Note: Descriptions are shown in the official language in which they were submitted.
~ wo gs/33823 2 I q ~ ~ ~ ' ' Pcrluss4/13782
--1--
METHODS FOR SELECTIVELY STIMULATING
PROLIFER~TION OF T CELLS
p~ ' nf " - T
The dc ~ ~IV,UIII~,IIL of techniques for ,ulu~uat5aLil~g T cell pU,UUIdtiulls in vitro has been
crucial to many of the recent advances in the ~ l~ L ~ ~ - . l; "p of T cell recognition of antigen
and T cell activation. The du~,luu~ t of culture methods for the generation of human
antigen-specific T cell clones has been useful in defining antigens expressed by pathogens
10 and tumors that are recogm~d by T cells to establish methods of ;~ '.y to trcat a
variety of human diseases. Antigen-specific T cells can be expanded in vitro for use in
adoptive cellular i 1 - ~ in which infusions of such T cells have been shown to have
anti-tumor reactivity in a tumor-bearing host. Adoptive ;. ~ h ~ has also been used
to treat viral infections in ;- - ~ In~ ., . .l " Ulll;.~.,d individuals.
Techniques for expanding human T cells in vitro have relied on the use of ~cessory
cells and exogenous growth factors, such as IL-2. The use of IL-2 and, for exarnple, an anti-
CD3 antibody to stimulate T cell proliferation is knov~n to expand the CD8+, ~ ,u~...1-: ;....
of T cells. The IC ~ 11.,..' for MHC-matched antigen presenting cells as accessory cells
presents a significant problem for long-term culture systems. Antigen presenting cells are
20 relatively short lived. Thus, in a long-term culture system, antigen presenting cells must be
~ / obtained from a source and rerlf ni~h~d The necessity for a renewable supply
of accessory cells is u-ul,l~ a ic for treatment of; " .. l l In.1~ f ~ in which accessory
cells are affected. In addition, when treating viral infection, accessory cells which may carry
the virus may result in of the entire T cell population during long term culture.
25 An altemative culture method to clone and expand human T cells in vitro in the absence of
exogenous growth factor and accessory cells would be of significant benefit.
S of"-I
~ This invention pertains to methods for selectively inducing ex vivo expansion of a
30 population of T cells in the absence of exogenous growth factors, such as l~ l l .lll .nk ;. .~ ~, and
accessory cells. In addition, T ceU ~u~ulif~ Liu~ can be mduced without the need for amtigen,
thus providing an expanded T cell population which is polyclonal with respect to antigen
reactivity. The method provides for sustained ,ululif~,laLiu.. of a selected population of CD4+
or CD8+ T cells over an extended period of time to yield a multi-fold increase in the number
35 of these cells relative to the original T cell population.
According to the method of the invention, a population of T cells is induced to
proliferate by activating the T cells and stimulating an accessory molecule on the surface of
the T cells with a ligamd which binds the accessory molecule. Activation of a population of T
cells is -. ~ .. ~,I;~h.. 1 by contacting the T cells with a frrst agent which stimulates a
WO 95/33823 -2- PCT/US94/13782
TCR/CD3 complex-associatea signal in the T cells. Stimulation of the TCR/CD3 complex-
associated signal in a T cell is ~ ~ . ." ~ ' . A either by ligation of the T cell receptor
(TCR)/CD3 complex or the CD2 surface protein, or by directly stimulating receptor-coupled
sigmaling pathways. Thus, an anti-CD3 antibody, an anti-CD2 antibody, or a protein kinase
5 C ~tivator in conjl nrtinn with a calcium ionophore is used to activate a population of T
cells.
To induce ~luli~t;laliull, an activated population of T cells is contacted with a second
agent which stimulates an accessory molecule on the surface of the T cells. For example, a
population of CD4+ T cells can be stimulated to proliferate with an anti-CD28 antibody
10 directed to the CD28 molecule on the surf~e of the T cells. Plulif.laliull of a population of
CD8+ T cells is a 1 l ' ' by use of a " ,.~ antibody ES5.2D8 which binds toCD9, an accessory molecule having a molecular weight of about 27 kD present on activated
T cells. Alternatively, plvlif,laLiull of an antivated population of T cells can be induced by
stimulation of one or more intr~rPII ' signals which result from ligation of an accessory
15 molecule, such as CD28.
Following activation and stimulation of an accessory molecule on the surface of the T
cells, the progress of 1 ~ . 8; f. ~1;. . of the T cells in response to continuing exposure to the
ligand or other agent wbich acts; ~ d.y to simulate a pathway mediated by the
accessory molecule is monitored. When the rate of T cell plulirc~alivn decreases, the T cells
20 are re~tivated and ' 1, such as with additional amti-CD3 antibody and a co-
stimulatory ligamd, to induce further plulir~la~iv~1 In one I, . ,ho.l;., ~ the rate of T cell
plvlif~,laLivll is monitored by examining cell size. Altematively, T cell plulir~;~aLiu-- is
monitored by assaying for expression of cell surface molecules in response to exposure to the
ligand or other agent, such as B7-1 or B7-2. The monitoring and l~i ' of the T cells
25 can be repeated for sustained proliferation to produce a population of T cells increased m
number from about 100- to about I 00,000-fold over the original T cell population.
The method of the invention can be used to expand selected T cell l~uuuldLivll~ for use
in treating an infectious disease or cancer. The resulting T cell population can be genetically
transduced and used for ~ ' , y or can be used for in vitro analysis of infectious
30 agents such as HIV. r~ulif~,~divll of a population of CD4+ cells obtained from am mdividual
infected with HIV can be achieved and the cells rendered resistant to HIV infection.
Following expansion of the T cell population to sufficient numbers, the expanded T cells are
restored to the individual. Similarly, a population of tumor-infiltrating IYIIIIJI~V~ can be
obtained from an individual afflicted with cancer and the T cells stimulated to proliferate to
35 sufficient numbers and restored to the individual. In addition, ~ from cultures of T
cells expanded in accordance with the method of the mvention are a rich source of cytokines
and can be used to sustain T cells in vivo or ex vivo.
~ WO 95/33823 2 1 9 1 5 8 ~ v .. Ç I i PCTNS94113782
-3 -
l~riPf I~.c~ ~ of . ~ ~
Figure I depicts in vi~ro growth curves of CD4+ peripheral blood T cells in response
to culture with either an anti-CD3 antibody and interleukin-2 (IL-2) (---), an anti-CD3
antibody and an anti-CD28 antibody mAb 9.3 (0-0) or PHA only
5 (~
Figure 2 depicts the growth curve of CD4+ peripheral blood T cells cultured in fetal
calf serum and either anti-CD3 amtibodies and IL-2 (---) or an anti-CD3 amtibody and an
anti-CD28 antibody, mAb 9.3 (0-0).
Figure 3 depicts the growth curves of CD4+ peripheral blood T cells cultured in the
10 presence of phorbol myristic acid (PMA) and ionomycin with or without IL-2, or with an
anti-CD28 antibody, mAb 9.3. The symbols are as follows: PMA and ionomycin (P+l) is
rppre~p~Afpd by ([~); PMA, ionomycin and IL-2 (P+l+IL-2) is represented by (-); and PMA,
ionomycin and anti-CD28 antibody (P+1+9.3) is represented by (-).
Figure 4 is a schematic ~ ,a;,llLiivll of the selective expansion of CD4+ T cells
15 following CD28 stimulation in e~ ... to T cell stimulation with IL-2.
Figure 5 depicts fluorescent activated cell sorter analysis (FACS) in which cells were
stained after isolation (day 0, panel A), or after 26 days in culture with eitber CD28
stimulation (panel B) or IL-2 culture (panel C), with lJh ~v.,ly i' conjugated anti-CD3,
CD4,CD80rwithanlgG2acontrol ~' 'antibodyandn,....r~.A-equantifiedwitha
flow cytometer.
Figure 6 shows FACS analysis of the EX5.3D10 .. ,.. ~ antibody depicting
reactivity with CD28 im ~ to an anti-CD28 .". ~ .L.I~ ~ antibody 9.3. The
following cell lines were tested: Panel A, ~ ' CH0-DG44 cells; Panel B, CH0-
HH cells; Panel C, unactivated peripheral blood Iylll~ho~ , and Panel D, Jurkat No. 7
cells.
Figure 7 shows FACS analysis of the ES5.2D8 . ' ' amtibody depicting the
binding reactivity with the following cell lines: Panel A, CH0-DG44 cells; Panel B, CH0-
105A cells; Panel C, unactivated human peripheral blood Iy , ' - ~ ~." and Panel D, PMA
activated peripheral blood Iylll~JLul,~.
Figure 8 is a ~' , . ' depicting , . ~ analysis of detergent Iysates
of surface labeled human activated T cells indicating that ~ 1 antibody ES5.2D8
reacts with a 27 kD cell surface protein.
Figure 9 depicts the increases in mean cell volume of CD4+ T cells following
stimulation (Sl, S2, S3, S4, S5 and S6) with an anti-CD3 ' ' antibody and an anti-
CD28 -' ' antibody over days in culture.
Figure 10 depicts the cyclic expression of B7-1 on CD4+ T cells following
stimulation (Sl, S2, S3, S4, S5 and S6) with an anti-CD3 ' ' antibody and an anti-
CD28 .. ~ antibody over days in culture.
WO 9~/33823 2 1 9 1 5 8 6 ; ~ PCT/US94113782
Figure 11 is a bar graph depicting the amount of IL-2 produced by CD4+ T cells
following stimulation ~-vith an anti-CD3 mnnnrlnn,ql antibody and an anti-CD28 mrmn In-
antibody or IL-2 over days in culture
Figure 12 is a bar graph depicting the amount of granulocyte-llla,lu,uha~5., colony-
stimulating factor (GM-CSF) produced by CD4+ T cells follo~-ving stimulation ~-vith an anti-
CD3 """"~ antibody and an anti-CD28 - -' ' antibody or IL-2 over days in
culture
Figure 13 is a bar graph depicting the amount of tumor necrosis factor (TNF)
produced by CD4+ T cells follovving stimulation vvith an anti-CD3,, nrl ~1 antibody and
an anti-CD28,, ---~cl ~1 antibody or IL-2 over days in culture
Figure 14 is a bar graph depicting the T cell receptor (TCR) diversity in CD4+ T cells
followving stimulation ~-vith an anti-CD3, ",nrl ~ antibody and an anti-CD28 mnnn~ Inn
antibody at day I and day 24 of culture
Figure 15 depicts cell surface staining of CD4+ T cells obtained from an HIV
~LIUIl~,~ aLive individual follovving stimulation (S l, S2 and S3) vvith an anti-CD3 " ""r rl- ~ ~1
antibody amd an nti-CD28 n~ antibody over days in culture
Figure 16 depicts cell surface staining of CD4+ T cells obtained from an HIV
upua;Live individual follovving stimulation (Sl, S2 and S3) vvitb an anti-CD3 mnnnrlr~n
antibody and an anti-CD28 ""-~-n~ antibody over days in culture
Figure 17 depicts expansion of CD8+ T cells follo ving stimulation witb an anti~CD3
,, ~1 ",_1 antibody and an ' ' antibody ES5 7D8 at day 4 and day 7 of culture
Figure 18 shows FACS ~malysis of the - -- rl. . -1 antibody ES5 2D8 (panels C and
D) or a control IgG (panels A and B) depicting the binding reactivity with MOP cells
transfected with a plasmid encûding the CD9 antigen
n ~ of'-~ "
The methods of this invention enable the selective stimulation of a T cell population
to proliferate and expand to sigmficant numbers in vilro in the absence of exogenous growth
factors or accessûry cells Interaction between the T cell receptor (TCR)/CD3 complex and
antigen presented in c ; with either major L ' . ' ~' ~' ' ~y complex (MHC) class I
or class 11 molecules on an antigen-presenting cell initiates a series of l~;r b , -I events
termed antigen-specific T cell activation The term "T cell activation" is used herein to
define a state in which a T cell response has been initiated or activated by a primary signal,
such as through the TCRICD3 complex, but not necessarily due to interaction with a protein
antigen A T cell is activated if it has received a primary signaling event which initiates an
immume response by the T cell
T cell ~tivation can be . ' ' ' by stimulatmg the T cell TCR/CD3 complex or
via stimulation of the CD2 surface protein An anti-CD3 mnnn~lnnql antibody can be used to
~\W095133823 2 ~ 9 1 5 ~ 6 ~ , PCTNS94113782
_5
activate a population of T cells via the TCR/CD3 complex. Although a number of anti-
human CD3 ,.1...,..~1., . 1 antibodies are commercially available, OKT3 prepared from
hybridoma cells obtained from the American Type Culture Collection or ~, --1 ",,-1
antibody G19-4 is preferred. Similarly, binding of an anti-CD2 antibody will activate T cells.
5 Stimulatory forms of anti-CD2 ~mtibodies are known and available. Stimulation through
CD2 with anti-CD2 antibodies is typically a.,. . ' ' I using a ~ 1.l "1.;., ~ ;. ." of at least two
different anti-CD2 antibodies. Stimulatory r. ~ .C of anti-CD2 antibodies which have
been described include the following: the T11.3 antibody in ~,...~.1~;~...1;..~ with the Tl l.l or
T11.2 antibody (Meuer, S.C. et al. (1984) Cell 36:897-906) and the 9.6 antibody (which
recognizes the same epitope as Tl 1.1) in .. , . ,1.; .. -~ Nith the 9- 1 antibody (Yang, S. Y. et
al. (1986) ~ Immunol. 137:1097-1100). Other antibodies which bind to the same epitopes as
any of the above described antibodies can also be used. Additional antibodies, or
., ",,1.8. ~ of antibodics, can be prepared and identifed by standard techniques.
A primary activation signal can also be delivered to a T cell through use of a
15 . ,... ~ .. 'f ;"' . of a protein kinase C (PKC) activator such as a phorbol ester (e.g., phorbol
myristate acetate) and a calcium ionophore (e.g., ionomycin which raises cyL~ fic
calcium, _ ). The use of thcse agents bypasses the TCR/CD3 complex but
delivers a stimulatory signal to T cells. These agents are also known to exert a synergistic
effect on T cells to promote T cell activation and can be used m the absence of antigen to
20 deliver a primary activation signal to T cells.
Although stimulation of the TCRICD3 complex or CD2 molecule is required for
delivery of a primaly activation signal in a T cell, a number of molecules on the surface of T
cells, termed accessory or ' y molecules have been implicated in regulating the
transition of a resting T cell to blast ~ ;. .,. and subsequent proliferation and
25 .1. ~ n Thus, in addition to the primary activation signal provided through the
TCR/CD3 complex, induction of T cell responses requires a second,, ' y signal.
One such . ' y or accessory molecule, CD28, is believed to initiate or regulate a
signal ~ , 1 ;. " . pathway that is distmct from those stimulated by the TCR complex.
Iy~ to mduce an activated population of T cells to proliferate (i.e., a
30 population of T cells that has received a primary activation signal) in the absence of
exogenous growth factors or accessory cells, an accessor,v molecule on the surface of the T
cell, such as CD28, is stimulated with a ligand which binds the accessory molecule or with an
agent which acts; '. .~ -- l y to stimulate a signal in the T cell mediated by binding of the
accessory molecule. In one eli L - ' t, stimulation of the accessory molecule CD28 is
35 ~ by contactmg an activated population of T cells with a ligand which binds
CD28. Activation of the T cells with, for example, an anti-CD3 antibody and stimulation of
the CD28 accessory molecule results in selective proliferation of CD4+ T cells. An anti-
CD28 ...., ,-1.- ~1 antibody or fragment thereof capable of ... v~ ; ..g the CD28 molecule,
W095/33823 2 1 9 1 58~ PC'r/US94113782 ~
or a natural ligamd for CD28 (e.g., a member of the B7 family of proteins, such as B7-
I(CD80) and B7-2 (CD86) (Freedman, A.S. et al. (1987) J. ImmunoL 137:3260-3267;
Freeman,G.J.etal.(1989)JlmmunoL 143:2714-27~;Freeman,G.J.etal.(1991)J:l~p.
