CA2195238A1 - Antibodies that bind a conformationally altered cd4 molecule induced upon human immunodefficiency virus binding - Google Patents

Abstract

Antibodies that bind a conformationally altered form of CD4 induced on the surface of a CD4+ cell upon contact of the cell with human immunodeficiency virus (HIV), or an envelope protein thereof (e.g., gp120), but which do not substantially bind native CD4 on the surface of a cell prior to contact with HIV or an envelope protein thereof, are disclosed. Preferred antibodies of the invention are the monoclonal Fab fragments 3-47 and 3-51. Pharmaceutical compositions, and diagnostic, screening and therapeutic methods utilizing the antibodies of the invention are also disclosed. The antibodies of the invention are useful for detecting a conformationally altered form of CD4 on the surface of a cell, for identifying agents that inhibit or induce formation of this conformationally altered form of CD4 upon gp120 binding, and for inhibiting infection of CD4+ cells by HIV. Molecules that express at least one epitope expressed on a conformationally altered form of CD4 induced on the surface of a CD4+ cell upon contact of the cell with HIV, or an envelope protein thereof, are also encompassed by the invention. Such molecules can be used to produce antibodies of the invention and to induce an antibody response in a subject that inhibits infection of cells in the subject by HIV.

Description

21 9~238 W0 96/02647 r~ 114 MOLECULE INDUCED UPON HUMAN IMMUNODEFICIENCY VIRUS BINDING

R.... ~ ~ oftL- I
CD4 is a ~ " ,. . "I " .. ,e ~Iyuu,ulut. i.l, having a molecular weight of 55,000 to 62,000 Daltons, that is expressed on the surface of a subset of T Iylll,uhouy i., (referred to as CD4+ T
cells). The CD4+ subset of T cells identifies the T helper or inducer cell population. Upon, antigenic ctimnl~ti~n this population produces cytokines that promote the proliferation and ~li~tlcillLiaiiull of T cells and B cells, thereby inducing effector . ". . 1, ~ such as antibody 10 production and T cell ~;yluLu;~;u;Ly. In addition to T cells, CD4 can also be expressed on other cell types, such as l.la~,.u~,l.at~, and certain brain cells.
The primary target of certain infectious agents, including the human immumo-deficiency virus (HIV), is cells that express CD4 on their surface. This tropism of HIV for CD4+ cells is attributed to CD4 rl",. I;l",;"g as a membrane-anchored receptor for the virus.
15 Thus, HIV binding to CD4, mediated through a surface envelope protein of HIV, is believed to target HIV to CD4+ cells. The major envelope protein of HIV is produced as a precursor polypeptide (gpl 60), which in mature form is cleaved into an exterior membrane protein (gpl20) and a smaller 11,~l~.l,. ..,I-,.,..,e protein (gp41) (Ratner, L. et al. (1985) Nature ~:277-284). The binding of gpl20 to CD4 is thought to initiate infection of CD4+ cells (Dalgleish, A.G. et al. (1984) Nature 312:763-767; I~ t7m:1nn D. et al. (1984) Nature 767-768), and additionally may initiate membrane fusion of infected CD4+ cells with uninfected CD4+ cells (called syncytia formation), which may contribute to cell-cell a, , ~. " i~ . . . " of the virus and to its cytopathic effects (Habeshaw, J.A. and Dalgleish, A.G.
(1989) J. AIDS ?:457-468; Lifson, J.D. and Engleman, E.G. (1989) Immunol Rel . 109:93-1 1 7).
The nucleotide sequence of human CD4 cDNA, and the deduced amino acid sequence of human CD4 protein, have been reported (see Maddon, P.J. et al. (1985) Cell 42:93-104;
and the corrected sequence reported in Littman, D. et al. (1988) Cell 55:541). Structurally, the CD4 protein can be divided into an I~Ytr:~Plllll~r dornain (a,ulJIu~h~ ,ly amino acids 1-375), a membrane sparming domain (a,u~)lu~dlll.. -~,ly amino acids 376-395) and a ~:yLuula~ l;u domain (a,uului~hll_~,ly amino acids 396-433). CD4 is ~yllLll~ d as a precursor protein with a 25 amino acid signal sequence. The CD4 r~t~ r domain can further be divided into four tandem regions, termed V I, V2, V3 and V4, having homology to immlln~glob~llin VJ regions. The Vl region spans a,u,ulux~hl._t~ly amino acids 1-113, the V2 region spans a,uul~ y amino acids 114-180, the V3 region spans alJlulu~dlll~ y amino acids 181-297 and the V4 region spans a~,ul~ y amino acids 298-375 (see Maddon, P.J.
et al. (1985) cited supra). The V I region has been identified as the binding site for HIV
gpl20 (Arthros, J. et al. (1989) Cell 57:469-481; Mizukami, T. et al. (1988) Proc. Natl. Acad.

Sci. USA 85:9273-9277; Peterson. A. and Seed, B. (1988) Ceil ~L:65-72; Landua, N.R. et al.
(1988)Nature~:159-162;Clayton,L.K.etal.(1988)Nature~:363-366).
A number of anti-CD4 antibodies have been described that bind to particular regions of the native CD4 molecule. Examples of these antibodies include the CD4 Vl -specific antibodiesLeu3AandOKT4A(seee.g.,Sattentau,Q.J.etal.(1986)5cience~:1120-1123;
Jameson, B.A. et al. (1988) Science 2~:1335-1339; Peterson, A. and Seed, B. (1988) cited supra;Bates,P.A.etal.(1989)Prot.Engng.3:13-21;McDougal,J.S.etal.(1986)J.
Immunol. 137:2937-2944;andDalgleish,A.G.etal.(1987)~ancef~:1047-lOSO),the antibodies MT151, VIT4 and MT321, which bind CD4 epitopes distrnct from those bound by OKT4A and Leu 3A (see e.g., Sattentau, Q.J. et al. (1986) cited supra; Sattentau, Q.J. et al.
(1989)J.~p.Med. 170:1319-1334;Bates,P.A.etal.(1989)citedsupra;LandauN.R.etal.
(1988) cited supra; and MPrk~ncrhlagPr, M. et al. (1990) J: Immunol. 2:2839-2845), the CD4 V2-specific antibody OKT4B (see e.g., Kieber-Emmons, T. et al. (1989) Biochim. Biophys.
Acta98h7:281-300;McDougal,J.S.etal,(1986)citedsupra;Lundin,K.etal.(1987)J.
ImmunoL Methods ~1:93- 100; and Lamarre, D. et al. (1989) EMBO J. ~:3271 -3277), and the CD4 V3V4-specific amtibody OKT4 (see e.g.; Berger, E.A. et al. ~1988) Proc. Natl Acad.
Sci USA ~:2357-2361). Such antibodies have been studied with regard to their ability to block gp 120 binding, inhibit HlV-induced syncytia formation and/or function as potential therapeutic agents for treatment of AIDS, ARC or HIV infection. Antibodies that bind the Vl region of CD4, such as Leu3A and OKT4A, can cu~ LiLi~ly inhibit the binding of gpl 20 to CD4. However, such antibodies may be limited in their therapeutic utility by an inability to act on CD4 that has already bound HIV gpl20. Certain antibodies which bind other regions of CD4 have been found to have some effect on syncytia formation or viral infection while not snhcfanfiAlly inhibiting gpl20 binding to CD4. However, another potential limitation to the use of anti-CD4 antibodies that bind to native cell-surface CD4 is their i~ h~ c~ive activity. For example, both OKT4A and OKT4B have been reported to be ;l~ lh~ ca~ive (see e~g~ Lamarre~ D~ et aL (l989) cited supra)~
It has been suggested that CD4 p.ll i . not only in the initial binding event with gpl20, but also in virus fusion with the cell membrane (a necessary ~cL~uhcl~ L for viral entry into the cell) and HlV-envelope mediated syncytia formation. It further has been suggested that upon binding of gpl20 to CD4 on the surface of a CD4+ cell, the CD4 molecule may undergo a c~ .,.rl"",,.~;.,,.~l change that is an ;..:. . ,"~.1;~1~ step in the mechanism of viral entry and/or syncytia formation (see e.g., Healey, D. et al. (1990) J. Exp. Med 172:1233-1242;Celada,F.etal.(1990)J.Exp.Med. 172:1143-1150). Onestudyhas 35 reported the detection in HlV-infected individuals of autoantibodies that do not bind CD4+
cells alone but weakly bind CD4+ cells ~JIC;III,U~ ,d with purified rP~hmhinant gpl20 (Sekigawa, I. et al. (1991) Clin. Immunol and l .. , ' ' ~:145-153). However, two subsequent studies of autoantibodies to CD4 in HlV-infected individuals failed to confirm this observation (see Callahan, L.N. et al. (1992) J. Immunol. 149:2194-2202; and Chams, V.

.

0 96/02647 r ~ 2 ~ ~ 5 2 3 8 r~"l ~ ~114 ~W -3 -et al. (1991) ,41D5 5:565). Moreover, one of these latter studies (Callahan, L.N. et al. (1992) cited supra) attributed the observed binding of A~ ;hO~1ieC to gpl20-treated cells to an artifact in.the assay system used. At present, it has not been established whether a novel ~ cullru~ iullal form of CD4 is induced on the surface of CD4+ cells upon binding of HIV to 5 CD4, and a need exists for reagents that can recognize this putative altered conformational ~ form of CD4.

