CCA GCT GTC CTG CAG TCT GAC CTC TAC ACT CTG AGC AGC TCA GTG ACT 636
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
GTC CCC TCC AGC ACC TGG CCC AGC GAG ACC GTC ACC TGC AAC GTT GCC 684
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cy6 Asn Val Ala
195 200 205
CAC CCG GCC AGC AGC ACC AAG GTG GAC AAG AAA ATT GTG CCC AGG GAT 732
His Pro Ala Ser Ser Thr Lyu Val Asp Lys Lys Ile Val Pro Arg Aup
210 215 2Z0
TGT 735
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
5 10 15
30 Ser Val Lys Ile Ser Cys Lya ~la Thr 31y Tyr Thr Leu Ser Ser Tyr
20 25 30
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
50 55 60
Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Aup Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asp Tyr Gly Asn Tyr Gly Asp Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
115 120 325
Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Aun Ser Met Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val E~is Thr Phe Pro Ala
165 170 175
WO 95/15982 ~ 7 5 4 8~ PCT/US94/14106
76
al Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro
180 185 190
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
210 215 220
(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
5 10
CTG GGG CAG AGG GCC ACC ATC TCA TGC AGG GCC AGC CAA AGT GTC AGT 156
Leu Gly Gln Arg Ala Thr Ile Ser Cy8 Arg Ala Ser Gln Ser Val Ser
15 20 25 30
~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
35 40 45
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
50 55 60
CCT GCC AGG TTC AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC AAC 300
Pro Ala Arg Phc Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn
65 70 75
ATC CAT CCT GTG GAG GAG GAG GAT ACT GCA ACA TAT TAC TGT CAG CAC 348
Ile Eis Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln Eis
80 85 90
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
100 105 110
WO95llSg82 77 21 7~482 PCT/US94/14l06
AAA 3 9 9
~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
5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser
20 25 30
Arg Tyr Ser Tyr Met ~ Trp Tyr Gln Gln Ly~i Pro Gly Gln Pro Ala
35 40 45
Lys Leu Leu Ile Ly~ Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
ro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cy~ Gln ~Iis Ser Trp
85 90 95
35 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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
ATGGCC GAG GTC CAG CTG CAG CAG TCT GGA GCT GAG CTG ATG ATG CCT 108
WO95/15982 ?~15~ 78 PCT/US9q/14106
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Met Pro
5 10
GGG GCC TCA GTG AAG ATC TCC TGC AAG GCT ACT GGC TAC ACA TTG AGT 156
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser
15 20 25 30
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
35 40 45
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
65 70 75
GCC TAC ATG CAA CTC AGC AGC CTG ACA TCT GAG GAC TCT GCC GTC TAT 348
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
80 85 90
TAC TGT GCC AGA GGA GAC TAT GGT AAC TAC GGG GAC TAC TTT GAC TAC 396
Tyr Cys Ala Arg Gly Asp Tyr Gly Asn Tyr Gly ASp Tyr Phe Asp Tyr
95 100 105 110
TGG GGC CAA GGC CAA GCT CTC ACA GTC TTC TCA GCC AAA ACG ACA CCC 444
Trp Gly Gln Gly Gln Ala Leu Thr Val Phe Ser Ala Lys Thr Thr Pro
115 120 125
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
130 135 140
ATG GTG ACC CTG GGA TGC CTG GTC AAG GGC TAT CTC CCT GAG CCA GTG 540
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val
145 150 155
ACA GTG ACC TGG AAC TCT GGA TCC CTG TCC AGC GGT GTG CAC ACC TTC 588
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val ~Iis Thr Phe
160 165 170
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
175 180 185 190
GTC CCC TCC AGC ACC TGG CCC AGC GAG ACC GTC ACC TGC AAC GTT GCC 684
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
195 200 205
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
210 215 220
TGT 73 5
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
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Leu Ser Ser Ser
20 25 30
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
50 55 60
Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
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
100 105 110
Gln Gly Gln Ala Leu Thr Val Phe Ser Ala Lys Thr Thr Pro Ser Ser
115 120 125
Val Tyr Pro Leu Ala Ala Gly Ser Ala Ala Gln Thr Asn Ser Met Val
130 135 140
Thr Leu Gly Cy9 Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175
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
215 220
(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
ATGGCC GAC ATT GTG ATG ACC CAG TCT CCT GCT TCC TTA GCT GTA TCT 108
A5p Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
1 s lo
CTG GGG CAG AGG GCC ACC ATC TQ TGC AGG GTC AGG CAA AGT GTC AGT 156
Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Val Arg Gln Ser Val Ser
15 20 25 30
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
35 40 45
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
50 55 60
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
65 70 75
ATC CAT CCT GTG GAG GAG GAG GAT ACT GCA ACA TAT TAC TGT CAG CAC 348
Ile His Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His
80 85 go
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
95 100 105 110
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
115 120 125
GAG CAG TTA ACA TCT GGA GGT GCC TCA GTC GTG TGC TTC TTG AAC AAC 492
Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn
130 135 140
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
145 150 155
CGA CAA AAT GGC GTC CTG AAC AGT TGG ACT GAT CAG GAC AGC ADA GAC 588
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