Med. 174:625-631;Freeman,G.J.etal.(1993)5cience2k~:909-911;Az~ima,M.etal.(1993)
S Nature ~:76-79; Freeman, GvJ. et al. (1993) J~ p. Med. 178:2185-2192)) can be used to
mduce stimulation of the CD28 molecule. In addition, binding 1~ f~ $ ' of a natural
ligand, whether native or synthesized by chemical or li~UlllbUlo.ll~ technique, can also be used
in accordance with the invention. Ligands useful for stimulating an accessory molecule can
be used in soluble form or immflhili7f d on a solid phase surface as described herein. Anti-
10 CD28 antibodies of fragments thereof useful in stimulating proliferation of CD4+ T cellsinclude ,...~ antibody 9.3, an IgG2a antibody (Dr. Jeffery Ledbetter, Bristol Myers
Squibb l'flrrf rAtifm, Seattle, WA)"".. ~ antibody KOLT-2, an IgGI antibody, ISE8,
an IgGI antibody, 248.23.2, an IgM antibody and EX5.3D101 an IgG2a antibody.
A preferred anti-CD28 antibody is mnr f f lf~nqi antibody 93 or EX53D10~ The
EX5.3DI0 " " ,. - f 1.1~ antibody was derived from; " .. ., ,1. ,~ a Balb/c mouse with CHO
(Chinese hamster ovary) cells transfected with the human CD28 gene (designated CHO-hh).
Hybl;duul~ from the fusion were selected by whole cell ELISA screening against Jurkat
(human T leukemia) CD28 i ~ designated Jurkat #7. Reactivity of the EX5.3D10
with CD28 was further confirmed by fluorescent activated cell sorter analysis (FACS)
20 analysis in which it was tested side by side with the ' ' 9.3 (Figure 6). Neither
antibody bound to .,..~ d CHO-DG44 cells and their binding profiles were nearly
identical for the two CD28 .l~f,~,Luli lines, CHO-hh and Jurkat #7, as well as normal
humam peripheral blood ly . ' - y .~,.,. A hybridoma wmch produces the " .. . ~
antibody EXS.3D10 has been deposited with the American Type Culture Collection on June
25 4, 1993, at ATCC Deposit No. HB 11373.
In amother cll ~ " of the invention, an activated population of CD4+ T cells is
stimulated to proliferate by contacting the T cells with am agent which acts " ' '~y to
stimulate a signal in the T cell mediated by ligation of am accessory molecule, such as CD28.
The term "agent", as used herem, is mtended to encompass chemicals and other
30 pl , - ~ . "i. ,.1 compoumds which stimulate a . ' y or other signal in a T cell
without the ,c, ~ ..i for an interaction between a T cell surface receptor and ay molecule or other ligand. For ex~imple, the agent may act intr:lrPllnlqrly to
stimulateasignalassociatedwithCD281igation. Inonet".l.o.li ..1 theagentisanon-
IJuJt: ~ - v ~ compound. As the agent used in the method is intended to bypass the nahiral
35 receptor:ligand stimulatory mP.-h-~niem the term agent is not intended to include a cell
expressing a natural ligand. Natural ligands for CD28 include members of the B7 family of
proteins, such as B7-l(CD80) and B7-2 (CD86).
~ WO 95t33823 2 1 9 ~ 5 8~6 ~ ~ ' PCT/US9~113782
-7-
It is known that CD28 receptor stimulation leads to the production of D-3
hoa~ oim~ai~id~ ~ in T cells and that inhibition of the activity of l)Lu~,ulldlidylillu~:~vl 3-
kinase (PI3K) in a T cell can inhibit T cell responses, such as ly~ hvhille production and
cellular l~lulif~laiiull. Protein tyrosine phvalJllulylaiiull has also been shown to occur in T
5 cells upon CD28 ligation and it has been l , ",~c - d that a protein tyrosine kinase
inhibitor, herbimycin A, can inhibit CD28-induced IL-2 production (V! - I ' ,,' , P. et al.
(1992)J:~7.Med. 175:951-960;Lu,Y.etal.(1992)~/mmunoL 149:24-29). Thus,to
selectively expand a population of CD4+ T cells, the CD28 receptor mediated pathway can
be stimulated by contacting T cells with an activator of P13K or an agent which stimulates
10 protein tyrosine ~Jhoa~ ulylaiiull in the T cell, or both. An activator of PI3K can be identified
based upon its ability to stimulate production of at least one D-3 L~ 1r- in a T cell.
The term "D-3 ~ " is intended to include derivatives of l ' , ' lylinoailul
that are ,uLua~lllvlyL.8 l at the D-3 position of the inositol ring and ~ the
crlmrolmrlc~ oalJlldti~lyli~ ~1(3)-1.l....--l} .~I.h '~ (ptdIns(3)p)~lullua~ulldli~lylillo :ol(3,4)-
1 . ~ . ' (Ptdlns(3,4)P2), and ~JLui~JLaiidylillua;iul(3~4,5)-i ~, ~ -, '
(Ptdlns(3,4,5)P3). Thus, in the presence of a P13K activator, the amount of a D-3
1...~l.1...:.,..-:l;.l. intheTcellisincreasedrelativetotheamountoftheD-3~ 1-1.r~in the T cell in the absence ûf the substance. Production of D-3 l.l)-.~l .~. .:, .. .~:; ;,1. ~ (e.g.,
Ptdlns(3)P, Ptdlns(3,4)P2 and/or Ptdlns(3,4,5)P3) in a T cell can be assessed by standard
20 methods, such as high pressure liquid ~,LIl O , ' ~ or thin layer . Lll O , ' y, as
discussed above. Similarly, protein tyrosine l ' . ' ylatiull can be stimulated in a T cell,
for example, by contacting the T cell with an activator of protein tyrosine kinases, such as
p~.~ ' (seeO'Shea,J.J.etal.(1992) Proc.Natl.Acad Sci. USA 84:10306-103101;and
Secrist, J.P. (1993) J. BioL Clzem. ~:5886-5893). Alternatively, the T cell c~m be contacted
25 with an agent which inhibits the activity of a cellular protein tyrosine l.l ~r ~ , such as
CD45, to increase the net amount of protein tyrosine ~yhuaullvlylatiull in the T cell. Any of
these agents can be used to expamd am activated population of CD4+ T cells in accordance
with the methods described herein.
In order to induce ,ulvlif~..aiiull and expand a population of CD8+ T cells, an activated
30 population of T cells is stimulated through a 27 kD accessory molecule found on activated T
cells and recogmzed by the l ' antibody ES5.2D8. As described in Example 9, a
population of CD8+ T cells was ,ul~ f~ tially expanded by stimulation with an anti-CD3
rl.. ' I antibodyandtheES5.2D8.. r.. L. -1antibody. The.",. ~ antibody
ES5.2D8 was produced by i. . - - ~ of mice with activated human blood ly . ' y ~;,
35 and boosted with l human CTLA4 protein produced in ~. coli . The ES5.2D8
" , ~ amtibody is of the IgG2b isotype and specifically binds to cells transfected with
human CTLA4. HylJl;dvl~laa producmg CTLA4-specific antibody were identified by
screening by ELISA against human CTLA4 protein as well as by differential FACS against
W095/33823 219~58G - PCT/U594113782--
-8 -
wild type CHO-DG44 cells vs. CHO-lOSA cells, which are transfected with the human
CTLA4 gene. As shown in Figure 7, the ES5~2D8 clone re~ts strongly with both activated
human T cells and CHO-105A cells but not with CHO-DCA4 cells, indicating that it does
indeed bind to CTLA4. h~ u..J,ulc. ;p;~tiU~I of detergent Iysates of surface labeled activated
S human T cells revealed that ES5.2D8 also re~ts with a 27 kD cell surf~e protein (Figure 8).
A hybridoma which produces the mrnA~ rlnAl antibody ES5.2D8 was deposited on June 4,
1993 with the American Type Culture Collection at ATCC Deposit No. HB11374.
Accordingly, to expand a population of CD8+ T cells, an antibody, such as
mt~nAIrlrnAl antibody ES5.2D8, or other antibody which recognizes the same 27 kD ligand as
ES5.2D8 can be used. As described in Example 10, the epitope recognized by the
antibody ES5.2D8 was identified by screening a phage display library (PDL).
Antibodies which bind to the same epitope as the - ' ' antibody ES5.2D8 are within
the scope of the invention. Such antibodies can be produced by; . .. .; ,~ with a peptide
fragment including the epitope or with the native 27 kD antigen. The term "epitope", as used
15 herein, refers to the actual structural portion of the antigen that is; " ,. ~rlA~ ly bound by
an antibody combining site. The term is also used ~ I,L~I)ly with "antigenic
". A preferred epitope which is bound by an antibody or other ligand which is tobe used to stimulate a CD8+ T cell population includes or . . ~ , an amino acid
sequence:
(Xaal)n-Gly-Xaa2-Trp-Leu-Xaa3-Xaa4-Asp(Glu)-(Xaas)n (SEQ ID NO: 5),
wherein Xaa4 may or may not be present, Xaal, Xaa2, Xaa3, Xaa4 and Xaas are any amino
acid residue and n = 0-20, more preferably 0-10, even more preferably 0-5, and most
preferably 0-3. In a preferred e ' - ' t, Xaa2 is Cys, ne or Leu, Xaa3 is Leu or Arg and
Xaa4, if present, is Arg, Pro or Phe. As described in Example 10, the mA~nrrlrn'~l antibody
ES5.2D8, which specifically binds a 27 kD antigen on activated T cells was used to screen a
cDNA library from ~tivated T cells to isolate a clone encoding the antigen. Amino ~id
sequence analysis identified the antigen as CD9(SEQ ID NO: 6). In the native human CD9
molecule, epitope defrned by phage display library screening is located at amino acid residues
31-37 (i.e., G L W L R F D (SEQ ID NO: 7)). Accordingly, Xaal and Xaa4 are typically
additional amino ~id residues found at either the amino or carboxy side, or both the amino
and carboxy sides, of the core epitope in the hu~nan CD9 (the full-length amino acid sequence
of which is shown in SEQ ID NO: 6). It will be appreciated by those skilled in the art that in
the native protein, additional non-contiguous arnino ~id residues may also contribute to the
~" r. ,"..A~ ..Al epitope recogruzed by the antibody. Synthetic peptides ~ g the
epitope can be created which includes other amino ~id residues flanking the core six amino
~WO 95/33823 2 1 9 ~ 5 8 6 I ~ , ~ PCTIUS94113782
acid residues (i.e., Xaa can alt~ ,aii~.,ly be other amino acid residues than those found in the
native CD9 protein). These flanking amino acid residues can function to alter the properties
of the resulting peptide, for example to increase the solubility, enhance the;, . " " ~. ,r~ y
or promote ." of the resultant peptide. When the peptide is to be used as an
5 i " " . ", . .~ ,~. . " one or more charged amino acids (e.g., Iysine, arginine) can be included to
increase the solubility of the peptide and/or enhance the " "J of the peptide.
Alternatively, cysteine residues cam be included to increase the .l:.. ;,r:;. ~ ~ of the resulting
peptide.
Othe m .. .l ~ù~l; ". ., ~ of the invention pertain to expansion of a population of CD8+ T
10 cells by use of an agent which acts intrPrPI' ' Iy to stimulate a signal in the T cell mediated
by ligation of CD9 or other CD9-associated molecule. It is known that CD9 belongs to the
TM4 au,u~ ~uily of cell surface proteins which span the membrane four times (Boucheix, C.
et al. (1990) ~ BioL Chem. 266, 11 7-l~ and Lan~a, F. et al. (1990) ~ BioL Chem. 266,
10638-10645). Other members of the TM4 superfamily include CD37, CD53, CD63 and
15 TAPA- I . A role for CD9 in interacting with GTP binding proteins has been suggested
(Sechafer, J.G. and Shaw, A.R.E. (1991) Biochem. Biophys. Res. Commun. 179, 401-406).
As used herein the term "agent" ..... ,.. ~ chemicals and other pl.~
' which stimulate a signal in a T cell without the 1. 1 C~l.,llt for an mteraction
between a T cell surface receptor and a ligand. Thus, this agent does not bind to the
20 r~trPrPlllllPr portion of CD9, but rather mimics or induces an ;, .l . ,... lli .' - signal (e.g., second
messenger) associated with ligation of CD9 or a CD9-associated molecule by an appropriate
ligand. The ligands described herem (e.g., -' ' antibody ES5.2D8) can be used toidentify an ~ " ' signal(s) associated with T cell expansion mediated by contact of the
CD9 antigen or CD9-associated molecule with an appropriate ligamd (as described in the
25 E~xamples) amd examining the resultant ;"l. . . !'..1 -- signalling that occurs (e.g., protein
tyrosine 1 ' , ' yldtiull~ calcium influx, activation of a.,lk. ~'Lill~i ~ and/or tyrosine
kinases, pllc,a~/llatidyl inositol .. s~ , etc.). An agent which enhances an ;. ,l . .. Ili ~
signal associated with CD9 or a CD9 rori~tP~l molecule cam then be used to expand CD8+
T cells. Ar ~,Iy, agents (e.g., small molecules, drugs, etc.) can be screened for their
30 ability to inhibit or enhamce T cell expansion usmg a system such as that described im the
Examples.
In yet another aspect of the invention, methods for expanding a population of antigen
specific T cells are provided. To produce a population of antigen specific T cells, T cells are
contacted with an antigen in a form suitable to trigger a primary activation signal in the T
35 cell, i.e., the antigen is presented to the T cell such that a sigmal is triggered in the T cell
~rough the TCR/CD3 complex. For example, the antigen can be presented to the T cell by
an antigen presenting cell m conjuction with an MHC molecule. An antigen presenting cell,
such as a B cell" .ul.}. .,~,., monocyte, dendritic cell, Langerhan cell, or other cell which
WO 95/33823 2 1 9 1 5 8 6 1 ~ ' PCT/US94/13782
-10-
can present antigen to a T cell, can be incubated with the T cell in the presence of the antigen
(e.g., a soluble antigen) such that the antigen presenting cell presents the antigen to the T cell.
Alternatively, a cell expressing an antigen of interest can be incubated with the T cell. For
example, a turnor cell expressing turnor-associated antigens c_n be incubated with a T cell
5 together to induce a tumor-specific response. Similarly, a cell infected with a pathogen, e.g. a
virus, which presents antigens of the pathogen can be incubated with a T cell. Following
antigen specific activation of a population of T cells, the cells can be expanded in accordance
with the methods of the invention. For example, after antigen specificity has been
P~-s~ hP~l T cells can be expanded by culture with an anti-CD3 antibody and an anti-CD28
10 antibody according to the methods described herein.