S of '-l This invention pertains to a novel ennfnrm-~finnAI form of cell-surface CD4 that is 10 induced upon binding of HIV (or an envelope protein thereof), ligands that bind this novel form of CD4, including antibodies and antibody mimetic agents, isolated molecules expressing one or more epitopes exposed on this novel form of CD4, and uses therefor. One aspect of this invention relates to antibodies, or fragments thereof, that bind a conformA~ionAlly altered form of a CD4 molecule, preferably human CD4, expressed on the I S surface of a CD4+ cell upon contact of the cell with HIV, or an envelope protein thereof (e.g., gpl20), thereby providing evidence for an HlV-induced r...,r~ change in cell-surface CD4. The antibodies of the invention are n ~ d by an ability to bind this cnnfnrtnofinnolly altered forrn of CD4 on the surface of a cell upon contact of the cell with gpl20 and by an inability to 5--h~to-Atiolly bind native human CD4 on the surface of the cell prior to contact with gp 120. In a population of CD4+ cells contacted with gpl 20, preferably at least 30 % of the gpl 20+ cells are bound by an antibody of the invention. More preferably, at least 50 ~/O, even more preferably at least 70~/O to 90 ~/O, of the gpl 20+ cells are bound by an antibody of the invention.
In a preferred emho~lim-~nf, the antibody of the invention is a mnnnclnnAI antibody, preferably a human " ,. " .n- 1- ~ I antibody. Preferred antibodies of the invention are human mnnnAlnnAI Fabs designated 347 and 3-51, or antibodies that binds the same epitope recognized by either 3-47 or 3-51. Additionally, antibodies that bind other epitopes exposed, upon gpl20 binding, on the rn.,r~ IY altered form of human CD4 that also displays either the 3-47 or 3-51 epitope are rll~ I by the invention. The invention further 3û provides antibody mimetic agents, such as 3-47 or 3-51 mimetic agents, which are non-antibody ..., . ,l ,.." . ,.l~ having the same epitope binding specificity as an antibody of the invention. Pl~ - "~ AI r~"~ "~c comprising the antibodies~ or fragments thereof~ or antibody mimetic agents described herein are also within the scope of the invention.
Another aspect of the invention pertains to isolated nucleic acid molecules (e.g., 35 DNA) comprising a nucleotide sequence encoding the light chain or heavy chain variable region (VL or VH region) of the antibodies of the invention, preferably encoding the VL or VH region of "....,ncl. IIIAI Fab 3-47 or 3-51. In one l .,.ho.l;".. ,.i the nucleic acid encoding the VL region further encodes a CL region (i.e., a full-length antibody light chain). In another emho~1im~nf, the nucleic acid encoding the VH region further encodes a CHI region -~
2J:952'38 w0 96/02647 ~ 311 (i.e., the first constant domain of an il"",.,.~f~gl~ ulin heavy chain). In yet another ~-mhor'imPnt the nucleic acid er~coding the VH region encodes a full-length antibody heavy chain (e.g., includes CHI, CH2 and CH3 regions). The nucleic acid molecules of the invention can be ;III..V~ ' ' into expression vectors and introduced into a host cell (e.g., a bacterial or mAmmAiiAn cell). In one . .,,ho~ .,1 the ~ ,,r.~l.,.,Al Fab 3-47 is expressed in a host cell. In another r",l.-~ll;", ,1, the mnnrlrlr,nAI Fab 3-51 is expressed in a host cell. In yet another Pmho~1imPnt a full-length antibody having the epitope binding specificity (e.g., the VL and VH regions) of m~nr~rlnnql Fab 3-47 or 3-51 is expressed in a host cell.
Accordingly, the isolated nucleic acid molecules, expression vectors and host cells of the invention are useful for producing 1~ ."" ,h;, IA~ Il antibodies of invention, in particular Ih;I~ antibodies having the epitope binding specificity of Ill~ rlvllAl Fab 3-47 or 3-51.
The antibodies, and mimetic agents thereof, can be used to detect the presence of a . ~ l" rl " " ~ Ally altered form of cD4 on the surface of a cell~ for exarnple to monitor the 1~ course of HIV infection or the efficacy of a therapeutic regimen. To detect the r~nformAfir,nAIIy altered form of CD4 on a cell surface, the cell is contacted with an antibody of the invention and the antibody bound to the surface of the cell is detected.
The antibodies and mimetic agents of the invention are further useful for screening agents for their ability to inhibit or induce formation of a . .l ", r~ 'ly altered form of CD4 on a cell surface. To identify an agent that inhibits for nation of *e rnn~nrmqfionAIIy altered form of CD4, a CD4+ cell cam be contacted v~ith gp120 and an agent to be tested, further contacted with an antibody of the invention, and the amount of antibody bound to the cell surface ~PtP minP~I A reduced amount of binding of the antibody to the gpl 20-treated cell in the presence of the agent, as compared to the amoumt of antibody binding to a gp 120-treated cell in the absence of the agent, can be used as an indicator that the agent inhibits formation of a c~-ntfirmqti~ ly altered form of CD4 on the cell surface. Alternatively, to identify an agent that induces the formation of the ~.1 ", r. ,., 1, ~ y altered form of CD4, a CD4+ cell can be contacted with an agent to be tested, further contacted with an antibody of the invention, and the amount of antibody bound to the cell surface ~IPlPrminprl An increased amount of binding of the antibody to the CD4+ cell in the presence of the agent, as compared to the amount of antibody binding to a CD4+ cell in the=absence of the agent, can be~used as am indicator that the agent induces formation of a conformationally altered form of CD4 on the cell surface. Such agents which inhibit or induce expression of a ~""r." " - ;v~AIly altered forrn of cell-surface CD4 may be useful thPmAr~PIltirqlly to treat HIV infection.
The antibodies, and antibody mimetic agents, of the invention can also be used to inhibit infection of a cell by human imm~mrl~lPfiriPnry virus (HIV). To inhibit infection of a cell by HIV, the cell is contacted with a Ih. A~ y effective arnount of an antibody or mimetic agent of the invention. A cell can be contacted v~ith the antibody or mimetic agent .

' 2~95238 W0 96/02647 E~ ,lt4 in vifro, or alternatively, the antibody or mimetic agent can be adlll;ll;a~ d to a subjcct in vivo.
Yet another aspect of the invention pertains to isolated molecules that express at least one epitope expressed on a cnnf~lrm:~ti~n ~iy altered form of human CD4 induced upon 5 contact with HIV, or an envelope protein or peptide thereof. Preferably, this r~nfi~rm~til~n~lly altered form of humam CD4 is one which displays the epitope bound by either ",...,.~I.",i~l Fab 3-47 or monoclonal Fab 3-51. In one ~.,.,ho.1;~"..,l the isolated molecule expresses the same epitope bound by 3-47 or 3-51. In another rll,l~o.l;,." .,1 the molecule expresses another epitope(s) that is also exposed on this ~. ".r~ .nlly altered 10 form of CD4 upon gp 120 binding. The molecule can be a protein or peptide, such as a modified human CD4 protein, or peptide fragment thereof, or a non-human primate CD4 protein, or peptide fragment thereof (e.g., from a rhesus monkey or .,I ,;",l,r",.. ~).
Alternatively, the molecule can be an anti-idiotype antibody, or fragment thereof, that binds 3-47 or 3-S I . Moreover, the molecule can be a peptide mimetic that expresses the 15 ~ Jl;aL~: altered CD4 epitope. A molecule ofthe invention expressing an epitope(s) displayed by a cnnformqtil~n:~lly altered form of human CD4 can be i..cvl~ '.d into a mnpocitif~n preferably including a r~ Y acceptable adjuvant. A
mammal can be immuni~d with such a cull-~o,;lioll to elicit antibodies which bind a i,.,,~rO""~I...,.~llyalteredformofhumanCD4.
The molecules of the invention expressing at least one epitope of a c~-nfoTm~ti-~n~'ly altered form of CD4 can be used to induce am amtibody response in a subject that may inhibit infection of cells in the subject by HIV. Accordingly, still another aspect of the invention pertains to methods for inhibiting infection of a cell by HIV involving a-l ";. ~ . i . ,g to a subject a lh- . ~ y effective amount of a molecule that expresses at least one epitope 25 exposedonthe~,r~.",.-';,~1lyalteredformofhumanCD4suchthatanantibodyresponse against the epitope(s) expressed by the molecule is induced in the subject.
A bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the l".."- ~l. ,,,AI Fab 3-47 has been deposited under the provisions ofthe Budapest Treaty with the Americam Type Culture Collection, Rockville, MD, on July I9, 1994 and assigned ATCC Designal;on No. 69658. A bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the ., .~ l. .. ".l Fab 3-51 has been deposited umder the provisions of the Budapest Treaty with the Americam Type Culture Collection, Rockville, MD, on ~ August 25, 1994 and assigned ATCC Designation No. 69684.

35 J~ri~f D~crription of Tl- - L' Figure I is a graphic ll~ iUll of the titers of human rsCD4-specific antibodies in the serum of four human rsCD4-imn nni7~i, HlV-infected humans at the time of, and at various time points after, ;~ ;,,. ;ol ~ The arrows indicate ;" ", . ~ Al ;< n time points.