160 165 170
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
GAA CGA CAT AAC AGC TAT ACC TGT GAG GCC ACT CAC AAG ACA TCA ACT 684
Glu Arg Bis Asn Ser Tyr Thr Cyu Glu Ala Thr His Lys Thr Ser Thr
0 195 200 205
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
210 215
(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
5 10 15
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
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile ~Iis
65 70 75 80
Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln ~is Ser Trp
85 90 95
Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
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
130 135 140
55 Pro Lys Asp Ile Asn Val Lys Trp Ly~ Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
A3n Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
W09S/15982 ~1548~ 82 PCI~/IJS9V14106
165 170 175
yr Ser Arg Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
i~ Asn Ser Tyr Thr Cys Glu Ala Thr Uis Lys Thr 5er Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
0 210 215
(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
GGA GGG TCC CTG ADA CTC TCC TGT GCA GCC TCT GGA TTC ACT TTC AGT 156
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
CTC TAC CTG CAA ATG AAC AGT CTG ADG TCT GAG GAC ACA GCC ATG TAT 348
Leu Tyr Leu Gln.Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr
80 85 90
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
100 105 110
WO 95/15982 ~ ~ 7 S 4 8 2 PCT/I~S94/l.llOC
83
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
115 120 125
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
130 135 140
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
145 150 155
GGA TCC CTG TCC AGC GGT GTG CAC ACC TTC CCA GCT GTC CTG CAG TCT 588
Gly Ser Leu Ser Ser Gly Val Fris Thr Phe Pro Ala Val Leu Gln Ser
160 165 170
GAC CTC TAC ACT TTG AGC AGC TCA GTG ACT GTC CCC TCC AGC ACC TGG 636
Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp
175 180 185 190
TCC AGC GAG ACC GTC ACC TGC AAC GTT GCC CAC CCG GCC AGC AGC ACC 684
Ser Ser Glu Thr Val Thr Cys Asn Val Ala Bis Pro Ala Ser Ser Thr
195 200 205
AAG GTG GAC AAG A~A ATT GTG CCC AGG GAT TGT 717
Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys
210 215
(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
5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Asp Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 50
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
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
100 105 110
S Val Ser æer Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly ser Ala Ala Gln Thr Aqn Ser Met Val Thr Leu Gly Cyli Leu Val
130 135 140
Ly~ Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val Eli~ Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
yr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Ser Ser
180 185 190
20 Glu Thr Val Thr Cys Asn Val Ala ~is Pro Ala Ser Ser Thr Ly9 Val
195 200 205
Asp Lys Lys Ile Val Pro Arg ~sp Cys
210 215
(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
ATGGCC GAT GTT GTG CTG ACC CAG ACT CCA CTC TCC CTG CCT GTC AGT 108
Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser
5 10
CTT GGA GGT CAA GCC TCC ATC TCT TGC AGA TCT AGT CAG AGC CTT GTA 156
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
35 40 45
CAG TCT CCA AAG CTC CTG ATC TAC AAG GTT TCC AAC CGG TTT TCT GGG 252
WO9~;/15982 85 21 75482 PCT/IJS94/14106
ln Ser Pro Ly6 Leu Leu Ile Tyr Ly6 Val Ser A6n Arg Phe Ser Gly
50 55 60
GTC CCA GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GAT TTC ACA CTC 3 0 0
Val Pro A6p Arg Phe Ser Gly Ser Gly Ser Gly Thr A6p Phe Thr Leu
65 70 75
AAG ATC AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TAT TTC TGC TCT 348
Ly6 Ile Ser Arg Val Glu Ala Glu A6p Leu Gly Val Tyr Phe Cys Ser
ao 85 90
CAA AGT ACA CAT GTT CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG 396
Gln Ser Thr ~li6 Val Leu Thr Phe Gly Ala Gly Thr LyY Leu Glu Leu
100 105 110
AAA CGG GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA CCA TCC AGT 444
Lys Arg Ala Asp Ala Ala Pro Thr Vai Ser Ile Phe Pro Pro Ser Ser
115 120 125
GAG CAG TTA ACA TCT GGA GGT GCC TCA GTC GTG GGC TTC TTG AAC AAC 492
Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Gly Phe Leu A6n A6n
130 135 140
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
145 150 155
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
160 165 170
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
175 180 185 190
GAA CGA CAT AAC AGC TAT ACC TGT GAG GCC ACT CAC AAG ACA TCA ACT 684
Glu Arg ~is A~in Ser Tyr Thr Cy6 Glu Ala Thr ~i6 Ly6 Thr Ser Thr
195 200 205
TCA CCC ATT GTC AAG AGC TTC AAC AGG AAT GAG TGT TAA 723
Ser Pro Ile Val Lys Ser Phe A6n Arg A6n Glu Cys
210 215
~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
WO 95/15982 PCT/US94/14106
~154~ 86
Gly Gln Ala Ser Ile Ser Cy~ Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
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
50 55 60
0 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr L.eu Lys Ile
65 70 75 60
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cy~ Ser Gln Ser
85 90 95
Thr His Val Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105 110
Ala ABP Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser ser Glu Gln
11S 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Gly Phe Leu Asn Asn Phe Tyr
130 135 140
~5 Pro Lys Asp Ile Asn Val Ly~ Trp Lys Ile aSp Gly Ser Glu Arg Gln
145 150 155 160
sn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Ly~ Asp Ser Thr
165 170 175
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
210 215