The term "antibody" as used herein refers to;, . " ".,. ,nvl, ~b -'; . . molecules and
-' ~- 'Iy active portions of ~,' ' ' molecules, i.e., molecules that containan antigen binding site which specifically binds (;.I.~ .v.~G.,la with) an antigen, such as
CD3, CD28. Structurally, the simplest naturally occurring antibody (e.g., IgG) comprises
15 four polypeptide chains, two heavy IH) chains and two light (L) chains inter-connected by
disulfide bonds. It has been shown that the antigen-binding function of an antibody cari be
performed by fragrnents of a naturally-occurring antibody. Thus, these antigen-binding
fragments are also intended to be designated by the term ''antibody". Bxamples of binding
fragments , ' witbin the term antibody include (i) an Fab fragment consisting of20 the VL, VH, CL and CHI domains; (ii) an Fd fragment consisting of the VH and CHI
domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an
antibody, (iv) a dAb fragment (Ward et al., (1989) Nature ~1:544-546 ) which consists of a
VH domain; (v) an iâolated . ' ~J ~' g region (CDR); and (vi) an F(ab')2
fragment, a bivalent fragment comprising two Fab fragments linked by a disulf de bridge at
25 tbe hinge region. Fl ' , although the two domains of the Fv fragment are coded for by
separate genes, a syntbetic linker can be made that enables them to be made as a single
protein chain (known as single chain Fv (scFv); Bird et al. (1988) Science 2~2:423-426; and
Huston et al. (1988) PN~S ~:5879-5883) by .~.. 1.:.. ,l methods. Such single chain
antibodies are also ~ .I within the term "antibody". Preferred antibody fragments
30 for use in T cell expansion are those which are capable of ~,luaali. 'uhlg their target antigen,
e.g., bivalent fragments such as F(ab')2 fragments. Alternatively, an antibody fragment
which does not itself crosslink its target antigen (e.g., a Fab fragment) can be used in
with a secondary antibody which serves to crosslink the antibody fragment,
thereby .,., " ' ~ the target antigen. Antibodies can be fragmented using eullv.~lltiollGl
35 techniques as described herein and the fragments screened for utility in the same marmer as
described for whole antibodies. An antibody of the invention is further mtended to include
bispecific amd cbimeric molecules having a desired binding portion (e.g., CD28).
~WO 9!i/33823 ' ; PCT/US94/13782
2 1 9 1 ~ 8 6 ! .
The language "a desired binding specificity for an epitope", as well as the moregeneral language "an antigen binding site which specifically binds (h~ ullul~a~L ~ with)",
refers to the ability of individual antibodies to specifically hll,l~ ,l..a~l with a T cell surface
molecule, e.g. CD28. That is, it refers to a non-random binding reaction between an antibody
5 molecule and an antigenic d of the T cell surface molecule. The desired binding
specificity is typically determined from the reference point of the ability of the antibody to
differentially bind the T cell surface molecule and an unrelated antigen, and therefore
distinguish between two different antigens, pO Li~,ulafly where the two antigens have unique
epitopes. An antibody which binds specifically to a particular epitope is referred to as a
10 "specific antibody".
"Antibody combining site", as used herein, refers to that structural portion of an
antibody molecule comprised of a heavy and light chain variable and L.~ lJle regions
that specifically binds ( ~L, with) antigen. The term ''hlllllullul~ '' or "reactive
with" in its various forms is used herein to refer to binding between an antigenic .1. l.., .. ;. ,,...l -
15 containing molecule amd a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereo~
Although soluble forms of antibodies may be used to activate T cells, it is preferred
that the amti-CD3 antibody be im~nnhili7~-1 on a solid phase surface (e.g., beads). An
antibody can be immnhili7rd directly or indirectly by, for example, a secondary antibody, to
20 a solid surface, such as a tissue culture flask or bead. As an illustrative ~. . .l .o.l;, . .. ', the
following is a protocol for immllhili7inF an anti-CD3 antibody on beads. It should be
appreciated that the same protocol can be used to immobili_e other antibodies or fragments
thereof (e.g., an anti-CD28 antibody) to beads.
Protocols
I. Pre-absorbing Goat anti-mouse IgG with OKT-3
A) BioMag Goat anti-Mouse IgG (Advanced Magnetics, Inc., catalog
number 8-4340D) is incubated with at least 20011g of OKT-3 per 5 x 108
magnetic particles in PBS for I hour at 5~C.
B) Particles are washed three time in PBS with the aid of a magnetic
separation unit.
Note: Advanced Magnetics also has an anti-Human CD3 direcdy conjugated
(Catalog number 8-4703N) which will induce T-cell stim~ ti.~n
II. Pre-labeling Lylll~L~ with OKT-3
A) I x I o6 cells (PBMC) are incubated in PBS with 10~Lg/ml of OKT-3
for 15 minutes at room ~
B) Cells are washed twice with PBS.
WO95/33823 2 1 9 1 5 8 6 ; ~ i ' PCT/IJS91/13782
III. Binding Magnetic Particles to PBMC for Stimulation
A) PBMC surface labeled with OKT-3 are c~ltured with Goat anti-Mouse
IgG (see above) at one bead per cell following a 30 minute incubation at 20~C
with gentle agitation.
B) Goat anti-Mouse IgG beads which were previously absorbed to OKT-3
are incubated with PBMC (1:1) for 30 minutes at 20~C with gentle agitation
and cultured.
IV. Binding Magnetic Particles to PBMC for Separation
Same as above (Part III) except the bead to cell ratio is increased to 20:1 rather
than 1: 1.
To practice the method of the invention, a source of T cells is obtained from a subject.
The term subject is intended to include living organisms in which an immune response can be
15 elicited, e.g., mammals. Examples of subjects mclude humans, dogs, cats, mice, rats, and
transgenic species thereof. T cells can be obtained from a number of sources, including
peripheral blood leukocytes, bone marrow, Iymph node tissue, spleen tissue, and tumors.
Preferably, peripheral blood leukocytes are obtained from an individual by 1~ ' rl To
isolate T cells from peripheral blood leukocytes, it may be necessary to Iyse the red blood
20 cells and separate peripheral blood leukocytes from monocytes by, for example,
~ ~ ~ a . . rl ~ through a pERcoLLrM gradient~ A specific: bl . ' of T cells, such as
CD4+ or CD8+ T cells, can be further isolated by positive or negative selection techniques.
For example, negative selection of a T cell population can be a , ' ' ' with a
of antibodies directed to surface markers unique to the cells negatively selected.
25 A preferred method is cell sorting via negative magnetic - " which utilizes a
cocktail of IllUII~ antibodies directed to cell surface markers present on the cells
negatively selected. For example, to isolate CD~+ cells, a r~ n ~l antibody cocktail
typicallyincludesantibodiestoCD14,CD20,CDllb,CD16,HLA-DR,andCD8.
Additional, ...,"1.. - 1 antibody cocktails are provided in Table 1.
The process of negative selection results in an essentially 1.. , . ,~ .S,.. ,., populatiûn of
CD4+ or CD8+ T cells. The T cells can be activated as described herein, such as by contact
with a anti-CD3 antibody ;,..",.,l"l;, ~I ûn a solid phase surface ûr an anti-CD2 antibody, ûr
by contact with a protein kinase C activator (e.g., bryostatin) in, ; with a calcium
ionophore. To stimulate an accessory molecule on the surface of the T cells, a ligand which
35 binds the accessory molecule is employed. For example, a population of CD4+ cells can be
contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions
appropriate for stimulating ~ .l, f. ~;"" of the T cells. Similarly, to stimulate proliferation of
CD8+ T cells, an amti-CD3 antibody and the ' ' antibody ES5.2D8 can be used.
WO 95/33823 2 1 9 1 5 8 6 ~ PCT/US94/13782
Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal
Essential Media or RPMI Media 1640) which may contain factors necessary for proliferation
and viability, including animal serum (e.g., fetal bovine serum) and antibiotics (e.g.,
penicillin streptomycin). The T cells are maintained under conditions necessary to support
growth, for example an appropriate tc~ c (e.g., 37~C) and atmosphere (e.g., air plus
5% CO2).
To maintain long term stimulation of a population of T cells following the initial
activation and StimlllDtinn it is necessary to separate the T cells from the activating stimulus
(e.g., the anti-CD3 antibody) after a period of exposure. The T cells are maintamed in
10 contact with the co-stimulatory ligand throughout the culture term. The rate of T cell
l.lolif~ iivll is monitored p~,.iov;cOlly (e.g., daily) by, for example, examining the size or
measuring the volume of the T cells, such as with a Coulter Counter. A resting T cell has a
mean diameter of about 6.8 microns. Following the initial activation and stimulation and in
the presence of the stimulating ligand, the T cell mean diameter will increase to over 12
15 microns by day 4 amd begin to decrease by about day 6. When the mean T cell diameter
decreases to ~ plv~ill._'cly 8 microns, the T cells are reactivated and ~ l 1l 5 d to induce
further ~lvlif~ liul~ of the T cells. Alternatively, the rate of T cell ~lvlif~ tivll and time for
T cell ~ ;.... can be monitored by assaying for the presence of cell surface molecules,
such as B7-1, B7-2, which are induced on activated T cells. As described in Example 5, it
20 was determined that CD4+ T cells do not initially express the B7-1 receptor, and that with
culture, expression is mduced. Further, the B7-1 expression was found to be transient, and
could be re-induced with repeated anti-CD3 1 J;""~ ;..,. Accordmgly, cyclic changes in
B7- 1 expression can be used as a meaDs of monitoring T cell p, . .1; 1~ where decreases
in the level of B7-1 expression, relative to the level of expression following an initial or
previous stimulation or the level of expression in an I ' ' cell, indicates the time for
1. ~;;"",1,.;",l
For inducimg long term stimulation of a population of CD4+ or CD8+ T cells, it may
be necessary to reactivate and restimulate the T cells with a anti-r~n3 Dn~ihvdy and an anti-
CD28 amtibody or ' ' antibody ESS.2D8 several times to produce a population of
CD4+ or CD8+cells mcreased m number from about 10- to about l ,000-fold the original T
cell population. Usimg this methodology, it is possible to get increases in a T cell population
of from about 100- to about 100,000-fold an original resting T cell population. Moreover, as
described in Example 6, T cells expanded by the method of the invention secrete high levels
of cytokines (e.g., IL-2, IFNr, IL-4, GM-CSF and TNFa) mto the culture ~ For
example, as compared to stimulation with IL-2, CD4+ T cells expanded by use of anti-CD3
and anti-CD28 ~ ' secrete high levels of GM-CSF and TNFa into the culture
medium. These cytokines can be purified from the culture 1 or the ~
cam be used directly for ~ ~ cells in culture. Similarly, the T cells expanded by the
WO 95/33823 2 1 9 1 ~ 8 6 ' ' PCTIUS94/13782
-14-
method of the invention together with the culture supernatant and cytokines can be
allllhl;~t~.Ld to support the growth of cells in vivo. For example, in patients v~ith tumors, T
cells can be obtained from the individual, expanded in vitro and the resulting T cell
population and supernatant. including cytokines such as TNFa, can be .. -.1,";": a~ lc;d to the
5 patient to augment T cell growth in vivo.
Althougb the antibodies used in the methods described herein can be readily obtained
from public sources, such as the ATCC, antibodies to T cell surface accessory molecules, the
CD3 complex, or CD2 can be produced by standard techniques. MPtho~logiPe for
generating antibodies for use in the methods of the invention are described in further detail
I 0 below.
I. Antih~dy Pr~ ~nrti~n
A. ThPI.,.",.. ,,,.. Theterm "i~ n~ isusedhereintodescribeac~ u~
containing a peptide or protein as an active ingredient used for the preparation of antibodies
15 against an antigen (e.g., CD3, CD28). When a peptide or protein is used to induce antibodies
it is to be umderstood that the peptide can be used alone, or linked to a carrier as a conjugate,
or as a peptide polymer.
To generate suitable antibodies, the ~ ~, should contain an effective,
., amount of a peptide or protein, optionally as a conjugate linked to a carrier.
20 The effective amoumt of peptide per unit dose depends, among other things, on the species of
animal inoculated, the body weight of the animal and the chosen regimen as is
well known in the art. The ~ preparation will typically contain peptide
cr~nrP-~tir.ne of about 10 ~ O to about 500 milligrams per ~ ~ dose,
preferably about 50 ~ UolCUl~.~ to about 50 milligrams per dose. An ;...., , ~
25 preparation cam also mclude an adjuvant as part of the diluent. Adjuvants such as complete
Freund's adjuvant (CFAJ, incomplete Freund's adjuvant (IFA) and alum are materials well
known m the art, amd are available ~,wll..~ ,;ally from several sources.
Those skilled m the art will appreciate that, instead of usmg natural occurring forrns
of the antigen (e.g., CD3, CD28) for ~ ~ ~ synthetic peptides can ' ' ~ be
30 employed towards wbich antibodies cam be raised for use in tbis invention. Both soluble and
membrane bound forms of the protein or peptide fragments are suitable for use as an
~ O and can also be isolated by ~ ~ ~y purification as well. A purified form
of protein, such as may be isolated as described above or as known in the art, can itself be
directly used as an ~ ~ or ~t~.lllati~ly ~ can be linked to a suitable carrier protein by
35 I;UIl~,lltiU~l~l techniques~ including by chemical coupling means as well as by genetic
o. ;,~g using a cloned gene of the protem. The purified protein can also be covalently or
null~,ù~ , modified with non-plu~acc~v~i~ materials such as lipids or ~,alboll.~d to
enhance ~ ,, ~hy or solubility. Altematively, a purified protem can be coupled with
_wossi33823 2 1 9 ~ 5 8 6 PCT/Uss4/l3782
-15-
or ill ,UI~/UI~ d into a viral particle, a replicating virus, or otuer LUi~lUUl~ULi~lll in order to
enhance; ~ g. ..;- ;;y. The protein may be, for example, chemically attached to the viral
particle or Illh.lUUlo~ul;i~lll or an ;.,.... ~ ,~, ..,;~ portion thereof.
In an illustrative c~hu~ , a purified CD28 protem, or a peptide fragment thereof5 (e g., produced by limited proteolysis or, r~ I DNA techniques) is conjugated to a
carrier which is ~ in animals. Preferred carriers include proteins such as
albumins, serum proteins (e.g., globulins and li~u~u~,h~), and polyamino acids. Examples
of useful proteins include bovme serum albumin, rabbit serum albumin, thyroglobulin,
keyhole limpet L~ uoUy~ egg ovalbumin and bovine gamma-globulins. Synthetic
10 polyamino acids such as polylysine or polyarginine are also useful carriers. With respect to
the covalent attachment of CD28 protein or peptide fragments to a suitable; . " .. ,., .~ r
carrier, t'uere are a number of chemical cross-linking agents that are known to those skilled in
the art. Preferred cross-linking agents are 1,. 1. . ol .; r, . -- d . ., .~1 cross-linkers, which can be used
to link proteins in a stepwise manner. A wide variety of L.,t~,l..l.; r, .... I ;.., .~1 cross-linkers are
15 known in the art, including ~u~ hulllidyl 4-(N-... 8 ...:.1.. ,. II,yl) cy~ albuAy~
(SMCC),ml''' ' ' ~yl-N-Ly~u~y~.l ;l- Il;L ester(MBS);N-~uc.iuLlu,idyl(4-
iodoacetyl) - - ;----1,- ~ ,-~ ~ (SIAB), ~. Iyl 4~(p 1 ~ r~ yl) butyrate (SMPB),
I-ethyl-3-(3-dil.~.,;l.yl~,,il,u~,ul,yl) .,~uI,udiiu,,ilc l,.~u.,Llu,idc (EDC); 4~ ~
u~y~,ulbuuyl-a-methyl-a-(2-~ lylu;lLio)-tolume (SMPT), N-~u~iL2Luidyl 3-(2-
20 pyridyldituio) propionate (SPDP), ~u~,~,iulhll;dyl 6-[3-(2-1Jylidyld;L' uo) propionate] hexanoate
(LC-SPDP).
In may also be desirable to simply immunize an animal with whole cells which
express a protein of interest (e.g., CD28) on their surface. Various cell Imes can be used as
;..--, -.-~0. - ~ to generate . - ..~C~ antibodies to an antigen, including, but not limited to T
25 cells. For example, peripheral blood T cells can be obtained from a subject which
CUIL~LiLUiLi~,ly express CD28, but c~m be activated in vi~ro with anti-CD3 antibodies, PHA or
PMA. Alternatively, an antigen specific (e.g., alloreactive) T cell clone can be activated to
express CD28 by l.,c of antigen, together with a ' y signal, to the T cell.