~f ~5z38 w.096/02647 P~~ 50~ll~

Figure 2 is a schematic ~ a~illtaLion of the phagemid pComb3 used to construct a~ riAl human hlullullo~lobulhl library. Arrows indicate the restriction sites used for cloning. Fab expression is induced through the lacZ promoter with IPTG. The pelB leader sequence directs heavy and light chains to the p~flulaallli~, space of induced bacterial cells, 5 where Fab assembly occurs. The heavy chain is expressed as a fusion protein with the M 13 coat protein encoded by gene III, which directs the expression of Fab molecules on the virion surface.
Figures 3A-J are a series of flow cytometric profiles depicting the binding of various antibodies to human peripheral blood IyllluLvcytcs })l~hl~,ubat~d with PBS (panels A-E) or 10 with 1~l.",1,;.,,..,l gpl20 (panels F-J). The following antibodies are depicted: control FITC-labelled goat anti-human secondary antibody (panels A and F), 19thySD7 (specific for the gpl20 binding site of CD4) vpanels B and G), L736523 (specific for the V3 loop domain of gpl20) (panels C and H), Fab clone 3-47 (panels D and I) and Fab clone 3-51 (panels E and J).
Figure 4 depicts the reactivity of Fab clone 3-47 on a Western blot with both human rsCD4 and CD4 from a human peripheral blood l~ JI,ouyt~ Iysate. The lanes show the reactivity of: an irrelevant gpl20-specific antibody (L736523) with human rsCD4 (lane 1), a CD4-specific control antibody (humanized SA8) with human rsCD4 (lane 2), the mr nr clrmAI
Fab 3-47 with rsCD4 (lane 3), the gpl20-specific antibody (L736~i23) with a human PBL
Iysate (lane 4) and the " .. ."~,rl. " ,~1 Fab 3-47 with a human PBL Iysate (lane 5). Molecular weights are indicated in kilr~ t~n~
Figure ~ is a ~hu~ur,la,uh of an h~ullullu~ ;ui~a~ion PYpPrimDnt depicting the ability of Fab 3-47, but not a negative control . ", ., .c rl~ 1 antibody, to hll~ ulu~ ;~t~ a 55 kD
cell surface protein (cvllc;auvll.lhlg to the molecular weight of CD4) from H9 cell Iysates.
Figures 6A-~1 is a series of flow cytometric profiles depicting the binding of Fab 3-47 (panels D and H), control Fab 2-36 (panels C and G), a control anti-HIV-I gpl20 antibody (panels B and F) or no antibody ("PBS", panels A and D) to human PBLs preincubated with HIV- I rgpl 20 at 4 ~C (panels A-D) or human PBLs ~, ~; ' ' with HIV- I rgp 120 at 37 ~C vpanels E-H), .1 ...",.~ , that Fab 3-47 binds to human PBLs ~ hn,ub_t~d with HIV-I rgpl20 at 37 ~C but not 4 ~C.
Figures 7A-D are a serles of flow cytome~ic profiles depicting the binding of Fab-347 (panel D), control Fab 2-36 (panel C?, a control anti-CD4 antibody ("Hu5A8", panel B) or no antibody ("PBS", panel A) to H9 cells l"c ' ' with live HIV-I~ .g that Fab 3-47 binds to H9 cells ,ul~hn,ub..tcd with live HIV-I.~5 of -l This invention pertains to a .v"rvl",aiiv"ally altered form of a cell-surface CD4 molecule expressed on a CD4+ cell upon binding of HIV, or an envelope protein thereof, to the cell. As described herein, this cv~rvl~l~aiiollally altered form of CD4 can be induced by ~W096102647 P~l/IJ~.._ ..14 contacting a CD4+ cell with isolated gpl20 (e.g., soluble ~ .",h;"..,.l gpl20). Various aspects of the invention relate to ligands, including antibodies, antibody fragments and antibody mimetic agents, that bind novel epitopes exposed on CD4 upon contact with HIV or a portion thereof (e.g., gpl20), as well as isolated molecules that express these novel epitopes exposed on CD4, and used therefor. The antibodies of the invention are capable of binding CD4 on the surface of a CD4+ cell following contact of the cell with gp 120, but do not nlhet~nti:-lly bind CD4 on the surface of the cell prior to contact with gpl20. Accordingly, these antibodies are specific for epitopes exposed on the CD4 molecule upon gpl 20 binding.
In a population of CD4+ cells contacted with gpl20, preferably at least 30 % of the gpl20+
cells are bound by an antibody of the invention. More preferably, at least 50 % of the gpl 20+ cells are bound by an antibody of the invention. Even more preferably, at least 70 %
to 90 % ofthe gpl20+ cells are bound by an antibody ofthe invention.
Preferred antibodies of the invention are human ~"nl~n~l. ."~l Fab fragments designated 3-47 and 3-51. These Fab fragments were isolated from a random ~,v~lbhl~Lul;al immlmnglnbulin phage display library prepared from an HlV-infected individual that had been immlmi7r~1 multiple times with soluble ~ hurnan CD4 in incomplete Freund's adjuvant. The light and heavy chain variable regions of mnnnclnn:ll Fabs 3-47 and 3-51 differ, ~L ~ e that they are distinct antibodies. In a preferred c~ ~ ~hu~ 1 a cnnfnrm~tinn~lly alte~ed form of cell-surface CD4 induced upon binding of HIV, or an envelope protein thereof, is defined by expression of an epitope recognized by 3-47 or 3-51 (referred to herein as the 3-47 and 3-51 epitopes"~ ly) on the CD4 molecule.
Accordingly, one aspect of this invention relates to antibodies, or fragments thereof, that bind the 3-47 or 3-51 epitopes or that bind other epitopes that are exposed on the ~nnfnrm~innsllly altered form of CD4 that also expresses the 3-47 and/or 3-51 epitope. The discovery of the 3-47 and 3-51 epitopes provides evidence for an HlV-induced rnnfnrm~tinn ~l change in the CD4 receptor that can be targeted for diagnostic, screening and therapeutic purposes, as described herein.
The term "antibody" as used herein refers to immllnnglnbulin molecules and imm~nnlngir~lly active portions of immlmngloblllin molecules, i.e., molecules that contain an antigen binding site wbich specifically binds (il~ Ul~ i with) an antigen, such as a n..,,r~ llyalteredformofCD4. Theinventionpertainstopolyclonaland,more preferably, mnnnrlnn:~l antibodies. In one ~.. bodi.. ~.i, a mnnnçlnn~l antibody of the invention is a human l ....".~ l antibody. Additionally, .~c.... ~ l antibodies, such as chimeric and humanized mnnnrl~ n~31 antibodies, comprising both human and non-human 35 portions, are within the scope of the invention.
Structurally, the simplest naturally occurring antibody (e.g., IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally-occurring antibody. Thus, these antigen-binding wo96/02647 21 95238 ~ ~l/L~

fragments are also intended to be designated by the term "antibody". Examples of binding fragments c,.. ~ .1 within the term antibody include (i) an Fab fraBment consisting of the VL, VH, CL and CHI domains; (ii) an Fd fragment consisting of the VH and CH1domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody,(iv)adAbfragment(Wardetal.,(1989)Nature341:544-546)whichconsistsofa VH domain; (v? an isolated rnmplimrnt~rity ~1. r, ....;.,;.,g region (CDR); and (vi) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linlced by a disulfide bridge at the hinge region. Antibody fragments, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using cullv~l.Liulldl techniques, such as papain or pepsin digestion, 10 respectively, of whole antibodies. Moreover, antibody fragments can be obtained using standard lcc.7.,.1.;..~ .I DNA techniques, as described herein. Furthermore, although the two domains of the Fv fragment are çoded for by separate genes, a synthetic linlcer can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al. (1988) Sc~ence 242:423-426; and Huston et al. (1988) PN~S 85:5879-5883) by 15 ~ .1 methods. Such single chain antibodies are also rll~ cd within the term "antibody". An antibody of the invention is further intended to include bispecific and chimeric molecules having a binding portion that recognizes a cnnforrn~tinnsllly altered form of CD4. 1 ~ : E : ~
The term "antibody combining site", as used herein, refers to that structural portion of 20 an antibody molecule comprised of heavy and light chain variable and hyu~l val;ablc regions that specifically binds (hlllllullul~,a~,b with) antigen. The term "hlllllullulca~.L" or "rqactive with" in its various forms is used herein to refer to binding between an antigenic 11~lr~
containing molecule and a molecule containing an antibody combining site, such as a whole antibody molecule or a portion thereof. The term "epitope", as used herein, refers to the 25 actual structural portion of the antigen that is imml-nnlngirally bound by an antibody combining site. The term is alsQ used iulltlcllallg~,ably with "antigenic rl.1.. ;,.~.. 1". When a particular epitope is present on a molecule and available for immnnnlogi~ l recognition (e.g., binding by an antibody), the epitope is said to be "expressed" or "displayed" by the molecule.
The epitopes of particular interest with regard to this invention are not expressed 30 cul~LiLuLi~,ly on the native cell-surface CD4 molecule but rather become expressed (i.e., exposed) on the ~.. r~.. - ;.. ,.~lly altered form of CD4 upon gpl20 binding, as described herein. Such epitopes that are exposed on a molecule upon a cnnfnrmsltinn ~I change in the molecule are often referred to in the art as "neo-epitopes". The term "epitope binding specificity" of an antibody, as used herein, refers to the reactivity of an antibody for a specific 35 epitope, i.e., antibodies that bind the same epitope are referred to as having the same epitope binding specificity. To determine whether two antibodies bind the same epitope on a particular antigen (e.g., the 3-47 epitope on the cnnfnrn~qtinn~lly altered form of CD4 described herein), the ability of one antibody to cv.l.l.~.iLivcly inhibit the binding of the other antibody to the antigen can be determined by conventional techniques.

:

.. ... . . . =

21 ~5238 ~Wo 96/02647 '- ~ ' r~ 5'~114 g Various aspects of the invention are described in further dctail in the following cnhcPctionc 1. p,~",.,..l;.~. and IdPnfifirs'tion of Anfihorii~c s A. Tmmnni7~tion An amtibody of the invention is typically prepared by immlmi7inp a suitable subject with an ~,u~-u,u-i...~, CD4 imm--nogPn and isolating an antibody having the .,1,~ t~
described herein. In a preferred ~, . ,ho~ . .1 " .. ~ r l~ 1 antibodies are isolated by 10 screening a eoml ' i " " .., ...ngl. ,1 " 11; l l library prepared from the immunized subject, although ll,..,., c.l",.~l antibodies may also be isolated by screening cullv~ tiulldl antibody-secreting hybridomas. Alternatively, it may also be possible to isolate a ""."orl,l"iil antibody having the desired epitope binding specificity by screening a ~ lob~ll;,, library prepared from an ".,;",.., ~..;,~ d subject (e.g., an HlV-infected subject or an uninfected subject).
In a preferred ~.. ,l o.l;,.. 1 the CD4 ;",.. ,.,."~,, .. is human .~ l;",.,.l soluble CD4 (rsCD4) and the immunized subject is an HlV-infected human. Alternatively, the immunized subject can be an uninfected subject (e.g., human). In addition, it may be possible to generate antibodies directed against a ~ . ,., r~ " " . ~ lly altered form of cell-surface CD4 by 20 immnni7ing a subject with soluble CD4 from another species. For exarnple, a non-hurnan primate, such as a rhesus monkey or a cl,;."l, ,,. ~, can be immunized with human rsCD4, or a human can be immunized with a non-human primate rsCD4 molecule (e.g., rhesus monkey or ~ l-""~ e CD4). In another . .~hbll;lll~ 11, a subject can be imrnunized with a molecule (e.g., a peptide) that expresses the 3-47 and/or 3-51 epitope(s), described in further detail 25 below. Alternatively, it may also be desirable to immunize an animal with a soluble CD4-gpl20 complex or with CD4+ cells (e.g., peripheral blood ly~ Jho. yt~;~) that have been treated (i.e., contacted with) soluble .c~ .... ,l ... - ,l gp 120. It may also be possible to raise antibodies as described herein by ;. ", .,....;, ;"g a mouse transgenic for human CD4 (and therefore tolerant to native human cell-surface CD4) with a CD4 i"",.,...r,, " ,. such as a 30 soluble form of a non-human primate CD4 molecule or an altered form of a soluble human CD4.
The unit dose of CD4 i.. ,.. "~.~,.. , and the il"".,......... ,;,~ regimen will depend upon the species of mammal immlmi7P~i, its immune status, the body weight of the mammal and the CD4 content ofthe CD4 immnnogen ad...;..ist~,.c;d. For ;....,. ~ .;, l;..,. the CD4 35 ;~ oy~ ., is typically dd...i..;,,.~l~d with an adjuvant, such as Freund's complete or incomplete adjuvant. In an illustrative rl I Iholl; ~ 1, I mg of human rsCD4 in incomplete Freund's adjuvant is injected i, ~ - ly into an HlV-infected individual (with absolute CD4 counts greater than 500) and the individual is boosted several times (e.g., five times) at regular intervals (e.g., every 3-5 weeks).

wo96/02647 21 95238 - lo r~l,., s ,~

Tllllllll.l;~AI;I",ofasubjectwithaCD4imml~A.A,genasdescribedaboveinducesa polyclonal anti-CD4 antibody response. The anti-CD4 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked uuuuuulbsbllJ~ assay (ELISA) using immnhili7Pd rsCD4. If desired, the polyclonal antibody S molecules can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chmlmAtngrArhy to obtain the IgG fraction. At an appropriate time after; I I I I ~ e.g., when the anti-CD4 antibody titers are highest, ~l~nl~lcl""AI antibodies can be prepared and screened.

10 B. I~ h;~ C..~ Anfihody LihrAAriPs In a preferred ~...bod;...~.L, mnnnAInnRI antibodies are prepared by ~,ullaLI u.lil.g a Ir."",l,",A,,I c~ l""i;" library, such as a Fab phage display library, using immlmngl~A~bulin light chain and heavy chain cDNAs prepared from mRNA derived from IYUI~J]IOI,YI.,~ ofthe immunized subject. Mr~ nl~ forpreparing and screening 15 such a library are described in detail in the Examples. Briefly, mRNA is isolated from a Iy~ llo~ ,-containing cell population, such as bone marrow Iyl~ llo~ . First-strand cDNA is ~yuLll~ .;~d using primers specific for a constant region ofthe heavy chain (e.g., CH3) and the constant region of each of the K and ~ light chains. Using primers specific for the variable and constant regions, the heavy and light chain cDNAs are amplifed by the 20 polymerase chain reaction (PCR). The amplified DNA is then ligated into appropriate vectorsforfurthermAnir..lAtiA,ningeneratingalibraryofdisplaypackages. Ol;~,.,...l.l.~l;~iP
primers useful in Amplifi~AAtiA,n protocols may be unique or degenerate and may incorporate inosine at degenerate positions. Restriction ~ ...1"". ., 1~ A~ recogmtion sequences may also be 'ul~w,uul~:L~d into the primers to allow for the cloning of the amplified fragment into a vector in a ,ul~llrlrl 111;11~ d reading frame for expression.
The immnnnglnblllin library, e.g., a Fab library, is expressed by a population of display packages, preferably derived from filAmPntmlc phage, to form an antibody display library. Ideally, the display package comprises a system that allows for the sampling of a large, diverse antibody display library, rapid sorting after each affinity separstion round, and easy isolation of the antibody genes from the purified display packages. In addition to ~,uuull.,l~,;dlly available kits for generating phage 'display libraries (e.g., the Pharmacia Rec,,...bi,~...i Phage ~ntibody S~stem, catalog no. 27-9400-01; and the Stratagene Sur,~Z4PTM phage display kit, catalog no. 240612), examples of methods and reagents ,u~Li.,ul_ly amenable for use in generating antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. TntPrnAfinnAI Publication No.
WO 92/18619; Dower et al. 1,.:. .. I,-l;,l..AI Publication No. WO 91/172il; Winter et al.
TntPrnAtinnAI Publication WO 92/20791; Markland et al. TntPrnAfi~AInAl Publication No. WO
92/15679; Breitling et al. In~PrnqtinnAI Publication WO 93/01288; McCafferty et al.
TntPrnqtinnAl Publication No. WO 92/01047; Garrard et al. Illlrl " -I;IIIIAI Publication No. WO

..

2 ~ 95238 ~w.o 96/02647 P~ 4 92/09690; Ladner et al. Int~rnRtionAl Publication No. WO 90/02809, Fuchs et al. (1991) Bio/TechnoloS~,~ 2:1370-1372; Hay et al. (1992) Hum Antibod Hybridornas ~:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) ~MBO J 12:725-734; Hawkins et al.
~ (1992)JMolBiol~2~:889-896;Clacksonetal.(1991)Nature352:624-628;Grametal.
(1992~ PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 2: 1373-1377; Hoo~Pnhonm etal.(l991)NucAcidResl9:4133-4137;andBarbasetal.(l991)PNAS88:7978-7982.
In certain cl~bodh~ , the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFv gene ~ 1 ly 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 amtibody, joined by a flexible (Gly4-Ser)3 linker can be used to produce a single chain antibody expressed on the surface of a display package, such as a filornPntl~lle phage.
Once displayed on the surface of a display package (e.g., filomPntouc phage), the antibody library is screened to identify and isolate packages that express an antibody that binds a r~nf~lrmAti~n~311y altered form of CD4. In a preferred (..lllbUdilll~,lll, the primary screening ofthe library involves panning with imm~hili7rd rsCD4, or, more preferably, am immr~hili7Pd rsCD4/gpl20 complex (described in further detail in Example 3). Display packages expressing antibodies that bind immobili_ed CD4, or, more preferably, the rsCD4/gpl20 complex, are selected. Soluble forms ofthe selected antibodies canthen be generated (as described in Example 3) amd the soluble antibodies further selected in secondary screenings, e.g., by ELISA, ,~:n~ y and/or flow cytometry (FACS
analysis). For example, an amtibody that binds a cr.nformAti~-nAlly altered form of CD4 is preferably selected based upon its ability to bind CD4+ cells contacted with ,~e ~ " "h" - ,1 gpl20, while not s~hctAnti~llly binding to CD4+ cells prior to gpl20 treatment (see Example 4). Preferably FACS analysis is used to determine whether an antibody binds cell-surface CD4 in the presence or absence of gpl20 treatment. FACS analysis can also be used to determine the percentage of gpl20+, CD4+ cells that bind the antibody.
Reagents useful for screening antibodies of the invention have been described indetail in the art andlor are cullllA.,~,;ally available. For example, full-length and truncated, soluble forms of human CD4 useful in the above-described screening assays are disclosed in PCT patent application PCTIUS88/02940 and Fisher, R.A. et al. (1988) Nature 331 :76-78.
Reference is also made to Littman (1988) Cell 55:541, which describes the correct signal sequence cleavage site of pre-human CD4 and which was published after the filing date of PCTIUS88/02940. R.e~,.. "l,;l.,".l soluble human CD4 is commercially available (e.g., from ABT, ('Amhri~lgP, MA). HIV gpl20 for use in the above-described assays can be, for example, gpl20 isolated from HIV or, more preferably, lccvlllL,hlalll gpl20 expressed by host cells transfected with the gene for HIV gpl20. Preferably, a purified, soluble HIV gpl20 isolated from a unicellular host transfected with a truncated gp 160 gene encoding HIV gp 120 wo96/02647 ~ . 5'0~l14 is used. Purified ~ ",h;"A~,l HIV gpl20 is commercially available (e.g., from Repligen, Cambridge, MA or from Celltech, Berkshire, United Kingdom). Cells producing l~C~ Il l lh;~ gpl 20 are described, for example, in Lasky et al. (1986) Science 2~:209-212.
CD4+ cells for use in screening assays include peripheral blood Iymphocytes (either 5 nnfrPrti( ~ or a selected CD4+ subpopulation) and tissue culture cells transfected with DNA encoding full-length human CD4 and expressing CD4 on their surface. Suitabletransfected CD4+ tissue culture cells are described in Fisher, R.A. et al. (1988) Nature ~1:76-78. ~ ~
Following screening and isolation of a . ,..., .nrl. ,.. l antibody of the invention from a 10 lr~,,..l,;..A.,I ;,,"~",.,nglrl~"~lin display library, 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 l~ ~ . " . ,1,; . . - - . I DNA techniques. The nucleic acid can be further I ~ rd (e.g., linked to nucleic acid encoding additional ;~ 8 b~
domains, such as additional constant regions) and/or expressed in a host cell, as described in 15 further detail below.