Whole cells tbat can be used as " to produce CD28 specific antibodies also
30 include ,c ~ ~ . r ' For example, COS and CHO cells can be . ~ ....t; I . It. ~1 by
~ r ~ W ith a CD28 cDNA to produce cells expressing CD28 on their surface. Thesei, ~. .- f ~ ' - .I cells can then be used as O to produce amti-CD28 antibodies. Other
examples of 1 . ,... f ~ cells are known, particularly eukaryotic cells able to glycosylate the
CD28 protein, but any procedure that works to express transfected CD28 genes on the cell
35 surf~e could be used to produce the whole cell ;I~ Ih~,...l
Alternative to a CD28 c,.~ , .iu.g cell or am isolated CD28 protein, peptide fragments
of CD28 or other surface antigen such as CD9 can be used as ~ ~ to generate
antibodies. For example, the CD9 epitope bound by the ES5.2D8 ' I antibody
WO 95/33823 2 1 9 1 5 8 6 PCT/US94/13782
-16-
comprises an amino acid sequence: (Xaal)n-Gly-Xaa2-Trp-Leu-Xaa3-Xaa4-Asp(Glu)-
(Xaas)n (SEQ ID NO 5), whe}ein Xaa4 may or may not be present Xaal, Xaa2, Xaa3, Xaa4
and XaaS are any amino acid residue and n = 0-20, more preferably 0-10, even more
preferably 0-5, and most p}eferably 0-3. In a preferred ~".l,o.l;.". .,1 Xaa2 is Cys, Ile or Leu,
S Xaa3 is Leu or Arg and Xaa4, if present, is Arg, Pro or Phe. Thus, a peptide having the
amino acid sequence of SEQ ID NO: S can be used as an; " ", - ",. . . Accordingly, the
invention further ~ an isolated CD9 peptide comprising an amino acid sequence:
(Xaal)n-Gly-Xaa2-Trp-Leu-Xaa3-Xaa4-Asp(Glu)-(Xaas)n (SEQ ID NO: 5), wherein Xaa4may or may not be present, Xaal, Xaa2, Xaa3, Xaa4 and Xaas are any amino acid residue
and n = 0-20, more preferably 0- 1 0, even more preferably 0-5, and most preferably 0-3. In a
preferred ~ ..,l ,n.l', Iq Xaa2 is Cys, Ile or Leu, Xaa3 is Leu or Arg and Xaa4, if present, is
Arg, Pro or Phe. Alternatively, it has been found that the ES5.2D8 " ,. ,.~ 1 antibody
cross-reacts with a number of other peptide sequences (determined by phage display
techmology as described in Example 3). Examples of these other peptide sequences are
shown below:
2D8#2(SEQIDNO:l) HQFCDHWGCWLLRETHIFTP
2D8#4 HQFCDHWGCWLLRETHIFTP
2D8#10 HQFCDHWGCWLLRETHIFTP
2D8#6(SEQIDNO:2) LRLVLEDPGIWLRPDYFFPA
G C W L L R E (phage 2D8#2, 4, 10; SEQ ID NO: 3)
G I W L R P D (phage 2D8#6; SEQ ID NO: 4)
G L W L R F D (CD9 sequence; SEQ ID NO: 7. )
Any of these peptides, or other peptides containing a stretch of seven amino acids bracketed
in bold type (lc~lca~ hlg the epitope bound by the antibody) possibly flanked by alternative
amino acid residues, can also be used as O to produce an antibody for use in the
methods of the invention and are . .... I~ by the invention. For use as g
30 peptides can be modified to increase solubility and/or enhance ~ J as described
above.
B. polyrl~.nDl Anrihn~liPc Polycolonal antibodies to a purified protein or peptide
fragment thereof can generally be raised in animals by multiple ' (sc) or
35 ~ 1 ' (ip) injections of an appropriate ,, such as the PYtrAA~Il ' domain
of the protem, and an adjuvamt. A polyclonal antisera can be produced, for eY~ample, as
described in Lindsten, T. et al. (1993) J. Immunol. 151:3489-3499. In an illustrative
t, animals are typically immunized against the ~L - protein, peptide or
~ WO 951338t3 2 1 9 1 5 8 6 ~ ~ . , PCTIUS94/1378t
derivative by combining about Illg to I mg of protcin with Freund's complete adjuvant and
injecting the solution inrrR~;Prrnqlly at multiple sites. One month later the animals are boosted
with 1/5 to 1/10 the original amount of i,.. ,.. n~,~ ., in Freumd's complete adjuvant (or other
suitable adjuvant) by ~,~ injection at multiple sites. Seven to 14 days later, the
5 animals are bled and the serum is assayed for amti-protein or peptide titer (e.g., by ELISA).
Animals are boosted until the titer plateaus. Also, &OO,~Oa~hlg agents such as alum can be
used to enhance the immune response.
Such ", "", li, .-produced pu~uuladulla of antibody molecules are referred to as"polyclonal" because the population comprises antibodies with differing ;~ ; r~
10 and affinities for the antigen. The antibody molecules are then collected from the mammal
(e.g., from the blood) amd isolated by well known techniques, such as protein A
~,1... O . ' .y~ to obtain the IgG fraction. To enhance the specificity of the antibody, the
antibodies may be purified by - ~ ~J ~lu~ ", . ~ .YI "~ y using solid phase-affixed
;,"" ~." ,~;. ., The antibody is contacted with the solid phase-affixed ;.,.",- O. ., for a period
15 of time sufficient for the i"" " ~ ."O. ,. to with the antibody molecules to form a
solid phase-affixed ;..., ~ The bound antibodies are separated from the complex
by standard techniques.
C. Mnnorl~nqlAntihn~iiPc Theterm"...n .rl.~. ~1 antibody"or",-,-, ~l"~-I
20 amtibody c~ ", as used herein, refers to a population of amtibody molecules that
contain only one species of an antigen binding site capable of; " ." - ~ i. ,g with a
particular epitope of am amtigen. A .... .~ l antibody rn~pcr;~i thus typically displays
a single bindmg affinity for a particular protein with which it ~ ~,a~la. Preferably, the
rl.l.l~.l antibody used in the subject method is further . l - ,-- ~ ;,- ~ as ~a~
25 with a protein derived from humans.
~ ,. .n~ l antibodies useful in the methods of the invention are directed to an
epitope of an antigen(s) on T cells, such that complex formation between the antibody and the
amtigen (also referred to herein as ligation) induces stimulation and T cell expansion. A
" ,. ,. ,. ,~l. ,. .~1 antibody to an epitope of an antigen (e.g., CD3, CD28) can be prepared by using
30 a technique which provides for the production of antibody molecules by continuous cell lines
in culture. These include but are not limited to the hybridoma technique originally described
by Kobler and Milstein (1975, Nafure 256:495-497), and the more recent human B cell
hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), EBV-hybridoma technique
(Cole et al. (1985), Mnn(lrl. 'A.ntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-
35 96), and trioma tecbniques. Other methods which can effectively yield " ~ n~amtibodies useful m the present invention include phage display techniques (Marks et al.
(1992) J Biol Chem 16007-16010).
W095/33823 -18- PCT/US94113782 --
In one I .,.1"~.1;",. .a, the antibody preparation applied in the subject method is a
~...."nrl..,.~l amtibody produced by a hybridoma cell line. Hybridoma fusion techniques were
first introduced by Kohler and Milstein (Kohler et al. Nature (1975) 256:495-97; Brown et al.
(1981) J. Immunol~:539-46; Brown et al. (1980) JBiol Chem 2~:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J Cancer 2~:269-75). Thus, the
' antibody .. ~l ~r.~ of the present invention can be produced by the following
method, which comprises the steps of:
(a) T..... :,;.,g an animal with a protein (e.g., CD28) or peptide thereof. The
is typicaTly ~ ., .l; ~ by , . . .; . .g the; ~ ~ to an
10 i- . - .- - ,nlng;~ _lly competent marnmal in an ' g 'ly effective amount, i.e., an
amount sufficient to produce an immune response. Preferably, the mammal is a rodent such
as a rabbit, rat or mouse. The mammal is then maintained for a time period sufficient for the
mammal to produce cells secreting antibody molecules that hl~ lul~ with the
;....,.- .n~,~... Such hlll~ lul~a~liull is detected by screening the antibody molecules so
producedfori~.. ,.. u.~livil~withapreparationofthe;.,.,.. ~,,.. ,protein. Optionally,it .
may be desired to screen the antibody molecules with a preparation of the protein in the form
in which it is to be detected by the antibody molecules in an assay, e.g., a membrane-
associated form of the antigen (e.g., CD28). These screening methods are well known to
those of skill in the art, e.g., enzyme-linked ~ ' assay (ELISA) and/or flow
20 cytometry.
(b) A suspension of antibody-producing cells removed from each imrnuni~ed marnmal
secreting the desired antibody is then prepared. After a sufficient time, the mouse is
sacrificed amd somatic antibody-producing 1~ A are obtained. Antibody-producing
cells may be derived from the Iymph nodes, spleens and peripheral blood of primed animals.
Spleen cells are preferred, and can be .. ~ .;. Ally separated mto individuaT cells in a
~Jh.1 aiOlog;~lly tolerable mediurn using methods well known in the art. Mouse l~...ul.c,. .1 ~O
give a higher percentage of stable fusions with the mouse myelomas described below. Rat,
rabbit and frog somatic cells cam also be used. The spleen cell l,LIl encoding
desired .' ' " are ~ - - --- .. i-l; . ~1 by fusmg the spleen cells with myeloma cells,
generally in the presence of a fusing agent such as pol.~ ; glycol (PEG) Any of a
number of myeloma cell lines may be used as a fusion partner according to standard
techniques; for example, the P3-NSI/I-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma
Imes. These myeloma lines are available from the American Type Culture Collection
(ATCC), Rockville, Md.
The resulting cells, which include the desired h.yl)lillull~ are then grown in aselective medium, such as HAT medium, m which unfused parental myeloma or Iymphocyte
cells eventually die. Only the hybridoma cells survive and cam be grown under limiting
dilution conditions to obtain isolated clones. The . of the h.~blilu~l_o are
~W095/33823 2 ~ 9 1 5 8 6 PCT/US9-1/13782
.: j r
-19-
screened for the presence of antibody of the desired specificity, e.g., by ;.1.., ., .. ,. ~A~-~,y
techniques using the antigen that has been used for:, .., ., I . : ,-1 ;.... Positive clones can then
be subcloned under limiting dilution conditions and the m~nrrlrnRI antibody produced can
be isolated. Various cul.v ~ Lion~l methods exist for isolation and ~ ; r~ of the
5 mnnr~rlonRl antibodies so as to free them from other proteins and other ~.. ."1,., " ;",.., ~
Commonly used methods for purifying ",..." ~ antibodies include ammonium sulfateprecipitation, ion exchange ~,L~ .Y, and affinity ~,LI.. ,.l.......... ~ .l.. y (see, e.g., Zola et
al. in Mnno( lorinl Hybridoma Antibodies: Techniques And A~lic.~iu,.." Hurell (ed.) pp. S I -
52 (CRC Press 1982)). Hybridomas produced according to these methods can be propagated
10 in vitro or in vivo (in ascites fluid) using techniques known in the art.
Generally, the individual cell line may be propagated in vitro, for example in
laboratory culture vessels, and the culture medium containing high .. ..,... ,u ,.1;.. ~ of a single
specific ' ' antibody can be harvested by (iPr~nt~tir~n~ filtration or ~ .; r, .~;~: ;....
Alternatively, the yield of " .-.. ,r.~8 ~ -I antibody can be enhanced by injecting a sample of the
hybridoma into a l . :~l.. r ~ animal of the type used to provide the somatic and
myeloma cells for the origmal fusion. Tumors secreting the specific, . .,1. .., I antibody
produced by the fused cell hybrid develop in the injected animal. The body fluids of the
animal, such as ascites fluid or serum, provide, .. 8.. ,,1 antibodies in high i.. 1. ,.1 ;
When humam L.yblidul~a or EBV-LylJIidulll~ are used, it is necessary to avoid rejection of
the xenograft injected into animals such as mice. T - ' ~ or nude mice may be used
or the hybridoma may be passaged first into irradiated nude mice as a solid ~1.1.~ 5~. v~
tumor, cultured in vitro and then injected LILI~IJ. 'Iy into pristane primed, irradiated
nude mice which develop ascites tumors secreting large amounts of specific human.1.... ..l,.~l .1.~l antibodies.
Media and animals useful for the preparation of these ~ are both well
known m the art and . "y available and include synthetic culture media, inbred mice
amd the like. An exemplary synthetic medium is Dulbecco's minimal essential medium
(DMEM; Dulbecco et al. (1959) ViroL 8:396) ~ t. ~I with 4.5 gm/l glucose, 20 mM
glutamme, and 20% fetal caf serum. An exemplary inbred mouse strain is the Balb/c.
D. C. ' ~ ~ ' Antihn~ c Monoclonal antibody o.. l.. ~:l;.. ~ ofthe invention
can also be produced by other methods well known to those skilled in the art of Ir~ . .."1.:., - .1
DNA technology. An alternative method, referred to as the "~ antibody display"
method, has been developed to identify and isolate antibody fragments having a particular
35 antigenspecificity,andcanbeutilizedtoproduce,.,.."..l.. ~lantibodies(for~Per-irtirn~of
~ ' antibody display see e.g., Sastry et al. (1989) PNAS~:5728; Huse et al.
(1989) Science 246:1275; and Orlandi et al. (1989) PNAS 86:3833). After ;.. .. .g an
animal with an appropriate ~ ~~, (e.g., CD3, CD28) as described above, the antibody
W095/33823 2 1 9 1 58~ PCTAUS94113782
-20-
repertoire of the resulting B-cell pool is cloned. Methods are generally known for directly
obtAining the DNA sequence of the variable regions of a diverse population of
;,. " "", ,ngh.l" ~ molecules by using a mixture of oligomer primers and PCR. For instance,
mixed nli~ ul i~lr primers .,ullc~l,vll~ g to the S' leader (signal peptide) sequences
5 and/or framework I (FRI ) sequences, as well as primer to a conserved 3' constant region
primer can be used for PCR ~nAplifir ~tinn of the heavy and light chain variable regions from
anumberofmurmeantibodies~Larricketal.(1991)~ir~t,~ 152-156). Asimilar
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 Enzymolo~
0 _:106-110).
In am illustrative ~ ,. ',o~ .,1 RNA is isolated from activated B cells of, for example,
peripheral blood cells, bone marrow, or spleen ~ iull~, using standard protocols (e.g.,
U.S. Patent No. 4,683,202; Orlandi, et al. PN~S (1989) ~:3833-3837; Sastry et al., PNAS
(1989) 86:5728-5732; and Huse et al. (198g) Science 246:1275-1281.) First-strand cDNA is
l S synthesized using primers specific for the constant region of the heavy chain(s) and each of
the IC and ~ light chains, as well as primers for the signal sequence. Using variable region
PCR primers, the variable regions of both heavy and light chains are amplified, each alone or
in c~ l -, and ligated into appropriate vectors for further . ' in generating
the display packages. Olig.,.. Ir~.l;rlf- primers useful in ,l,l;.',. - ;.", protocols may be
20 unique or degenerate or incorporate inosine at degenerate positions. Restriction r~ ,fl. 1. . .1~ 1. A~-
recognition sequences may also be; ~ .1 into the primers to allow for the cloning of
the amplified fragment into a vector in a ~ ' reading frame for exprcssion.
The V-gene library cloned from the ;- : ;, . derived antibody repertoire can be
expressed by a population of display packages, preferably derived from ''' phage, to
25 form an antibody display library. Ideally, the display package comprises a system that allows
the samplmg of very large variegated antibody display libraries, rapid sorting after each
affinity separation round, amd easy isolation of the antibody gene from purified display
packages. In addition to Cuuull~ I.y available kits for generating phage display libraries
(e.g., the Pharmacia R. ' : Phage Antibody System, catalog no. 27-9400-01; and the
Stratagene SurJZ~PTM phage display kit, catalog no. 240612), examples of methods and
reagents particularly amenable for use in generating a variegated antibody display library can
be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. 1" . ., .: ;. ."