C. Hyhri~lnmAc Alternative to screening of an antibody display library, a mnnnclnnAI antibody of the invention can be prepared and isolated using a technique which provides for the production of 20 antibody molecules by continuous cell lines in culture. These include, but are not lirnited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981) J: Immurol 127:539-46; Brown et al. (1980~ JBiol Chem 255:4980-83,Yehetal.(1976)PNAS76:2927-31;andYehetal.(1982)1nt.J. Cancer _:269-75), and the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), EBV-hybridoma technique (Cole et al. (1985), Monn~lnnAI Antihn~ c An~ ('AnAPr Ther~,ny, Alan R. Liss, Inc., pp. 77-96), and trioma techniques.
l~he technology for producing mnnnrlnns31 antibody hybl;dolllds is well known (see generally R. H. Kenneth, in l\~fnnnrlnnol Antih~ c A New Dim,oncir-n Tn gini~i~AAI
Analyses, Plenum Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., ~L:387402; M. L. Gefter et al., ~1977) Somaric Cell Genet., ;~:231-36).
Briefly, an immortal cell line (typically myeioma cells) is fused to Iy~ ho.,y ..~ (typically locy~.,s) from a mammal immunized with a CD4 i,,"."."n~,~ ,. as described above, and the culture ~ ~1'' . .A I A .1~ of the resulting hybridoma cells are screened, as described above for screening of l~ n. . .h;, IA ~1 immlmnglnblllin libraries, to thereby identify an antibody of the 35 irlvention.
Any ofthe many well known protocols used for fusing IyllllJho~.yi. ~ and immortalized cell lines can be applied for the purpose of generating an antibody of this invention (see, e.g., G. Galfre et al., (1977) Nature ~i:55052; Gefter et al., somatic Cell GeneL, cited suFra;
Lerner, Yale J. Biol Med., cited supra; Kenneth, MonnclonAI Antihn~ c cited supra).

~ W096/02C47 2 1 95238 P~ 0r~-l4 Moreover, the ordinary skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same ~ ;-.l species as the Iy~ JLoL,yi~i. For example, murine hybl ;du~l~as can be made by fusing Iy~ ho.,y t., from a mouse immumi~d with a CD4 5 immlm~lprn with an hlllllultaL.~,d mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hy~u~alltL;l-e, ,u' ;1 l and thymidine ("HAT medium"). Any of a number of myeloma cell lines maybe used as a fusion partner according to standard techniques, e.g., the P3-NSI/I-Ag4-1, P3-x63-Agg.653 or Sp2/O-Ag 14 myeloma lines. These myeloma lines are available from the 10 American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse S~ IO~Y .." using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and umproductively fused myeloma cells (unfused splenocytes die after several days because tbey are not ~ r,.. d). Alternatively, human Lyl~l;dulllds can be made using 15 human Iylll~Lo~,yL~,i (e.g., from an HlV-infected individual immuni~d with soluble human CD4, as described above) and human B cell- or EBV-hybridoma techniques.
Hybridoma cells producing a m~ .. ,o~ antibody of the invention are detected by screening the hybridoma culture ~u~,llla~ll:. using the screening assays described above.
For example, a primary screen can be performed to select antibodies that bind immobilized humam rsCD4 or, more preferably, an immnbili7Pd rsCD4/gpl20 complex (e.g., by ELISA).
A secondary screen can then be performed to identify antibodies that bind CD4+ cells treated with Ir~ ....1.; ._ -1 gpl20 but which do not c--hct~lntis~lly bind CD4+ cells prior to gpl20 treatment. This secondary screen is preferably performed by FACS analysis.
Hybridoma cells that test positive in the above screening assays can be cultured in a 25 nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the ..,...,,~ 1 antibodies into the culture medium, to tbereby produce whole antibodies. Tissue culture techniques and culture media suitable for hybridoma cells are well known (see, e.g., Lerner, Yale J Biol. Med. and Kenneth, Mrm~ nnsll Antih-~ip~ cited supra). C.-.n~litinnPd hybridoma culture ~ a containing the antibody can then be 30 collected. Alternatively, the desired antibody may be produced by injecting the hybridoma cells into the peritoneal cavity of an .. ; .. - l; ,~1 mouse. The hybridoma cells proliferate in the peritoneal cavity, secreting the antibody homolog, which àCI ' as ascites fluid (see Lerner, Yale J. Bioi. Med. and Kenneth, Monorlf~n~l Antihn~ cited supra). The antibody is harvested by wiLhLllaw;llg the ascites fluid from the peritoneal cavity with a syringe.
35 Accordingly, it will be understood by the ordinary skilled worker that monnf~ nAI antibodies of the invention rnay be purified with ease from cf n~iti~nrd hybridoma culture ~u~u."ll~llallt or from ascites.
A mon~ n~l antibody prepared from a murine (or other non-human) hybridoma has the d;~ld~all~a~, that the antibody will be recogni_ed as foreign in a subject ûf anûther Wo 9C102C47 ~2 1 9 5 2 3 8 r~l" 'J~.14~

species (e.g., a human). One approach to circumventir;g this problem is to engineer a l~ .",l,;"~ chimeric or humanized antibody derived from the original non-human mnnn- ]~n~l antibody, as described in further detail below. As an alternative to h~ lg a non-human Illnl~n~ 1 antibody, a human mnnn~lnn~l directed against a human protein can 5 be generated in transgenic mice carrying human antibody repertoires (see, e.g., Wood et al.
PCTpublicationWO91/00906,Kn.h~rl:~r~tietal.PCTpublicationWO91/10741;Lonberg et al. PCT publication WO 92/03918; Kay et al. PCTpublication 92/03917; Lonberg, N. et al.
(1994) Nature 368:856-859; Green, L.L. et al. (1994) Nature Genet. 1:13-21; Morrison, S.L.
etal.(l994~Proc.NatLAcad.Sci. USA81:6851-6855;Rn.pP:~m~netal.(1993)Year Immunoll:33-40; Tuaillon et al. (1993) PNAS 90:3720-3724; Bruggeman et al. (1991) EurJ
Immunoll:1323-1326). A human antibody-transgenic mouse can be immunized with a CD4;, . " . ", ~nc,~ . I as described above and ~ u.,, t~.~ from these immunized transgenic mice can then be used to create hyb~;dulllas, which are then screened to identify an antibody of the invention as described above. Additionally, double transgenic animals, e.g., transgenic both 15 for human CD4 and human antibody genes, can be immunized with a CD4 immunogen and Iylll~hu~.rt~s therefrom used to generate human mnnnrl.-nsll antibodies of the invention.

D. ChimPric rn~l Hnnnsmi7.. ~i Antiho~ c The antibodies of the invention further encompass 1~ .., .h;. ~ forms of antibodies, 20 such as chimeric and humanized antibodies. When antibodies produced in non-human subjects are used ih~-r:lr~ ti~ lly in humans, they are recognized to varying degrees as foreign and an immune response may be generated in the patient. One approach for " ;;; ,;" ", ;. ,g or eliminating this problem, which is preferable to general h ~ ,;ull. is to produce chimeric antibody derivatives, i.e., antibody molecules that combine a non-human arlimal 25 variable region and a human constant region: Such antibodies retain the epitope binding specificity of the original, .. ~rll ." ~ antibody, but may be less h ~ n& ~;~ when - ' c;d to humans, and therefore more likely to be tolerated by the patient.Chimeric .,....~..,l....~l antibodies can be produced by l~..",h;"..,.l DNA techniques known in the art. For example, a gene encoding the constant r~egion of a non-human antibody 30 molecule is substituted with a gene encoding a human constant region. (see Robinson et al., Tnt~nn~tinn~l Patent Publication PCT/US867Q2269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al.. European PatentApplicationl73,494;Neubergeretal.,PCTApplicationWO86/01533;Cabillyetal.
U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al.
(1988 Scien~ce 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J.
Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Canc.
Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancerlnst 80:1553-1559).
.

..... : . : , .

W0 96/02c47 ~ r~ .114 A chimeric antibody can bc furthcr l'Lu..~ .d" by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions.
General reviews of "I.ul.l_l;~.,d" chimeric antibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207 and by Oi et al. (1986) BioTechn~ques _:214. Such methods include 5 isolating, mqnip~ ting" and expressing the nucleic acid sequences that encode all or part of an immnn~-globulin variable region from at least one of a heavy or light chain. The cDNA
encoding the humami~d chimeric antibody, or fragment thereof, can then be cloned into an a,UIJlUI expression vector. Suitable '~LU.~A ~d antibodies can be alternatively produced by CDR or CE~A, ~ ..,.. (see U.S. Patent 5,225,539 to Winter; Jones et al.
(1986) Nature ~1:552-525, Verhoeyan et al. ( 1988) Science 239 :1534; and Beidler et al.
(1988) J. ImmunoL 141:4053-4060).

F Deriv,qfi7~ ntihn~lirc In another .. ,.h~.1:.. ,1 this invention provides a derivati~d antibody in which an 15 antibody ofthe invention is functionally linked (by chemical coupling, genetic fusion or otherwise) to one or more other molecular entities, such as another antibody of the invention, a mimetic agent of the invention (described below), a detectable agent, a cytotoxic agent amd/orapl.A,."~ ;.Al agent.
One type of derivati~d antibody is produced by ~,luaalil~illg two or more antibodies 20 (of the same type or of different types). Suitable ~luaalh~tla include those that are h~t~,luh;rl~ nql having two distinctly reactive groups separated by am appropriate spacer (e.g.,m-maleimidobenzoyl- N-hydluAy~ ester) or h~ h; r~ lA1 (e.g., ~1 _.-. . ;..;,,,;(lyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL.
Useful detectable agents include fluorescent ~ u.~ xemplary fluorescent detectable agents include fluorescein, fluorescein iau ll;o~y , rh~rlqminr 5-dilll~lllyllllille-l- r ' ~ ~ yl chloride"uLy~,u.,.y~ll;ll and the like.
An antibody may also be derivati~d with detectable enzymes, such as alkaline Pl~ hA A~ h~..~..,..l;~l. peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent h.,l ,- ,--1; ~1. peroxidase is present, the addition of hydrogen peroxide and b~ line leads to a colored reaction product, which is detectable. An antibody may also be derivatized with biotin, and detected through indirect Ill.,aaulc;lll.lll of avidin binding.
The invention also provides antibodies linked to one or more ~ agents.
Useful l~ agents include biologically active peptides, polypeptides and proteins, such as an antibody specific for a human polypeptide other than CD4. Other useful l.hAl ~ ,.l;. AI agents include non-~lu..,hld~euua pl, ~ "l;Aql agents such as HIV reverse inhibitors (e.g., 3'-azido-2',3'-dideu,~yll.~.l.idine ("AZT") and l j' ''-2-1 q5238 wo 96102647 16 , ~ s .114~
_ 2',3'-did~u~yillo~;lle ("DDI") and other antiviral cnmrolm~c or immnnn~;v~ agents (e.g., ~y~lO~IJUl;l~ or FK506).

F. ~mihnf¦y IVTimetic ~entc The invention further .. , .. "~ non-antibody molecules that mimic the epitope binding specificity of the antibodies described herein. These agents are referred to herein as "antibody mimetic agents". The antibody mimetic agents of the invention are non-antibody compounds that bind an epitope exposed on a l,ollrullllaL;ùnàlly altered form of human CD4 upon binding of HIV, or an envelope protein thereof, to CD4. Accordingly, these uullll~uul~d~
lO bind CD4+ cells upon incubation with gpl20 but do not substantially bind CD4+ cells prior to gpl20 treatment. Preferred antibody mimetic agents ofthe invention bind an epitope recognized by the " .l ,. ~nrl- ~~ IAI Fab 3-47, referred to herein as a "3-47 mimetic agent", or an epitope recognized by the mnnrrlnn:~l Fab 3-51, referred to herein as a "3-51 mimetic agent" .
Preferred amtibody mimetic agents, e.g., 3-47 or 3-51 mimetic agents, inhibit infection of ~ l S CD4+ cells by HIV. The most preferred antibody mimetic agents of the invention display the propertiesofoneormoreantibodiesofthisinvention(e.g., """,n~l.".Al Fabs3-47Or3-51).
An antibody mimetic agent of this invention may be produced by synthesizing a plurality of peptides (e.g., 5-20 amino acids in length), semi-peptidic culll,uuullds or non-peptidic, organic .~ u~ and then screening those ~ for their ability to 20 bind CD4+ cells upon treatment of the cells with gpl 20, using assays described herein. For general ~Ircrrirtinnc of peptide library Cull:~u U~,l;UII and screening see U.S. Patent No.
4,833,092; Scott, J.K. and Smith, G.P. (1990) Science~12:86-90; Devlin, J.J. et al. (1990) Science 2~L2:404-407. Alternatively, the agents can be screened for their ability to competitively inhibit binding of an antibody of the invention to gpl 20-treated, CD4~ cells.
25 For example, a 3-47 mimetic agent can be identified based upon its ability to inhibit the binding ofthe monoçlnn6~l Fab 3-47 to gpl20-treated CD4+ cells. Preferably, FACS analysis is used to determine whether an antibody mimetic agent can Culll~ ~liLi~.,ly inhibit the binding of an antibody of the invention to gpl20-treated CD4+ cells.

30 IT Rr-~nmhin:lnt Fyrreccinn of Antihn~ c In one ~",l,o.l;,". .,1 an antibody ofthe invention is produced in quantity by ~ "",l,;.,A.,lexpressionofimmnnngloblllinlightandheavychaingenesinahostceil. Toexpress an antibody IC~ ' ~I "T,;, ,-. .ITy, a host cell is transfected with DNA encoding the immlmnplobulin light and heavy chains of tke antibody in a form suitable for expression of 35 the light and heavy chains in the host cell. Re~ " "l ,; ".., ,1 antibodies may be produced by well known genetic rl ~ Ig techniques (see, e.g., U.S. Patent No. 4,816,397).
When an antibody (or antibody fragment) of the invention is isolated from a 1~ .,,l,h;l.,,.ll immnnnglnblllin display library, as described above, DNA encoding the light and heavy chains of a selected antibody of interest can be recovered from the display package ~ WO 96/02647 ~ 1 9 5 2 3 8 P~ .114 (e.g., from the genome of the fil~nn~ntm ~c phage) and, if desired, further ~ Such ;u~ ~ may involve conversion of a partial antibody chain to a full-length antibody chain. For example, when a Fab expression library is screened, the isolated DNA encoding the heavy chain of the Fab can be converted to a full-length heavy chain gene by operatively 5 linking the DNA to another DNA molecule encoding the additional heavy chain constant regions. In this manner, the monnrl nn~l Fab fragments 3-47 and 3-51 described herein can be converted to full-length, whole antibodies having the same epitope binding specificity of 3-47 or 3-51. Similarly, if a scFv library is screened, the portions of the isolated DNA
encoding the linked VL and VH regions of the scFv can be separated and the separate VL-10 and VH-encoding DNA molecules can then be operatively linked to other DNA molecules encoding the ~ ul light and heavy chain constant regions to produce full-length antibody genes.
Alternatively, when an antibody of the invention is isolated by screening hrb~;du~l~ds, as described above, cDNA or genomic DNA encoding the immnnnglclblllin light and heavy 15 chains of a selected antibody, or a portion thereof, can be isolated from the hybridoma cell by standard molecular biology techniques.
Following isolation, and, if desired, further ' nn cDNAs or genomic DNAs encoding partial or full-length light or heavy chains are inserted into expression vectors so that both genes are operatively linked to their own ~ . ,;l,lin.~l and l","~l~a;",.~l control 20 sequences. The expression vector and expression control sequences are chosen to be uulll~dtilJle with the expression host cell used. Typically, both genes are inserted into the same expression vector. For expression of the light and heavy chains, the expression vector(s) is transfected into a host cell by standard techniques. r~uhhlruL;c or eukar,votic host cells may be used. The terms '~ . I;""" or "Llall~r~ .d into" are intended to encompass a 25 wide variety of techniques commonly used for the hlLIudu~,l;un of exogenous DNA into a ~uhhlruLic or eukaryotic host cell, e.g., elcl~Llul~ul~Lion~ calcium-phosphate ~ ;LdLiull~
DEAE-dextran ~ f ~ ... and the like. Expression of antibodies in eukaryotic host cells is preferred because such cells are more likely than ~luh~uLic cells to assemble and secrete a properly folded and ;.,.... ~ ,oln~ lly active antibody. However, any antibody produced that 30 is inactive due to improper folding may be renaturable according to well known methods (see e.g., P. S. Kim and R. L. Baldwin (1982) Ann. Rev. BioclZe~n. 51.45989).
Host cells can also be used to produce portions of intact antibodies, such as light chain dimers or heavy chain dimers, which are ~ ;I by the terrn "antibody" as used herein. It will be understood that variations on the above procedure are within the scope of 35 the present invention. For example, it may be desirable to transfect a host cell with DNA
encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. Re~ DNA technolog,v may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to a ..., . r... ,..~ lly altered form of CD4. The molecules expressed from such truncated DNA

W096/02647 - 18 - r~

molecules are also r~ 1 by the antibodies of the invention. In addition, hifimrt;-lnql antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than CD4, or another epitope of CD4.
In a preferred embodiment, the m~-nf~nlf~nql Fab 3-47, or a full-length antibody having the same epitope binding specificity of 3-47 (e.g., the VL and VH regions of 3-47), is produced Ir~ h; ~ ly in a host cell. The partial nucleotide sequence of an isolated nucleic acid molecule (e.g., DNA) encoding the light chain variable region (VL) of 3-47 is shown in SEQ ID N0: 15. The partial nucleotide sequence of an isolated nucleic acid molecule (e.g., DNA) encoding the heavy chain variable region (VL) of 3-47 is shown in SEQ ID N0: 16. A
bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the m~ rl~nql Fab 3-47 has been deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, MD, on July 19, 1994 and assigned ATCC
Designation No. 69658. Olig~nlu~lqoti(l~ primers described in Example 2 can be used to amplify DNA encoding the light or heavy chain of 3-47 by the polymerase chain reaction using this plasmid as template DNA.
Inanotherpreferredrll,h-~.l;",. .~l~them~n~nl~nq~Fab3-5l~orafull-lengthantibody having the same epitope binding specificity of 3-51 (e.g., the VL and VH regions of 3-51), is produced IC~ ly in a hbst cell. The partial nucleotide sequence of an isolated nucleic acid molecule (e.g., DNA) encoding the light chain variable region IVL) of 3-51 is shown in SEQ ID N0: 17. The partial nucleotide sequence of an isolated nucleic acid molecule (e.g., DNA) encoding the heavy chain variable region (VL) of 3-51 is shown in SEQ ID N0: 18. A
bacterial host cell carrying a plasmid encoding the light and heavy chain genes of the mr~nrml~nql Fab 3-51 has been deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, MD, on August 25, 1994 and assigned ATCC Designation No. 69684. Oligu~u~,le~JIide primers described in Example 2 can be used to amplify DNA encoding the light or heavy chain of 3-51 by the polymerase chain reaction using this plasmid as template DNA.
A nucleic acid molecule of the invention may encode only the i " " " . ~ bulil, light and/or heavy chain variable region of an antibody of the invention, e.g., the 3-47 or 3-51 Fab.
More preferably, the nucleic acid molecule also includes nucleotide sequences encoding at least one immlm~ bnlin constant region operatively linked to the variable region-encoding DNA (i.e., the VL region can be linked to a CL region or the VH region can be linked to one or more CH regions). The nucleic acid molecules of the invention can be inserted into expression vectors and transfected into host cells to express the mon~lrl~nql Fab fragments 3-47 or 3-51, or a portion thereof, or a full-length antibody having VL and VH regions derived from 3-47 or 3-51, as described above. In one r,~ O~ 1, the host cell is a bacterjal cell (e.g., E. coll~. In another, preferred, c",ho~l;",. 1 the host cell is a mqmms3liqn cell, such as a CH0 cell or a myeloma cell. To produce ml-nl-rlAnql Fab 3-47 or 3-51, or an antibody (e.g., wos6lo2c47 19 r~ ,.14 full-length _ntibody) having the epitope binding specificity of 3-47 or 3-51, the host cel;is cultured for a period of time suff cient to allow for expression of the antibody in the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell - is grown.