Publication No. WO 92/18619; Dower et al. T" . . I .A. ;~ I Publication No. WO 91/17271;
Winteretal. TnfPrr~inn~l PublicationWO 92/20791; Marklandetal.1, a~ A1;l~llAl
Publication No. WO 9~115679; Breitling et al. T, llr 1 l ~ Publication WO 93/01288;
McCafferty et al. 1"t ~ I Publication No. WO 92/01047; Garrard et al. I
Publication No. WO 92/09690; Ladner et al. T"~ ;., ,I Publication No. WO 90/02809;
Fuchsetal.(1991)Bio/~Aechnology2:1370-1372;Hayetal.(1992)XumAnfibodllyb,i~.
~wo 95/33823 2 1 9 1 5 8 6 Pcr/uss4/l3782
21 ~
3:81-85;Huseetal.(1989)5cience246:1275-1281;Griffthsetal.(1993)EMBOJ12:725-
734; Hawkins et al. (1992) JMolBiol ~:889-896; Clackson et al. (1991) Nature 352:624-
628; Grarnetal. (1992)PNAS89:3576-3580; Garradetal. (1991)Bio/Tecknolo~y~:1373-
1377;Hoogr~l)ol~llletal (199T)NucAcidResl9:4133-4137;andBarbasetaL(l991)PNAS
5 ~:7978-7982.
In certain ~ ' , the V region domains of heavy and light chains can be
expressed on the same polypeptide, joined by a flexible linker to form a smgle-chain Fv
fragment, and the scFV gene ~- h,~l.. lly cloned into the desired expression vector or phage
genome. As generally described in McCafferty et al., Nature (1990) ~:552-554, complete
VH and VL domains of an 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
antigen affinity. Isolated scFV antibodies h l~ ;YC with the antigen can ~ f ly
be formulated into a l~l . -- . -- ~ ... ;. -l preparation for use in the subject method.
Once displayed on the surface of a display package (e.g., fil~nnPnt~nl~ phage), the
antibody library is screened with the protein, or peptide fragment thereof, to identify and
isolate packages that express an antibody having specificity for the protein. Nucleic acid
encoding the selected antibody can be recovered from the display package (e.g., from the
phage genome) and subcloned into other expression vectors by standard .c ' DNA
techniques.
F. Tlyhri~l-mn:~c an~l IvlPthn~ of PrP~ fion Hybridomas useful in the present
invention are those . l. ~. i. . ;,. .] as having the capacity to produce a ' ' antibody
which will specifically ~ ~,~l with an antigen of interest (e.g., CD3, CD28). Methods
for generating hybl;dulllaa that produce, e.g., secrete, antibody molecules having a desired
. ~ ~ ~" e.g., having the ability to with the CD28 antigen, and/or an
identifiable epitope of CD28 are well known in the art. Particularly applicable is the
hybridoma technology described by Niman et al. (1983) PNAS 80:4949-4953; and by Galfre
et al. (1981) Meth En23~mol. 1~:3-46.
IT IICP~ ofthPMPth~ of ~hPInVPnti~n
The method of this invention can be used to selectively expand a population of CD4+
or CD8+ T cells for use in the treatment of infectious disease, cancer and ' . y.
As a result of the method described herein, a population of T cells which is polyclonal with
respect to antigen reactivity, but essentially h~.. ,.~.~,.... ~,. ,~ with respect to either CD4+ or
35 CD8+ can be produced. In addition, the method allows for the expansion of a population of
T cells in numbers sufficient to ~c~,ullalil~ an individual's total CD4+ or CD8+ T cell
population (the population of l.~ l.U~ , in an individual is a~ , 1011). The
resultmg T cell population can be genetically transduced and used for ~ ' . ~ or can
WO 95133823 _ PCTIUS9411378~ --
2 1 9 1 586
-22-
be used in methods of in vifro analyses of infectious agents. For example, a population of
tumor-infiltrating lylll,uho~ can be obtained from an individual afflicted with cancer and
the T cells stimulated to proliferate to sufficient numbers. The resulting T cell population can
be genetically transduced to express tumor necrosis factor (INF) or other factor and restored
to the individual.
One particular use for the CD4+ T cells expanded by the method of the invention is in
the treatment of HIV infection in an individual. Prolonged infection with HIV eventually
results in a marked decline in the number of CD4+ T l~ hb~ . Tnis decline, in turn,
causes a profound state of ~ fi~ nry, rendering the patient susceptible to an array of
life threatening U,UIIUI ~ iC infections. R ~F' ' g the number of CD4+ T cells to normal
levels may be expected to restore immune function to a sigmficant degree. Thus, the method
described herein provides a means for selectively expanding CD4+ T cells to sufficient
numbers to Ic~,ullaLi~uLc this population in an HIV infected patient. It may also be necessary is
to avoid infecting the T cells during long-term stimulation or it may desirable to render the T
cells ~ y resistant to HIV infection. There are a number of techniques by vhich T
cells may be rendered either resistant to HIV infection or incapable of producing virus prior
to restoring the T cells to the infected individual. For example, one or more anti-retroviral
agents can be cultured with CD4+ T cells prior to expansion to inhibit HIV replication or
viral production (e.g., drugs that target reverse l~ ,l r ' and/or other < ~ of the
viral machinery, see e.g., Chow et al. (1993) Nafure 361, 650-6~3).
Several methods can be used to genetically transduce T cells to produce molecules
which inhibit HIV infection or replication. For example, in one l ' t, T cells can be
genetically transduced to produce 'I ' inhibitors, which are mutated"~....r, ,;....
forms of normal HIV gene products. T 1 ~ ' inhibitors function to nl ;y~ ûr
25 compete for binding with the wild type HIV proteins. Several i 11 inhibitors have
been derived from HIV proteins including tat, rev, and gag. The function of tat is to enhance
the I . . . ;p1;~ . of viral genes by binding to the trans activation response element (tar) found
m the promûter region of mûst HIV genes. Rev, through binding to the rev response element
(RRE) found at the 5' end of unspliced HIV transcripts, facilitates the transport of
30 ~ . u.,.,~ d mRNA from the nucleus to the cytoplasm for packaging into virions. Gag is
first synthesized as a smgle poly~ Jti.lc and ~ l , bJ cleaved by a Yill._ e..~,od~,.l
protease to yield three structural proteins, plS, pl7, and p24. T ' inhibitors
derived from these gene products have been ~' ' to inhibit infection of cells
cultured with lab pet HIV isolates. One example of a i ' inhibitor which appears
35 toactbyformingn. ..,r. ~ multimerswithwild-typeRevisRevM10. RevM10
construct has blocked infection of CEM cells by HTLV-IIIB for up to 28 days (Malim et al.
J~M176:1197,Bevecetal.PN~S89:9870). Inthesestudies,RevMlOfailedto~'
~WO 95/33823 , ~ PCT/US9~/13782
adverse effect on normal T cell function as judged by the criteria of growth rate and IL-2
secretion.
In another approach T cells can be transduced to produce molecules known as
"molecular decoys" which are binding elements for viral proteins critical to replication or
assembly, such as TAR. High level retrovirus-mediated expression of TAR in CEM SS cells
has been found to effectively block the ARV-2 HIV isolate, as measured by RT assay
(Sullenger et al. Cell 63:601). I~ )u~ llly, it also blocked SIV (SlVmac251) infection,
suggesting that inhibition of HIV infection with molecular decoys may be generally
applicable to various isolates and thereby alleviate the problem of Lyp~ uiabilily. Further,
it has been .1. .~ .ar~l that TAR expression has no discernible effects on cell viability
(Sullenger et al. J. Virol. 65:6811). Another "molecular decoy" which T cells can be
transduced to produce is a soluble CD4 tagged at the carboxy terminus with a KDEL (Iysine-
aspartic acid-glutamic acid-leucine) sequence, a signal for ER retention (Buonocore and
Rose, PNAS 90:2695)(Nature 345:625). The sCD4-KDEL gene expression is driven by the
HIV LTR. H9 cells transduced with the sCD4-KDEL construct show up regulation of
expression of intrRrrlllllRr CD4 upon HIV infection. This strategy effectively blocked
production of HIV MN for up to 60 days post infection. The proposed advantage of this
inhibitor is that the virus should not be able to escape it's effect by mut_tmg because CD4
binding is essential for HIV infectivity.
T cells cam also be transduced to express antisense molecules and ribozyme whichblock viral replication or infection. Viral replication cam be inhibited with a variety of
amtisense strategies. One particular ribozyme which cleaves HIV mtegrase (Sioud and Drlica,
PN~S 88:7303), has been developed and may offer an approach to blocking infection as
opposed to merely viral production.
Another approach to block HIV infection involves ' ~ T cells with HIV-
regulated toxins. Two examples of this type of approach are the diphtheria toxin A gene
(Harrison et al. AIDS Res. H~m. Retro. 8:39) and the herpes simplex virus type I thyrmdine
kinase gene (HSV TK) (Caruso and Klatzmann, PNAS 89:182). In both cases" ~ a~
was umder the control of HIV regulatory sequences. While the diphtheria toxin is itself toxic,
the HSV TK requires the addition of acyclovir to kill infected cells. For example the use of
HSV TK followed by the addition of 10 ~m acyclovir for 17 days totally blocks HIV
infection of HUT 78 cells for up to 55 days of culture.
The methods for stimulatmg and expandmg a population of antigen specific T cellsare useful in therapeutic situations where it is desirable to upregulate an immune response
(e.g., induce a response or enhance an existing response) upon l ' ~ ~ of the T cells to
a subject. For example, the method can be used to enhance a T cell response against tumor-
associated antigens. Tumor cells from a subject typically express tumor-associated antigens
but may be unable to stimulate a ' y signal in T cells (e.g., because they lacks
=
W095/33823 2 ~ 9 1 586 -' - PCTIUS94/13782 --
-24-
expression of, ~ y molecules). Thus, tumor cells can be contacted with T cells frorn
the subject in vifro and antigen specific T cells expanded according to the method of the
inverition and the T cells returned to the subject. Alternatively, T cells can be stimulated and
expanded as described herein to induce or enhance IC:~,UUll~ ,a~ to pathogenic agents, such
5 as viruses (e.g., human i.,.", ~ s~ .'i....,. ~ virus), bacteria, parasites and fungi.
This invention is further illustrated by the following examples which should not be
construed as limiting. The contents of all references and published patent ~P~ cited
throughout this application are hereby hl~,ul,uul~l~tl by reference. The following
,... l~nrlr~lf~y described in the Materials and Methods section was used throughout the
10 examples set forth below.
MF,TI:IODS AND MATF.R-AT,.'~
Prep~-~tinn of T"..,..)l,ili,..l Anti-CD3 Antihr,fly
Tissue culture flasks were coated with anti-CD3 mn~nrlrln~l antibody. Although a15 number of anti-human CD3 ",...,r,rl.",.l antibodies are available, OKT3 prepared from
hybridoma cells obtained from the American Type Culture Collection was used in this
procedure. For any anfi-CD3 antibody the optimal ~..r~..,1.,.1;.... to be coated on tissue
cultured flasks must be determined CAP. ' ' 71~/ . With OKT3, the optimal ~ .. 1, ,-l i.. "
was determined to be typically in the range of 0.1 to 10 llli~lu~ ~ per milliliter. To make
coating solution, the antibody was suspended in 0.05 M tris-HCI, pH 9.0 (Sigma Chemical
Co., St. Louis, MO). Coating solution sufficient to cover the bottom of a tissue culture flask
was added (Falcon, Nunc or Costar) and incubated overnight at 4~ C. The flasks were
washed three times with phosphate buffered saline without calcium or magnesium (PBS wlo
Ca or Mg) and blocking buflfer (PBS w/o Ca or Mg plus 5% boYine serum albumin) added to
25 coYer the bottom of the flask and were incubated two hours at room t.,~ lt. c. After this
incubation, flasks were used directly or frozen for storage, leaving the blocking solution on
the flask.
I~nl 1if~n of Per~ph~r ~I Blood r ~ ncYtr~ (Psr .cd
Samples were obtained by !~ n~ of healthy donors. Using sterile conditions,
the leukocytes were transferred to a T80Q culture flask. The bag was washed with Hanks
balanced salt solution wlo calcium or magnesium (HBSS wlo) (Wluttaker Bioproducts, Inc.,
Walkersville, MD). The cells were diluted with HBSS wlo and mixed well. The cells were
then split equally behveen two 200 milliliter conical-bottom sterile plastic tissue culture
tubes. Each tube was brought up to 200 ml with HBSS wlo and spun at 1800 RPM for 12
minutes in a Beckman TJ-6 centrifuge. The supernatant was aspirated and each pellet
lf fl in 50 ml HBSS wlo. The cells were transferred to two 50 ml conical bottom
tubes and spum at 1500 RPM for eight minutes. Again the supernatant was aspirated.
~ wo 9s/33823 2, 9 ~ 5 8 6 - ~ PCTfUS94fl3782
-25-
To Iyse the red blood cells, the cell pellets were ~ ..de,d in 50 m. of ACK Iysing
buffer (Biofluids, Inc., Rockville MD, Catalog #304) at room ~ aL~ with gentle mixing
for three minutes. The cells were again pelleted by spinning at 1500 RPM for 8 minutes.
After aspirating the snrPnnRtRnt the pellets were combined into one 50 rnl tube in 32 ml
S HBSS w/o.
SeparationofthePBLsfrommonocyteswas .r.~ pll~l~ rlby ll~;rl~ through
a PERCOLLThf gradient. To prepare I liter of PERCOLLThf solution (PERCOLL~M-MO),716 ml of PERCOLLT~f (Pharmacia, Piscataway, NJ, Catalog #17-0891 -01) was combined
with 100 ml I .5 M sodium chloride, 20 ml I M sodium-HEPES, and 164 ml water. All
10 reagents must be tissue culture grade and sterile filtered. After mixing, this solution was
filtered through a sterile 0.2 ,um3 filter and stored at 4~ C. 24 ml of PERCOLLrM-MO was
added to each of two 50ml conical bottom tubes. To each tube 16 ml of the cell suspension
was added. The solution was mixed well by gently inverting the tubes. The tubes were spun
at 2800 RPM for 30 minutes without a brake. The tubes were removed from the centrifuge,~5 being careful not to mix the layers. The PBLs were at the bottoms of the tubes. Then, the
a was aspirated and the PBLs were washed in HBSS w/o by r. ,U; r~ ; "g for 8
minutes at 1500 RPM.
1'PIl Srrtin,~ V;R NP.~,RtiveMR,~nnPIjr ¦l"" .,f.~.f~ ...... f
The cell sorting via negative magnetic ;l........... ,. l. ~11.. c. ,.. f. must be performed at 4~ C.