- JTT Anfihody C.. "~,.. ~;l;.. ,.
The antibodies of this invention can be hlCUI,UI ' ' into l,l IA111IA. ~ . ~1 ;~ Al compositions that can be used prophylactically or ~ y in the prevention or treatment of diseases caused by infectious agents whose primary targets are CD4+10 Iymphocytes. Such diseases include AIDS, ARC and HIV infection in humans. The generic term "HIV" is intended to refer to ;~ isolates from AIDS patients and to laboratory strains derived therefrom. The term HIV is also intended to include viruses elsewhere identified as human T cell lylll,uhulluAullic virus type III (HTLV-III), lylll~had..lu~/ailly-associated virus (LAV) and AIDS-associated retrovirus (ARV). The rnmpncitinnc may also 15 be useful for treating or preventing AIDS-like diseases caused by other l~lluvi. u~cs, such as simian imml-nn~1rfirirnry virus.
Preferred pl .A ", . ~ ;rAI c..., .~ .Uc of this invention include an antibody having the epitope binding specificity of the " ,. ~ ~n~ Fab 3-47 or the epitope binding specificity of the mnnn~AInnAI Fab 3-51. The antibody may be, for example, 3-47 or 3-51 itself, or a whole antibody containing VL amd VH regions derived from 3-47 or 3-51 (as described herein).
The ~ of this invention may further comprise other lh~ for the ~lu~ull~laAi~ or treatment of AIDS~ ARC and HIV infectiom For example, an antibody of the invention may be used in c....,l.;"Al;nl, with allthc;Lluv;lal agents that block 25 reverse ~ l-, such as AZT, DDI, HPA-23, l~l,n~ r( , suramin, ribavirin and didcuAy~,ylidine, or with agents that inhibit the HIV protease. Additionally, the ,I,A,.,,~ ~llirA~ ofthis invention may further comprise anti-viral agents such as interferons (including alpha interferon, beta interferon and gamma interferon) or glllroci~lAc~
inhibitors such as . AJ - ~n~l~. ' Ill;llr, or i""..l...n~.ll,l"~,a~ , agents such as adrenal 30 cu.Licu~.t~,.uids, A~AIh~ ,lu;~UU-;-- orFK506.
Moreover, the antibodies of the invention may be adl~ ;st.,.cd in rnmhinotinn with anti-CD4 antibodies having differing antigenic .~ . ;r. ;1;. C than the antibodies described herein, such as anti-CD4 antibodies that bind native cell-surface CD4 in the absence of gpl20 binding (e.g., that are specific for the Vl, V2, V3 or V4 domain of native CD4). They may 35 also be ad...i.~ cd in r.nmhin-~finn vith antibodies (anti-idiotypic or otherwise) specific for HIV polypeptides such as gpl20 and gp41.
Fu. Lhcllllulc:~ one or more antibodies of the invention may be used in rnmhinAfinn with two or more of the foregoing therapeutic agents. Such uullllJ;lla~iull therapies may WO 96/02647 '~2 1 q 5 2 3 8 ~ 14~

aJval~Lagcuualy utilize lower dosages of the dd~ ;a~ cJ therapeutic agents, thus avoiding possible toxicities or rr~mplir~tir~nc associated with the various monotherapies.
Preferably, the 1~ c of the invention comprise an immnn~ au~JLi~ Dy effective amount of one cr more antibodies according to this 5 invention, orderivatized form(s)thereofand,preferably, al,l,- ..,~....l;..~llyacceptable carrier. By "tl~ lly effective amount" is meant an amount capable of lessening the spread, severity or; ..., . " .. ,n~ ulll;ahlg effects of AIDS, ARC or HIV infection, or of other diseases caused by infective agents whose primary targets are CD4+ lylllpllo~
Preferably, the ~ ly effective amount is capable of completely preventing infection 10 by such viruses when used ~lu~Jhyla~,Li~,ally.
The term '~ I ;r ~lly acceptable carrierl~ refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is adlll;.l;Jt~lcd.
Suitable l~ ,.lly acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as 15 i ..., .l .;. ,~l ;....c thereof pl ~ y acceptable carriers may further comprise rninor amounts of auxiliary substances such as wetting or emulsifying agents, ,UlCi~ Livca or buffers, which enhance the shelf life or ~ ,Li~ of the antibody.
The a~mrr'citir~nc of this invention may be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid 20 solutions, dispersions or ~..~llr.l~l.lllc, liposomes, i~u,u,uuaiLvl;c~, injectable and infusible solutions. The preferred form depends on the intended mode of A.l,,.;";~l,,.l;nn and therapeutic ~pFlir ~tir)n The preferred ~nmrr~citir~nc are in the form of injectable or infusible solutions. The preferred ~ cl l~ o~ lc of this invention are similar to those used for passive; ., .. ; ~ ~ 1 ;.. of humans with other antibodies. The preferred mode of 25 ~ a,.1;-." isparenteral(e.g.,hlLI~ ,IIvùa~ , v ' ;,,1.~
It will be apparent to those of skill in the art that the 1~ 1;, lly effective amount of antibody of this invention will depend, inter alia, upon the ~ 1, 1 ;. ." scheduie, the unit dose of antibody adll.;.l; ~l~d, whether the antibody is a.hll;ll;.t~lcd in r. ", .1.;. . -~ ;. ., . with other therapeutic agents, the immume status and health of the patient, and the therapeutic 30 activity of the particular antibody alllll;ll;alclcd. In " ,. ..~ .. "I'Y for treatment or prophylaxis of HIV infection, ARC or AIDS, 1~ ;. -lly effective amounts per unit dose of an antibody which is an intact antibody typically range from about 0.5 to about 5 mg/kg patient weight, preferably about 2 to about 3 mg/kg patient weight. Unit doses should beaJlll;ll;at~lcJ from once each 3 days to once each 28 days until an antiviral effect is observed, 35 preferably once each week for an indefinite period oftreatment. The antiviral effect may be measured by a variety of methods, including assessment of viral load, lylll,uhoc~ .~ counts and clinical signs and symptoms. It will be apparent to the skilled artisan, however, that lower or higher dosages and other i~.l"~ l;nn schedules may be employed. Treatment regimens =21 95238 wo 96102647 ~ 5.'~9114 for antibodies that are not intact sntibodies (e.g., Fab fragments) may differ, depending on their size a nd ~ ;l Al properties.
It should be a~ Led that the antibody mimetic agents of the invention can also be ;II~,VI~ into l~h~ c.,l;. Al r~ in accordance with the foregoing ~1Pcrrirtinn 5 Treatment regimens for antibody mirnetic agents may differ, depending on their size and l,hA....A~,,I;. Al properties. Additionally, an antibody mimetic agent ofthe invention can be used in combination with an antibody of the invention.
The antibodies and antibody mimetic agents of this invention are also useful in diagnostic . olll~o~;L;~ and methods, such as for the diagnosis of diseases involving changes 10 in helper T cell number. For example, they may be used to monitor the course of, or the efficacy of treatment for, HIV infection. Accordingly, this invention ~....,., ,I,A~. ~
nmrncitinnc, kits and methods .1, A. 1~,;, . d by a ~lisgnnctirAlly effective amount of an antibody or antibody mimetic agent according to this invention. A ~ ly effective amount" of antibody, or mimetic agent thereof, refers to an amount sufficient for monitoring 15 the course of HIV infection or the efficacy of treatment for HIV infection. For example, an amount of antibody or mimetic agent sufficient to allow qnsntitAtinn of CD4+ cell number in a subject or sample obtained from a subject or to determine the quantity of CD4+ cells expressinga~ ..r~"...-~i--..AllyalteredformofCD40ntheirsurfaceinasubjectorsampleobtained from a subject.
IV. U.cPc of Antihn~iPc ofthP Inventinn Theamtibodiesoftheinventionidentifyanovel~.. r.. An.~ lformofcell-surface CD4 that can be targeted for diagnostic, screening or therapeutic purposes. Accordingly, in various emho~limPntc the antibodies of the invention are useful in methods for detecting cells 25 expressing on their surface a ,r, -~ y altered form of a human CD4 molecule, for inhibiting infection of a cell by HIV (e.g., by interfering with viral entry into the cell) and for identifying agents that inhibit or induce the formation of this ~A.nnfnnnAtinnslly altered form of CD4 on the cell surface (which agents themselves may be useful in treating HIV
infection).
In one emho~limPnr the invention provides a method for detecting a cell expressing on its surface a ~- -. . r .. ~ Ally altered form of a human CD4 molecule induced upon binding of HIV, or an envelope protein thereof (e.g., gpl20) to the cell. This method involves contacting the cell with an antibody of the invention and detecting the antibody bound to the cell surf. ce to thereby detect a nnnfnrmAtinnslly altered form of a human CD4 35 molecule expressed on the cell surface. Preferred antibodies for use in the method are the rnnnnrlnnAI Fabs 3-47 and 3-51, or an antibody having the same epitope binding specificity as 3-47 or 3-51. The antibody bound to the cell surface may be detected directly, e.g., the antibody can be directly labelled with a detectable substance (e.g., a derivatized antibody of the invention, as described above, labelled with a fluorescent label or a l~liuis~lu~e), or W096/02647 2195238 22 r~ o,~