The washed cell pellets obtained from the PERCOLL~M gradients described above were
d m coating medium (RPMI-1640 (BioWhittaker, Walkersville, MD, Cataog #
12-167Y), 3~/O feta. ca f serum (FCS) (or 1% human AB- serum or 0.5% bovine serum
albumin) S mM EDTA (Ouality Biological, Inc., (' ' ' ~ MD, Cat_og # 14-117-1),2
mM L-glutamine (BioWhittaker, Walkersville, MD, Catalog # 17-905C), 20 mM HEPES
(BioWhittaker, Wa kersville, MD, Catalog # 17-757A), 50 ugfml gentamicin (BioWhittaker,
Walkersville, MD, Catalog # 17-905C)) to a cell density of 20 x I o6 per ml. A cocktail of
lll" nrl.~ l antibodies directed to cell surface markers was added to a fina. ofI llgfml for each antibody. The ~ of this cocktail is designed to enrich for either
CD4+ or CD28+ T cells. Thus, the cocktail will typically include antibodies to CD14, CD20,
CDllb,CD16,HLA-DR,and(forCD4+cellsonly)CD8. (SeeTable I foralistofsorting
, ..., .~ l., - ~l antibody cocktails.) The tube containing cells and amtibodies was rotated at 4~
for 30-45 minutes. At the end of this incubation, the cells were washed three times with
coating medium to remove unbound antibody. Magnetic beads coated with goat anti-mouse
IgG (Dynabeads M-450, Catalog #11006, P&S R~ C ' ' , MD) and
prewashed with coating medium were added at a ratio of three beads per cell The cells and
beads were then rotated for 1-1.5 hours at 4O C. The antibody-coated cells were removed
using a magnetic particle: accordmg to the ~ directions (MPC-I,
WO 9~/33823 2 1 9 1 ~ 8 6 ~ ; PCT/US94113782
-26-
Catalog # 12001, P&S Ri- ' ' 's, (' ' ' ~, MD). The n..,~ h. .~1 cells were
washed out of the coating medium and ~ dcd in an appropriate culture medium.
TART.F,1: Sort~ M..,..~ Antihr~ly Crmkt:~ilc
5 (Italicized mAbs are available from the ATCC)
l; v~ rn Ahc
rt-A CD14 63D3 (IgGlJ, 20.3 fIgM)
CD20 IF5 (IgG2a), Leu-16 (IgGI)
CD16 FC-~2 (IgG2b), 3G8 (IgGI)
HLA-DR 2.06 (IgGI), BlOa (IgG)
ail ~ Rr~ ;Ye mAhe
rT-B CD14 63D3 (IgGI), 20.3 (IgA~)
CD21 HB5 (IgG2~)
CD16 FC-2.2 (IgG2b), 3G8 (IgGI)
HLA-DR 2.06 (IgGI), HBlOa (IgG)
f;y~ rnAhc
r9.3-A CD14 63D3 (IgGI), 20.3 (IgM)
CD20 IF5 (IgG2a), Leu-16 (IgGI)
CDl lb OKMl (lgG2~, 60~ gG2b)
CD16 FC-2.2 (IgG2b),3G8 (IgGlJ
HLA-DR 2. 06 (IgGI), HB I Oa (IgG)
'; Y~; I Ahc
r9.3-B CD14 63D3 (IgGI), 20.3 (IgM)
CD21 HB5 (IgG2~
CDl lb OKMl (lgG2~), 60.1 (IgG2b)
CD16 FC-2.2 (IgG2b),3G8 (IgGI)
HLA-DR 2. 06 (IgGI), HB l Oa (IgG)
~ktail ~r~ Rr~ ;v~ rnAbs
~W095/33823 2 1 9 1 S 8 6 PCTNS94/13782
, ~; .,
rCD4-A CD14 63D3 ~IgGI), 20.3 (IGM)
CD20 IF5 (IgG2a). Leu-16 (IGgl)
CDl lb OKMl (lgG2~), 60~ gG2b)
CD16 FC-2.2 (IgGb), 3G8 ~IgGI)
HLA-DR 2. 06 (IgGl), HB I Oa (IgG)
CD8 51.1(1gG2), G10-l.l(qgG2a),
0Rl'8, (IgG2a)
jy~ mAh~
rCD8-B CD14 63D3 (IgGI), 20.3 (IgM)
CD20 IF5 (IgG2a), Leu-16 (IGgl)
CDl lb OKMl (lgG2~), 60.1 (1SG2b)
CD16 FC-2.2 agG2b), 3G8 (IgGI)
HLA-DR 2.06 (IgGI), HBlOa (IgG)
CD4 G17-2.8 (IgGI)
~ F,~ iv~ rnAh~
rMO CD2 35.1 (IgG2a), 9.6 (IgG2a)
CD20 IF5 (IgG2a), Leu-16 (IGgl)
WO 95/33823 ' i ~ é PCTNS94/13782
21 91~86
-28 -
;V~ m~,h~
rB CD2 35.1 (IgG2a), 9.6 agG2a)
CD14 63D3 (IgGI), 20.3 (IgM)
CDl lb OKMI (lgG2b), 60-1 (IgG2b)
CD16 FC-2.2 (IgG2b),3G8 (IgGI)
T.nn~ Tern S
Tissue culture flasks precoated with anti-CD3 mnnnrlnnPl antibody were thawed and
washed three times with PBS. The purified T cells were added at a density of 2 X 106/ml.
Anti-CD28 I,..,l,nrl,,.._l antibody mAb 9.3 (Dr. Jeffery Ledbetter, Bristol Myers Squibb
t~nrpnrPtinn Seattle, WA) or EX5.3D10, ATCC Deposit No. HB 11373 (Repligen
Corporation, Cambridge, MA) was added at a ~ . ..1,..1;..,, of I ~lg/ml and cells were
10 cultured at 37'~ C overnight. The cells were then detached from the flask by forceful pipetting
and transferred to a fresh untreated flask at a density of 0.5 x 106/ml. Thereafter, the cells
were . ' ' every other day by forceful pipetting and diluted to 0.5 x 106/ml. The
meam diameter of the cells was monitored daily with a Coulter Coumter 2M mterfaced to a
Coulter t'l 'y~l Resting T cells have a mean diameter of 6.8 microns. With this
15 stimulation protocol, the mean diameter increased to over 12 microns by day 4 and then
begam to decrease by about day 6. When the mean diameter decreased to about 8 microns,
the cells were again stimulated overnight with anti-CD3 and anti-CD28 as above. It was
important that the cells not be allowed to return to resting diameter. This cycle was repeated
for as long as tbree months. It can be expected that the time between lC ' will
20 ~luKl~i~..;c~,ly decrease.
Examplel: 1 T- Gro~h~'CD4+TrPllcWithA ~ CD3
~1)28 A "- ~ '
Previous known methods to culture T cells in vitro require the addition of exogenous
25 feeder cells or cellular growth factors (such as interleukin 2 or 4) and a source of antigen or
mitogenic plant lectin. Peripheral blood CD28+ T cells were isolated by negative selection
using magnetic ~ -' ' and mnnnrlnnql antibodies as described in the Methods and
Materials section above. CD4+ cells were further isolated from the T cell population by
treating the cells with anti-CD8 mnnnrlnrql antibody and removing the CD8+ cells with
30 magnetic ;.. , . .. ,1,. ~ Briefly, T cells were obtained from l~,lJku~h~,lca;a of a normal
donor, and purified vvith FICOLLTM density gradient . ~ . ,l,; ru~ followed by magnetic
-' ' sorting. The resulting CD28+, CD4+ T cells were cultured in defined medium
(X-VivolO contair;mg gentamicin and L-glutamme (Whittaker Bioproducts) at an initial
~wo 9~/33823 2 1 ~ i 5 8 6 . ~ I ~ PCT/US94/13782
-29-
density of 2.0 x 106/ml by adding cells to culture dishes containing plastic-adsorbed Goat
amti-mouse IgG (Kirkega. rd and Perry T qhrlr,qt~rire, ~ thrr.~b.lrg MD) and anti-CD3 mAb
G19-4. After 48 hours, the cells were removed and placed in flasks containing either hIL-2
(5~/0, CalBiochem) or anti-CD28 mAb (500 ng/ml). The cells cultured with IL-2 were fed
5 with fresh IL-2 at 2-day intervals. Fresh medium was added to all cultures as required to
maintain a cell density of 0.5 x 106/ml. Cells were u ' ' at .3r ' ' ~y weekly
intervals by culture on plastic-adso}bed anti-CD3 mAb for 24 hours, the cells removed and
placed at I .0 x 106/ml in fresh medium in flasks containing either IL-2 or anti-CD28 mAb.
In the example shown in Figure 1, the culture vessel initially contained 50 x lo6
10 cells, amd the cells were cultured in an optimal amount of mitogenic lectin PHA, or cultured
withcyclicstimulationofplastic;,..,.-.l.;l;,.danti-CD3mAbinthepresenceofinterleukin2
or anti-CD28 mAb 9.3. The cells cultured in PHA alone did not proliferate, with all cells
dying by about day 20 of culture"' ~ l_ the functional absence of accessory cells. In
contrast, the cells grown in anti-CD3 with IL-2 or anti-CD28 entered a logarithmic growth
15 phase, with equal rates of growth for the frst three weeks of culture. However, the anti-CD3
cultures began to diverge in growth rates during the fourth week of culture, with the IL-2 fed
cells entering a plateau phase after a ~2.81og l o expansion. In contrast, the cultures grown in
the presence of anti-CD28 remained in logarithmic growth until the sixth week of culture, at
which time there had been a -3.81Og1o expansion. Thus, CD28 receptor ~timnlqtif~n perhaps
20 by anti-CD28 ulo ,~lhll~ g, is able to stimulate the growth of CD4+ T cells in the absence of
fetal calf serum or accessory cells, and fl~ ..uu.c, about 10-fold more cells can be obtained
using anti-CD28 as opposed to addition of exogenous IL-2. In repeated . ~1.. . ;". .'~, CD4+
T cell expansion usimg anti-CD28 antibody Cu-~ Ll~ yielded more CD4+ T cells than
expansion using IL-2 (e.g., up to 1000-fold more cells). This system has the added advantage
of not requiring the presence of accessory cells which may be a.lv _ in clinicalsituations where accessory cells are limiting or defective.
Example 2: L~ T~ G,row~h of Ar~i-CD2~-TI ~ ' T rPIlc ln r~
F~t~ r s
Another series of 1~ tested whether the growth advantage of CD28 receptor
stimulation was due to I~ ll~lL of factors normally present in fetal calf serum. T cells
were obtained from I ' r ~ c~h> of a normal donor, and purified with FICOLLTM density
gradient rrntrjfil~,qtinn~ followed by magmetic ;.~ 1, - 1 sorting. The resulting CD28+,
CD4+ T cells were cultured at an initial density of 2.0 x 106/ml in medium (RPMI-1640
containing 10% heat-inactivated fetal calf serum [Hyclone, Logan, Utah] and gentamicin and
L-glutamine) by adding cells to culture dishes containing plastic-adsorbed OKT3. After 48
hours, the cells were removed and placed in flasks containing either hIL-2 (10~/o final
c~ "-~ Rir rhf~m) or anti-CD28 mAb 9.3 (800 ng/ml). The cells were fed with
wossl33823 21 91 586 Pcr/uss4/l3782 --
- -30-
fresh medium as required to maintain a cell density of 0.5 x 106/ml, and ~C~lLilll l._'~.i at
alJ~lL~hll_8,1y weekly intervals by culture on plastic adsorbed anti-CD3 mAb for 24 hours.
As shown in Figure 2, the cells entered logarithrnic growth phase, with equal rates of
growth for the first three weeks of culture. However, the anti-CD3 cultures began to diverge
5 in growth rates during the fourth week of culture, with the IL-2 fed cells entering a plateau
phase after a ~4.01Oglo expansion. In contrast, the cultures grown in the presence of anti-
CD28 remained in logarithmic growth umtil the fifth week of culture, at which time there had
been a ~5.llog1o expansion. Thus, CD28 stimulation resulted in a ~l~S,000-fold expamsion
of the initial culture while IL-2 feeding resulted in a 10,000-fold expansion of cells.
Example 3: l _ Tl Gro~h of T rf~llc ~ Phorbol r~
' A~ ~D28-A ~ ~ ~ T r~ollc
Further . ~l~ tested whether alternative methods of activating T cells would
also permit CD28 stimulated growth. Pl.A. ,., ~ Og;L activation of T cells with PMA and
15 ionomycin is thought to mimic antigen receptor triggering of T cells via the TCR/CD3
complex. T cells were obtained from l . L . ,1,1. . c~ ~ of a normal donor, and purified with
sequential FICOLLlM and PERCOLL~M density gradient ~ ....s; r~ , followed bymagnetic i.,.,, Ir~b~ All sorting. The resulting CD28+, CD4+ T cells were cultured at an
initial density of ~o x 106/ml by adding cells to culture dishes containing phorbol myristic
acid (PMA 3 nglml, Sigma) amd ionomycm (120 ng/ml, ~ rh~r.A. lot #3710232). After
24 hours, the cells were diluted to 0.5 x 106/ml and placed in flasks containing either rlL-2
(50 IU/ml, Boerhinger Mannheim, lot #11844900)) or amti-CD28 mAb (I ug/ml). The cells
were fed with fresh medium as required to maintain a cell density of 0.5 x 106/ml, and
' I cyclically at ap,UIUAil~ ,y weekly intervals by readdition of PMA and
ionomycin. Fresh IL-2 was added to the IL-2 containing culture at daily intervals.
The results of this experiment are shown m Figure 3. T cells that were purified of
accessory cells did not grow in cell nurnbers in the presence of PMA ("P" in th-e Figure) and
ionomycin ("I" in the Figure), with or without IL-2. The cells clumped amd enlarged, as
indicated by size analysis, mdicating the cells had been induced to enter the Gl phase of the
cell cycle but did not progress to DNA synthesis and cell division. In contrast, addition of
CD28 mAb to PMA plus ionomycin treated cells resulted in logarithmic cell growth. Thus,
anti-CD3 mAb is not required to provide T cell activation. It should be appreciated that other
activators of protein kinase C, such as bryostatin or dia.,~4;1y~,~,lul can be used in place of
PMA.
~WO 95/33823 2 1 9 1 5 8 6 i PCT/US94/13782
Example 4: 1 t of Cr~l1C C ' ' With A ' cm
S' ' ' ~ ' '"' ot'TI,-2 ~r~r'i-CD?51 ~h
To examine the subsets of T cells that are expanded, PBL were propagated for 16
days using either amti-CD3 and IL-2 or anti-CD3 and anti-CD28. Figure 4 .1. . ,.1 ~ u. s ~ the
5 selective enrichment of CD4 cells from peripheral blood Iyu.~lloc~ . Mnn~nn.~ r cells
wereisolatedfrombloodbyficollhypaquedensitygradient...,uir.~ ." Thecellswere
stained with CD4 and CD8 " .. ."nr1. ~lrl antibodies, and analy~d for the percent positive cells
on day 0. The cells were then cultured on plastic i.. ,.n1.;1i,. J anti-CD3 .. ,.nrl .. , 1
amtibodyG19-4plusIL-2Orplastic;.,....n1.,1;,.Janti-CD3mnnn.1nn~1antibodyG19-4plus
anti-CD28 1l... nrln.. A1 antibody 9.3 (0.5 llg/ml). The cells were isolated from culture on day
16, and repeat staining for CD4 and CD8 antigens was done by flow cytometry. Data was
gated on the IYU~ U~ , population by forward angle light scatter amd side scatter. By this
analysis, the % CD4 and CD8 cells were 8.0% and 84.5% in the cells grown in IL-2, amd
44.6% and 52.5% in the cells grown in CD28. These results suggest that CD28 expansion
15 favors the CD4+ cell, in contrast to the well c .LUl,li .l.~l observation that CD8+ cells
pl1 ' in cells grown in IL-2 (for example, see D.A. Cantrell and K.A. Smith, (1983),
JE~;p Med 158:1895andGullberg,M.andK.A.Smith(1986)JEi~p.Med 163,270).
To further test this possibility, CD4+ T cells were enriched to 98% purity usingnegative selection with ...- ...nrl....~1 antibodies and magnetic ' ' as described
20 above Fluorescent Activated Cell Sorter (FACS) Analysis was used to examine the
phenotype of the T cells cultured with anti-CD3 ~md anti-CD28. Cells were pelleted by
~ ~r nn and ~ L .1 in PBS/1% BSA. The cells were then washed by repeatingthis procedure twice. The cells were pelleted and ~ ..L .~ in 100 111 of primary amtibody
solution, vortexed, amd kept on ice for one hour. After washing twice in PBS/1% BSA, the
cells were ~ ........ L ;i im 100 111 of n ~ I ~ ~ ~ goat-anti-mouse IgG amd mcubated
for 30 minutes on ice. At the end of this incubation, the cells were washed twice m PBS and
1 in 500 ~.d 1% ~ .yd~ in PBS. The labeled cells were analy~d on an
Ortho Cy~unuulu6.~pll. Cells were stained after isolation, or atter 26 days in culture, with
~L~,u~ly i' conjugated anti-CD3 (Leu-4), CD4 (Leu-3A), CD8 (OKT8) or with IgG2acontrol - ' ' antibodies amd rl.. G~.. ..r qu~mtified with a flow cytometer. The cells
were cultured for one month using amti-CD3 and either IL-2 or amti-CD28 to propagate the
cells. There was equal expansion of the cells for the first 26 days of the culture (not shown),
however, as c m be seen m Figure 5, the phenotype of the cells diverged p~u6~..,.;c .,ly with
increasing time in culture so that at day 26 of culture, the I ' ~ cell in anti-CD28
culture was CD4+ while the cells in the IL-2 culture were I ' '.~ CD8+. Thus, CD28
receptor ' perhaps by .. ~ i.............. g is able to selectively expand T cells of the CD4
phenotype while the UUII~,ll.,iUUal method of in vi~ro T cell culture yields cells of the CD8
phenotype. Additional CAIJ. ~ ' have been conducted with similar results, indicating that
W095/33823 2 1 9 1 5 8 6 . . ! : PCT/US94/13782
-32 -
CD28 stimulation of initially mixed populations of cells is able to yield cultures containing
,ulclullfL._'~,ly or exclusively CD4 T cells, and thus one c_n expand and "rescue" the CD4
cells that were initially present in limiting amounts.
5 l~xarnple 5: Use nf CPII S ~ or C ' F. ~ of B7 on CD4+ T ~ to r~
T CPII i . ,~ -
To determine the time of T cell, ~ changes in cell volume were monitored
usmg a Coulter Counter ZM interfaced with a Coulter. CD28+, CD4+ T cells were isolated
as described by magnetic ;11111111..., ,. 1. ,,1;. ",, and cultured in the presence of amti-CD28 mAb
9.3 (0.5 ~Lg/ml) and lC ' ' with plastic i~rAA,hili7.'~i anti-CD3 mnnn~AlnnAl amtibody
G194 as indicted. Figure 9 ,l...,....~ the cyclic changes in cell volume during six
CVl~ Ve l~ ("Sl " to "S6") performed essentially as described in Example 1.
Briefly, cells were expanded with anti-CD3 and anti-CD28 over three weeks in culture. Cells
were changed to fresh medium at each l~ IA ;1111 with anti-CD3 antibody. !~8 ", .
were spaced at ten day intervals. The cells were . ' ' whenever cell volume
decreased to <400 fl.
In anot_er ~ r t, cyclic expression of the B7-1 antigen was used to determine
the time for T cell ,, ' The cells obtained from the experiment shown in Figure 10
were stained with a CAl~A-41g fusion protein (obtained from Repligen t'nrrnrAtinn see also
Linsley P.S. et al. (1991) J. Exp. Med. 174, 561-569) and analyzed by flow cytometry to
measure B7-1 receptor expression. It was determined that CD4+ T cells do not initially
express the B7-1 receptor, and that with culture, expression is induced. Further, the B7-1
expression was found to be transient, and to be re-induced with repeated anti-CD3
Esarnple 6: r of C ' ~ ~ by T t~llc F " ~ & Ar~i-CD28 C~ - ' "
F~l,. . ;".. .,l~ were conducted to analy~ the cytokines produced by T cells following
anti-CD28 s~ lAtinn CD28+/CD4+ T cells were isolated as described in the previous
examples. The cells were stimulated with plastic; --- .i.;l;,. ;i anti-CD3 mAb amd IL-2 (200
30 U/ml), or anti-CD3 and anti-CD28 without added l~ L.l~i. The cells were lca-ill.J!~b,d
with anti-CD3 antibody as determined by changes in cell volume as described in Example 5.
Cell culture supernatant was removed at the time points indicated and amaly~d for IL-2
(Figure 11), GM-CSF (Figure 12), and TNF-a (Figure 13). IL-2 was determined by bioassay
on CTLL-2 cells while TNF-a and GM-CSF were measured by ELISA accordir g to
35 ...A.. r~ ~ U~,livl~ (TNFa, GMCSF:R&D Systems, Mimneapolis, MN). The data
shownforthevariouscytokinesarefromseparate,~l,..;- ..,~ Inother..p..; ..I~(not
shown) anti-CD3 plus anti-CD28 stimulation was shown to cause high levels of IL-4 and IL-
~~VO 9~;/33823 2 ~ 9 1 5 8 6 ~ ~ PCT/US94113782
-33 -
5 in culture ~ after a,u~l~ ly day 10 of culture, although only small amoumts
of these cytokines were present during the early period of culture.
The pattems of cytokine secretion with cells expanded by several rP~timnlotil~neaccording to the protocol described in the examples was compared to cells expanded with
5 amti-CD3 plus IL-2 over three weeks in culture. Cells were changed to fresh medium at each
r. ~tim~ tinn with anti-CD3 antibody. Stim~ofi--ns were spaced at ten day intervals. After
24 hours of further culture, an aliquot of cell culture supernat nt was removed for assay.
ELISA assays for individual cytokines were performed with kits from various suppliers (IL-
2:T Cell Diagnostics, Cambridge, MA; IFN-1~ Endogen, Inc., Boston, MA; IL~, TNFa,
10 GMCSF:R&D Systems, ~.~i. ", ap.~ , MN) according to directions supplied with the kits. As
can be seen from the results of a l~,ul~,s~,llLaLivc experiment shown in Table 2, the two
protocols result in very similar levels of IL-2 and IL-4 secretion. The higher levels of GM-
CSF and TNFa secretion with anti-CD3 and anti-CD28 ~ timnloti~m suggests that tne
plulirclaLivc capacity of this ~ , of stimuli may be due in part to its ability to
15 stimulate an autocrine loop.
Table 2
C~ , ~ of cytokines secreted by T cells expanded with anti-CD3
and IL-2 versus T cells expanded with anti-CD3 and anti-CD28.
C.., . .'i,.18 ~.. of IyllluLokille in pg/ml
S ' Costimulus IL-2 IFN-y IL4 GM-CSF TNFa
cycle
Sl IL-2 20714 1458 16 2303 789
aCD28 13794 2211 14 3812 3387
S2 IL-2 20250 16600 964 51251 3221
aCD28 28411 56600 1030 138207 13448
S3 IL-2 21282 8617 1153 86418 2899
aCD28 14129 12583 1044 120418 5969
W0 95133823 2 1 9 1 5 8 ~ PCIIUS94113782
-34-
Example7: r ~ ~ of TCellsF " ~ A ~D28S- '
The po~ ull~l;Ly of a population of T cells following stimulation with an anti-CD3
and an anti-CD28 antibody as described in the preceding examples was ~Ptrrminrd
CD28+/CD4+ T cells were isolated as described in the previous examples. The cells were
S stimulated with plastic immr~hili7Pd anti-CD3 mAb and anti-CD28 mAb and FACS analysis
conducted essentially as described in Example 4 using a panel of anti-TCR antibodies (V135a,
V,B5b, V~5c, V~6a, V~8a, V!312a and V~2a) obtained from r~ ~ The polyclonality
of the T cell population was determined before (Day 1) and after stimulation (Day 24). As
shown in Figure 14, tbe TCR diversity of a population of T cells stimulated through CD28 is
maintained at day 24.
Example 8: t~ ~' of CPll e ~ ~ ~ of T CPIIC f l~TTV+ ' IllV-
l ' ' ' F -' ~ A ' ~DZ8 8' ' '
Another series Of ~ - was conducted to determine the expression of various T
cell surf~e markers on cells from HIV :~ClU~/Ua;LiVC and 7~lull~,t;adve individuals expanded
~cording to the procedures described in the previous examples. CD28+/CD4+ T cells were
obtained as described herein. In these . . the anti-CD3 mAb was labeled with a
first label (e.g., rhodamine) and the appropriate second antibody (e.g., anti-CD28, anti-CD4,
anti-CD8) was labeled with a second label (e.g., fluorescein). T cells were stimulated with
plastic immt hili7r~1 amti-CD3 m--Ab and anti-CD28 mAb as described herein and the percent
of T cells expressing a variety of cell surface markers at differenct ' (i.e., Sl, S2
and S3) determined by FACS analysis. As shown in ~igures 15 and 16, the overall cell
surface marker distribution on T cells obtained from HIV s~,~v~u:~iLi~, and :~CI~ ,, ve
mdividuals is ~ lu~ihl._-~ly the same throughout the stimulation assay. It is noteworthy that
the presence of one cell surface marker, CD45RA, which is a marker for naive T cells,
declines over the com se of CD28 stimulated T cell expansion. In contrast, the percent of T
cells expressing-the memory T cell surface marker, CD45RO, increases with CD28
stim~ tilm Thus, T cell expansion through CD28 stimulation preferentially expands
memory T cells or converts naive T cells to memory T cells. It should be noted that the
decline m the percent of T cells expressing CD28 is an artifact of the experiment due to the
presence of anti-CD28 amtibody in the T cell culture throughout the assay. The presence of
anti-CD28 amtibody prevents staining of the CD28 antigen.
Example g: 1 _ T~ Growth of CD8+ T rPIlc With A ~1)~
r~ ' ' A ' ' - ~ 2rl8
FYpPrir- Pnte were conducted to determine whether a population of CD8+ T cells
could be ,ulef~lc;ll~ lly expanded by stimulation with an anti-CD3 mAb and a
amtibody 2D8. CD28+ T cells were obtained essentially as described m Example I . To assay
~W0 95133823 2 1 9 1 5 ~ 6 ~ PCTIUS94/13782
-35-
for CD8 expression, a primary anti-CD8 antibody and a labeled appropriate secondary
antibody were used in FACS analysis to detennine the percent positive cells. As shown in
Figure 17, at day 7 following stimulation of T cells with the anti-CD3 mAb G19-4sp and the
mAb 2d8, the CD8+ fraction had increased from ~ u~hllat~ly 20% to over 4û%. Another
5 ,"..".~,l. ."~l antibody ER4.7GI I (referred to as 7GI 1) was also found to stimulate CD8+ T
cells. This antibody was raised against ~ human CTLA4 and has been deposited
with the ATCC on June 3, 1994 at Accession No. 11642. This result indicates that binding of
either a distinct region of CTLA4 or of a cross-reactive cell surface protein selectively
activates CD8+ T cells.
E~amplclO~ F4- of -rl - ' 'A ' 1, 2n~ ~cr ~ _ -
CDg ~
To detemine the epitope of the ".. .",.,l. I..AI antibody 2D8, epitope mapping was
performed by phage display library (PDL) screening and was confirmed using synthetic
15 peptides. A random 20 amino acid PDL was prepared by cloning a degenerate
,~lig~.. " .. l. . ,l if lr imto the fUSE5 vector (SCOK~ J.K. and Smith, G.P. (1990) Science 2~ 386-
390) as described in Cwirla, S.E. et al. (1990) Proc. NatL A.cad. Sci. U~ 87:6378-6382. The
PDL was used to identify short peptides that specifically bound mAb 2D8 by a lll;~
technique described in Jellis, C.L. et al. (1993) Gene 137:63-68. Individual phage clones
werepurifiedfromthelibrarybyvirtueoftheiraffmityfori".,.,.-l,!i,~.lmAbandtheramdom
peptide was identified by DNA ~Pq -Pnf inFI Briefly, mAb 2D8 was coatcd onto Nunc
Maxisorp 96 well plates amd incubated with 5 x 101~ phage l~ 8 x 106 different
phage displaying random 20 amino acid peptides. S, ~~ "~, bound phage were eluted,
amplified, then incubated with the antibody a second time. After the third round, 7 phage
were isolated, and DNA was prepared for ~eql~Pn~ inp
Sequence analysis of these clones ,l ' that three of the seven sequences
were identical amd a fourth was similar:
2D8#2(SEQIDNO: I) HQFCDHWGCWLLRETHIFTP
2D8#4 HQFCDHWGCWLLRETHIFTP
2D8#10 HQFCDHWGCWLLRETHIFTP
2D8#6(SEQIDNO:2) LRLVLEDPGIWLRPDYFFPA
Based on this data an epitope of G X W L X DIE (SEQ ID NO: 8) was proposed.
In addition to CTLA4, a second antigen for mAb 2D8 was discovered usmg cDNA
expression cloning.
wO gs/33823 2 1 9 1 5 8 6 PCTIUS9~/13782
-36-
A. C-.nctr~rti-~n of a ~AnNA F.~rrcci~-n T ihrary
A cDNA library was constructed in the pCDM8 vector (Seed, (1987) Na~ure ~:840)
using poly (A)+ RNA isolated from activated T cells as described (Aruffo et al. (1987) Proc.
~latl. Acad. Sci. USA ~L:3365). To prepare total RNA, T cells were harvested from culture
5 and the cell pellet 1~ ,;, . d in a solution of 4 M guanidine thiocyanate, 0.5 ~/O sarkosyl,
25 mM EDTA, pH 7.5, 0.13 % Sigma anti-foam A, and 0.7 % ~ v~ nl RNA was
purified from the 1-."... ,~;. - '- by c~ ntrifi~g,Atil~n for 24 hour at 32,000 rpm through a solution
of 5.7 M CsCI, 10 mM EDTA, 25 mM Na acetate, pH 7. The pellet of RNA was dissolved in
5 % sarkosyl, I mM EDTA, 10 mM Tris, pH 7.5 and extracted with two volumes of 50 %
phenol, 49 % chloroform, I % isoamyl alcohol. RNA was ethanol ~c~ ;L~L~d twice. Poly
(A)+ RNA used in cDNA library c~ ;" - 1;.~,, was purified by two cycles of oligo (dT)-
cellulose selection.
DNA was synthesized from 5.5 ,ug of poly(A)+ RNA in a reaction
containing 50 mM Tris, pH 8.3, 75 mM KCI, 3 mM MgC12, 10 mM .~ 11, 500 IlM
dATP, dCTP, dGTP, dTTP, 50 ~lg/ml oligo(dT)12 18, 180 units/ml RNasin, and 10,000
units/ml Mol~ I\~Y reverse 1~ - in a total volume of 55 ~LI at 37 ~C for I hr.
Following reverse n - ;~ the cDNA was converted to double-stranded DNA by
adjusting the solution to 25 mM Tris, pH 8.3, 100 mM KCI, 5 mM MgC12, 250 ~LM each
dATP, dCTP, dGTP, dTTP, 5 mM ' ' ' c .1 250 units/ml DNA polymerase I, 8.5
units/mlrih.~n.~ Ac~Handincubatingat 16~Cfor2hr. EDTAwasaddedto 18mMand
the solution was extracted with an equal volume of 50 % phenol, 49 % chloroform, I %
isoamyl alcohol. DNA was ~1~ , ' with two volumes of ethanol in the presence of 2.5
M ammonium acetate amd with 4 ~ of linear ~U~ n as carrier. In addition,
cDNA was synthesized from 411g of poly(A)+ RNA in a reaction containing 50 mM Tris, pH
8.8, 50 ~Lg/ml oligo(dT)12 18, 327 units/ml RNasin, and 952 units/ml AMV reversein a total volume of 1001l1 at 42 ~C for 0.67 hr. Following reverse u. - . ;~
the reverse ~ was inactivated by heating at 70 ~C for 10 min. The cDNA was
converted to double-stranded DNA by adding 320 ~LI H20 and 80 111 of a solution of O.lM
Tris, pH 7.5, 25 mM MgC12, 0.5 M KCI, 250 llg/ml bovine serum albumm, and 50 mM
liLvLLl~ 1, and adjusting the solution to 200 IlM each dATP, dCTP, dGTP, dTTP, 50
units/ml DNA poly.ll.,.~., I, 8 units/ml l i ' H and mcubating at 16 ~C for 2 hours.
EDTA was added to 18 mM and the solution was extracted with an equal volume of 50 %
phenol, 49 % chloroform, I % isoamyl alcohol. DNA was precipitated with two volumes of
ethanol in the presence of 2.5 M ammonium acetate and with 4 lll;~.l~J~;l~.l.~ of linear
pol~lyLI.ide as carrier.
The DNA from 4 ,ug of AMV reverse t. . and 2.0 llg of Moloney MLV
reverse u A- '- ' 11~ 1 were combined. Non 5 1 r. . ~ Y BstXI adaptors were added to
the DNA as follows: The double-stranded cDNA from 61~g of poly(A)+ RNA was incubated
~W095133823 2 1 9 1 5 8 6 1; PCI/US94113782
-37-
with 3.6 !lg of a kinased ~ gnn~ poti~lp of the sequence CTTTAGAGCACA (SEQ ID NO:
5) and 2.4 llg of a kinased .1 ir~ fU~ P of the sequence CTCTAAAG in a solution
containing 6 mM Tris, pH 7.5, 6 mM MgC12, 5 mM NaCI, 350 llg/ml bovine serum albumin,
7 mM ~ iu~ -1, 0.1 mM ATP, 2 mM d;:L;uLLc:iLul, I mM cpPrmi~inP and 600 units
S T4 DNA ligase in a total volume of 0.45 ml at 15 ~C for 16 hours. EDTA was added to 34
mM and the solution was extracted with an equal volume of 50 % phenol, 49 ~/O chloroform,
I % isoamyl alcohol. DNA was ,u~, , ' with two volumes of ethanol in the presence of
2.5 M :lmm~-nil-m acetate.
DNA larger than 600 bp was selected as follows: The adaptored DNA was redissolved
in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCI, 0.1 ~~c sarkosyl and .,L, ~ ' ' on
a Sepharose CL-4B column in the same buffer. DNA in the void volume of the column
(containing DNA greater than 600bp) was pooled and ethanol ,u.c .
The pCDM8 vector was prepared for cDNA cloning by digestion with BstXI and
IJ. " ~ ;. .11 on an agarose gel. Adaptored cDNA from 6 llg of poly(A)+RNA was ligated to
2.25 ~Lg of BstXI cut pCDM8 in a solution containing 6 mM Tris, pH 7.5, 6 mM MgC12, 5
mM NaCI, 350 llg/ml bovine serum albumin, 7 mM 1ll~ u~ u - --,~1 0.I mM ATP, 2 mM
dilliuLLc;Lol~ I mM . ~ ' and 600 units T4 DNA ligase in a total volume of 1.5 ml at
15 ~C for 24 hr. The ligation re~tion mixture was then I ","~ r ., . ~ into competent E.coli
DHIOB/P3 by standard techniques.
Plasmid DNA was prepared from a 500 ml culture of the original I, "" r " .~;,," of
the cDNA librOEy. Plasmid DNA was purified by the alkaline Iysis procedure followed by
twice bamding in CsCI P~lnilihrillTn gradients (Maniatis et al, Molecular Cloning: A
Laboratory Manual, Cold Spring HOEbor, NY (1987)).
B. fllmir~T ProrP~ e
In the cloning procedure, the cDNA expression librOEy was introduced into MOP8
cells (ATCC No. CRL1709) using 1;~ and the cells screened with mAb 2D8 to
identify ~. . f . 1 . t' expressing a 2D8 ligand on their surface. In the first round of
screening, thilty 100 mm dishes of 50 % confluent COS cells were transfected with 0.05
llg/ml activated T cell librOEy DNA using the DEAE-Dextram method (Seed, B. et al. (1987)
Proc. Nafl. Acad. Sci. U~4 84:3365). The cells were trypsinized and re-plated after 24 hours.
After 47 hours, the cells were det~hed by incubation in PBS/0.5 mM EDTA, pH 7.4/0.02 %
Na azide at 37 ~C for 30 min.
Detached cells were treated with 10 ~Lg/ml mAb 2D8. Cells were incubated with the
1".. .~ 1 OEntibody for 45 rninutes at 4~C. Cells were washed and distributed into pOEming
dishes coated with affinity-purified goat anti-mouse IgG antibody amd allowed to attach at
room i , c. After 3 hours, the plates were gently washed twice with PBS/0.5 mM
EDTA, pH 7.4/0.02 % Na azide, 5% FCS and once with 0.15 M NaCI, 0.01 M Hepes, pH
WO 9~133823 2 ~ 9 1 5 8 6 , ~ I ~ PCTIIJS94/13782
-38-
7.4,5 % FCS. Unbound cells were thus removed and episomal DNA was recovered from the
adherent panned cells by conventional techniques.
Episomal DNA was ~ .", . ;l into E. coli DHl OB/P3. The plasmid DNA was re-
introduced into MOP8 cells using I ;~ f I - - -; 1 If ~ and the cycle of expression amd panning was
5 repeated twice. Cells expressing a 2D8 ligand were selected by panning on dishes coated
with goat anti-mouse IgG antibody. After the third round of screening, plasmid DNA was
prepared from mdividual colonies and transfected mto MOP8 cells by the DEAE-Dextran
method. Expression of a 2D8 ligamd on transfected MOP8 cells was analyzed by indirect
immunonuulcDc.,u.,e with mAb 2D8 (See Figure 18).
DNA from one clone (mpS) identified as positive by FACS analysis was sequenced
using standard techniques. FASTA analysis of the arnino acid sequence of mpS identified a
matching protein, CD9~ in the GCG data banks. The full amino acid sequence of CD9 is
shown below (SEQ ID NO: 6).
BESTFIT analysis of the phage epitopes of mAb 2D8 to the amino acid sequence of
15 CD9 revealed a close match:
G C W L L R E (phage 2D8#2, 4, 10; SEQ ID NO: 3)
G I W L R P D (phage 2D8#6; SEQ ID NO: 4)
G L W L R F D (CD9 sequence; SEQ ID NO: 7)
2û
FT DOMAIN 111 194 EXIRACELLULAR (PROBABLE)
FT TRANSMEM 195 220 POTENTIAL
FT DOMAIN 221 227 CYTOPLASMIC (PROBABLE)
FT CARBOHYD 51 51 POTENTIAL
FT CARBOHYD 52 52 POTENTIAL
FT CONFLICT 8 8 C ~ S (INREF. 1)
FT CONFLICT 66 66 G ~ A (IN REF. 1)
FT CONFLICT 193 193 MISSING (IN REF. I)
SQ SEQUENCE = ~7AA; 25285MW; 261251 CN;
Cd9_Human Length: ~7 May25,1994 14:10 Type: P Check: 1577
(SEQ ID NO: 6)
PVKGGTKCIK YLLFGFNFIF WLAGIAVLAI GLWLRFDSQT KSIFEQETNN
51 NNSSFYTGVY ILIGAGALMM LVGFLGCCGA VQESQCMLGL FFGFLLVIFA
101 IEIAAAI~VGY SHKDEVIKEV Qkl YKI) l Y~K LKTKDEPQRE TLKAIHYALN
~ WO 95/33823 2 1 9 1 ~ 8 6 PC~/US94/13782
-39-
1~1 CCGLAGGVEQ FISDICPKKD VLETFTVKSC PDAIKEVFDN KFHIIGAVGI
201 GL~WMIFGM IFSMILCCAI RRNREMV
EOUIVAT,T~ l~ITS
Those skilled in the art vill recogmzc, or be able to ascertain using no more than
routine ~ ~p ~ many equivalents to the specific .......... ,.,1"~ ofthe invention
described herein. Such equivalents are intended to be ~ l by the follo ving claims.
WO95/33823 2 ~ 9 1 58~ ;, . PCTr~S94/13782
-40-
SEQUENCE LISTING
(1) GENERAL l~ru~A'l lU~:
~i) APPLICANT:
(A) NAME: THE UNITED STATES OF AMERICA AS ~ l~ BY THE
SECRETARY OF THE NAVY
(B) STREET: BALLSTON TOWER ONE, 800 NORTH Q~INCY STREET
(C) CITY: ARLINGTON
(D) STATE: VIRGINIA
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 22217
(G) TELEPHONE:
(H) TELEFAX:
(i) APPLICANT:
(A) NAME: THE REGENTS OF THE U~l~Kbl'~l OF MICHIGAN
(B) STREET: 3003 SO~TH STATE STREET, WOLVERINE TOWER, ROOM 2071
(C) CITY: ANN ARBOR
(D) STATE: MICHIGAN
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 48109
(G) TELEPHONE:
(H) TELEFAX:
(i) APPLICANT:
(A) NAME: REPLIGEN CORPORATION
(B) STREET: ONE RENDALL SQUARE, B~ILDING 700
(C) CITY: CAMBRIDGE
(D) STATP: M~c.Q~Tc~TTS
(E) COUNTRY: ~SA
(F) POSTAL CODF (ZIP): 02139
(3) TELEPHONE: (617)225-6000
(H~ TELEFAX: ~617)494-1975
(i~ APPLICANT:
(A) NAME: DANA-FARBER CANCER INSTITUTE
~B) STREET: 44 BINNEY STREET
~C) CITY: BOSTON
~D) STATE: "~CcD~Tc~TTs
~B) CO~NTRY: USA
~F) POSTAL CODE ~ZIP): 02115
~G) TELEPHONE:
~H) TELEFAX:
~ii) TITLE OF INVENTION: NETHODS FOR SELECTE~ELY a~ TTNr
PROLIFERATION OF T-CELLS
~iii) N~MBER OF SEQUEN OES: 8
~iv) UU~I ADDRESS:
~A) ADDRESSEE: LAHIVE ~ COCXFIELD
~B) STREET: 60 STATE STREET, SUITE 510
~C) CITY: BOSTON
~D) STATE: MA
~E) COUNTRY: USA
~F) ZIP: 02109
~ W09~33823 2 1 9 1 5 8 6 ~ r ~ PCTrUS9~/13782
-41-
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Ploppy disk
(B) COMPUTER: IBM PC compatible
(c) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII TEXT
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILrNG DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/253,751
(B) FILING DATE: 4 ~UNE 1994
(A) APPLICATION NUMBER: US 0S/253,964
(B) FILING DATE: 4 JUNE 1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: r~r~r.~rr~Dq, AMY E.
(B) REGISTRATION NUMBER: 36,207
(C) REFERENCE/DOCXET NUMBER: RPI-002CPP2
(ix) Tr.~r.r..~.. 'NI~TION INFORMATION:
(A) TELEPHONE: (617) 227-7400
(B) TELEFAX: (617) 227-5941
(2) INFORMATION POR SEQ ID NO:l:
(i) SEQUENCE ~rD~rTRr~T.CTICS
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: li~ear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE ~r.~LKlrll~N.: SEQ ID NO:1:
HiB Gl~ Phe Cys Asp His Trp Gly CYB Trp Leu Leu Arg Glu Thr His
Ile Phe Thr Pro
4520
(2) lNrvKr~ll~N. FOR SEQ ID NO:2:
(i) SEQUENOE r~r~
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MoLECrJLE TYPE: peptide
W O95/33823 2 1 9 1 5 8 6 . ~ PCT~US94/13782 -
-42-
(xi) SEQUEN OE ~b - KlL''llVN: SEQ ID NO:2:
5 ~eu Arg Leu Val Leu Glu Asp Pro Gly Ile Trp Leu Arg Pro Acp Tyr
, 15
Phe Phe Pro Ala
(2) INFORM~TION FOR SEQ ID NO:3:
(i) SE9UENCE C~D~DrT~T.~TIcs:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECUBE TYPE: peptide
(xi) SEQUENCE LJKbUKl~llU~: SEQ ID NO:3:
Gly Cyc Trp Leu Leu Arg Glu
(2) lN~'~ ' lUL~ FOR SEQ ID NO:4:
(i) SEQUENCE rnD~D~ . ~ I ~Ll - b:
(A~ LENGTE: 7 amino acidc
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE L~:bUKlrllUL~: SEQ ID Nû:4:
Gly Ile Trp Leu Arg Pro Asp
(2) INFORMATION FOR SEQ ID NO:S:
(i) SEQUENCE r~ ~ D rT~ r.cTIcs
(A) LENGTU: 9 amino acid~
(B) TYPE: amino acid
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
... . . .. , ... . ... _
~ W O95/33823 2 1 9 i ~ 8 6 ':; i ' t - PCTiUS94/13782
~3-
(ix) FEATU~E:
(A) NAME/KEY: misc_~eature
(B) LOCATION: 8
(D) OTHER INFORMATION: /label=Xaa is Asp or Glu
S
(xi) SEQUEN OE J~:~O~l~ll~: SEQ ID N0:5:
Xaa Gly Xaa Trp Leu Xaa Xaa Xaa Xaa
s
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE r~T~T.CTICS:
(A) LENGT~: 227 amino acids
(B) TYPE: amino ac~d
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:6:
Pro Val Lys Gly Gly Thr Ly~ Cys Ile Lys Tyr Leu Leu Phe Gly Phe
5 10 15
Asn Phe Ile Phe Trp Leu Ala Gly Ile Ala Val Leu Ala Ile Gly Leu
20 25 30
Trp Leu Arg Phe Asp Ser Gln Thr Lys Ser Ile Phe Glu Gln Glu Thr35 40 45
Asn Asn Asn Asn Ser Ser Phe Tyr Thr Gly Val Tyr Ile Leu Ile Gly
50 55 60
Ala Gly Ala Leu Met Met Leu Val Gly Phe Leu Gly Cys Cy~ Gly Ala
65 70 75 80
Val Gln Glu Ser Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu Leu
85 90 95
Val Ile Phe Ala Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr Ser ~is
loo loS llO
Lys Asp Glu Val Ile Lya Glu Val Gln Glu Phe Tyr Lys Asp Thr Tyr
115 120 125
Asn Lys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys Ala
130 135 140
Ile ~is~Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val Glu Gln
145 150 155 160
Phe Ile Ser A~p Ile Cys Pro Lys Ly8 Asp Val Leu Glu Thr Phe Thr
165 170 175
W O9~/33823 2 1 9 1 5 8 6 ~ PCTrUS9~/13782 -
-44-
Val Ly6 Ser Cys Pro A6p Ala Ile Lys Glu Val Phe A6p Asn Lys Phe
180 185 ~ 190
Hi8 Ile Ile Gly Ala Val Gly Ile Gly Ile Ala~Val Val Met Ile Phe
195 200 205
Gly Met Ile Phe Ser Met Ile Leu Cy6 Cy6 Ala Ile Arg Arg Asn Arg
210 ~ 215 220
Glu Met Val
225
(2) INFORMATION FOR SEQ ID NO:7:
~i~ SEQ~BNCB r~o~rTRoT.cTIcs
(A~ LENGTH: 7 amino acid8
~B~ TYPB: amino acid
(D~ TOPOLOGY: linear
(ii~ MOLBCULB TYPB: peptide
(xi~ SBQUEN OE ~bU~l~llU~: SEQ ID NO:7:
Gly Leu Trp Leu Arg Phe A8p
(2~ INFORMATION FOR SEQ ID NO:8:
(i~ SEQUEN OE r~ ll b:
(A~ LBNGTH: 6 amino acid6
(B~ TYPE: amino acid
(D~ TOPOLOGY: li~ear
(ii~ MOLECULE TYPE: peptide
(ix~ FEATURE:
(A~ = /XBY: misc_~eature
(B~ Lor-ATIoN: 6
(D~ OTBBR 1~ -'TT~N: /laoel~Xaa is Asp or Glu
(xi~ SEQ~BN OE J~bU~l~llU~: SBQ ID NO:8:
Gly Xaa Trp Leu Xaa Xaa