aiternatively, the antibody bound to the cell surface can be detected indirectly, e.g. using a labelled secondary antibody that recognizes the antibody of the invention. Since the formation of this ~ . ", r~ " " ~ lly altered form of CD4 on the cell surface is thought to be an hlLtl 1 step in the m--nh~nicm of infection of CD4+ cells by HIV, this detection method 5 can be used to monitor the progress of HIV infection, e.g., during a therapeutic regimen.
In another embodiment, the invention provides methods for identifying an agent that inhibits or induces formation of a ~.. ", r, .., ., ,~ ;. ,, .Ally altered form of a human CD4 molecule induced on a cell surface. To identify an agent that inhibits formation of the ..... ,r.. ~ y altered CD4, CD4+ cells are contacted with a gpl20 UUIIIIlO~;LiUll and an 10 agent to be tested, and then further contacted with an antibody ofthe invention (e.g., the m~-n~ nsll Fabs 3-47 or 3-51, or an antibody having the same epitope binding specificity as 3-47 or 3-51). Sub~e~lu~lLly, the amount of antibody bound to the cells is determined (e.g., directly or indirectly, as described above). A reduced amoumt of binding of the antibody to the gp 120-treated cells in the presence of the agent (as compared to the amount of antibody 15 bound to gp 120-treated cells in the absence of the agent) is used as an indicator that the agent inhibits formation of a c. ." r~" " ,~ lly altered form of CD4 On the cell surface. To identify an agent that induces formation of the conformationally altered form of CD4 (i.e., an agent other than gpl20), CD4+ cells are contacted with an agent to be tested and then further contacted with an antibody of the invention. Sub~e~lu. .~lly~ the amount of antibody bound to 20 the cells is determined (e.g., directly or indirectly, as described above). An increased amount of binding of the antibody to the CD4+ cells in the presence of the agent (as compared to the amount of antibody bound to CD4+ cells in the absence of the agent) is used as an indicator thattheagentinducesformationofa.,.,,.rn"..,.~ ".llyalteredformofCD40nthecell surface.
CD4+ cells for use in these screening assays can be, for example, CD4+ lylll~Jhu.,.r l~s (e.g., from peripheral blood) or tissue cultured cells transfected with DNA encoding CD4, as described above. A gpl20 f ~ 11;.,., with which the celi is contacted to induce formation ofthe ~u,.r,.""~I;n"~ly altered form of CD4 onthe cell surface ispreferably ~ ,h;~
soluble gpl20, although HIV itself or cells infected with HIV may also be used to induce the conformational alteration of CD4 on the surface of the CD4+ cell. The amount of antibody binding to the surface of the CD4+ cells can be determined by FACS analysis or other suitable assays icnown in the art.
In yet another ~,,,ho~ . ,I the invention provides a method for inhibiting infection of a CD4+ cell by HIV (or a related CD4-tropic virus such as SIV), involving contacting the cell with an antibody (or antibody mimetic agent) of the invention. Preferred antibodies for use in the method are the ""."-).l. ."~1 Fab 3-47 and 3-51, or antibodies having the same epitope binding specificity as 3-47 or 3-51. In a preferred rll~hO~l;lll. .~1, the antibody is a~L~ui .Ltlcd to a subject to inhibit infection of cells by HIV in vivo. While not intending to be limited by mf-~h~ni~m it is thought that these antibodies may inhibit a requisite .: . . . ... ... . . ...

~ WO 96/02C47 2 1 9 5 2 3 8 1 ~.I/L ~ 4 -23 - ~ =

illt~,.lll 1' ' step in viral infection subsequent to the induction of a ~ l l r~ change in cell-surface CD4 that is necessary for viral membrane fusion and viral entry into the cell (i.e., antibodybindingtothe.,l..,r~,,,,.,.l;.".~llyalteredforrnofCD4mayinhibitsubsequent ~ processes necessa~ for viral infection). Additionally, since the antibodies of the invention 5 do not bind native CD4 on the surface of CD4+ cells but only bind CD4+ cells upon gpl20 binding, these antibodies are likely to be less; l l l l ~ c~ e than anti-CD4 antibodies that bind native cell-surface CD4.
A cell is contacted with a ll.. l,.. l;. ~lly effective amount of antibody that is sufficient to sl-hsf~nti~lly inhibit infection of cells by HIV. Preferably, "substantial inhibition 10 of infection" of a cell indicates at least an 80 % reduction in the infection of CD4+ cells by HIV in vitro. To deterrnine whether a particular antibody of the invention substantially inhibits infection of CD4+ cells by HIV, any indication of HIV infection and/or replication can be monitored. For example, inhibition of HIV infection can be deterrnined by comparing HIV p24 levels in the presence and absence of the antibody in HIV-infected CD4+ cell 15 cultures. HIV p24 levels may be ~Irt~rmin~rl for example, by ELISA, r~i;"~ y or the like. Other assays that can be used to determine the effect of the antibody on viral infectivity include reverse I l ~ d l~ activity assays, bone marrow cell colony forrnation assays and assays that measure in vitro viral infection of bone marrow ~a~,lu~ c~, each described in PCT patenF application PCT/US90/00358.
Additionally, the ability of the antibodies of the invention to block early events in HIV infection (e.g., viral entry into a CD4+ cell) can be measured in an assay which allows for only a singly round of virus replication. For example, CD4+ cells can be infected with a rcc.lmhin~nt HIV strain, either in the presence or absence of an antibody of the invention.
The ,~...,.l.;,"~ .I HIV strain used in the assay directs the expression of a detectable gene 25 product (e.g., .,1.1..., ..,~1...;~.ul acetyl transferase (CAT)) in infected cells and carries a deletion in the viral envelope gene. Such virions are capable of infecting target CD4+ cells, but do not direct the synthesis of infectious progeny virions as a result of the deletion in the envelope gene in the viral genome. The number of infected cells can be assessed by measuring the amount of detectable gene product (e.g., CAT) syll~ ;~l in cells exposed to 30 the virus. Thus, a reduced amount of detectable gene product (e.g., reduced CAT activity) in cells exposed the virus in the presence of an antibody of the invention, relative to that measured in the absence of the antibody, can be used as an indicator that the antibody blocks (e.g., inhibits) early events in HIV infection of a CD4+ cell.
Otber rh~ associated with HIV infection can also be assayed to determine the ~ 35 effect of an antibody of tbe invention on such l~l ,.. ,~ For exarnple, syncytia formation in the presence of the antibody can be exarnined. To deterrnine whether a particular antibody si~ll;rl.,all~ly influences (e.g., blocks) HIV-induced syncytia formation between CD4+ cells, any known syncytia assay may be used. Preferably, an HIV laboratory isolate or HIV-infected CD4+ tissue culture cells (e.g., H9) are added to cultures of C8 i 66 or W0 96/02647 2 1 9 5 2 3 8 - 24 - r~

CEMXI 74 cells, and varying amounts of the antibody are added to all but the control cultures. Alternatively, the antibody may be assessed for its ability to inhibit syncytia among tissue culture cells expressing the HIV env gene product (gpl60). Control cultures (negative controls) are ~ rd with nothing, or with an irrelevant antibody of the same isotype 5 as the antibody homolog. Afler incubation, all the cultures are scored by visual inspection for syncytia. In this way, the ability of the antibody to block syncytia formation is evaluated.
Additionally, agents that inhibit or induce expression of a r~mf~rmationally altered form of CD4 on the surface of a CD4+ cell (e.g., identified in screening assays as described above), may have therapeutic utility in treating HIV infection. Accordingly, such agents can 10 be formulated into P~ mp-~citi~nc and dd~ L~ d to a subject, e.g., to inhibit infection of cells in the subject by HIV

V. Mr~lPAIIIPC F~rPccir~ Altrred CD4 Fpi1~rPc ar ~i UsPc Thprefor Another aspect of the invention pértains to isolated molecules that express at least one 15 epitope exposed on a t~f~nf~-rmAti~-nally altered form of human CD4 induced on the surface of CD4+ cells upon contact with gpl20. Preferably, the altered r~mf~rmati~nal form is one that expresses the 3-47 epitope and or the 3-51 epitope (i.e., the epitopes bound by the monru~lonAl Fabs 3-47 and 3-51, ~ ,ly). Even more preferably, the isolated molecule itself expresses the 3-47 epitope and/or the 3-51 epitope. Alternatively, or additionally, the 20 molecule may express other epitopes that are exposed on the , r." ", - ;", lAlly altered form of CD4 induced upon gpl20 binding.
As used herein, the term "isolated" refers to a molecule substantially free of cellular material or culture medium when purified from a natural source or produced in a host cell by " ,1,;"- ,1 DNA techniques~ or 511hctanti~11y free of chemical precursors or other chemicals when chemically ~ylllh~;~d~ In one PmhoriimPnt the isolated molecule is a protein or peptide. For example, the molecule can be a modified human CD4 protein, or peptide fragment thereof, or a non-human CD4 protein, or peptide fragment thereof, such as a non-human primate CD4 protein or peptide (e.g., from a rhesus monkey om.l~ t).
Alternatively, the molecule can be an anti-idiotype antibody, or fragment thereof, that binds monoclonal Fab 3-47 or 3-51 (i.e., the molecule can be an antibody having specificity for the antigenic binding site of the 3-47 or 3-51 Fab). Alternatively, the molecule can be a peptide mimetic. The peptide mimetic may be a semi-peptidic compound or a non-peptidic, organic compound that mimics the r~nformAti~n of an epitope exposed on the conformationally alteredformofCD4upongpl20binding,e.g.,the3-47epitopeorthe3-51 epitope.
A molecule of the invention expressing an epitope exposed on a conft-rmati-.naliy altered form of CD4 can be identified, for example, by screening a library of ~ c with an antibody of the invention and selecting a compound(s) that binds the antibody. The library may be composed of, for example, modified (e.g., mutated) human CD4 proteins, non-human CD4 proteins (e.g., non-human primate CD4 proteins), peptides, semi-peptidic wo 96102C47 - 25 ~ '1.114 ..." ,I ,.," . "l~ or non-peptidic, orgamic ...." .1.. ..l~ In a preferred ~...l~o. l; ... . l a random peptide display library is expressed on the surface of a display package, e.g., filamentous phage, and the peptide library is screened with am antibody of the invention. The library can - be screened with the ~ L II IAI Fab 3-47 to identify and isolate peptides that bind 3-47 (i.e., express the 3-47 epitope). Altematively, the library can be screened with the m~-nnrlnn~l Fab 3-51 to identify and isolate peptides that bind 3-51 (i.e., express the 3-51 epitope). Techniques for preparing random peptide display libraries, and screening thereof v~ith antibodies, are known in the art (see e.g., Parmley, S.F. and Smith, G.P. (1988) Gene 73:305-318; Cwirla, S.E. et al. (1990) Proc. NatL Acad. Sci. USA 87:6378-6382, Devlin, J.J.
et al. (1990) Science 249:404-406; Scott, J.K. and Smith, G.P. (1990) Science ~5!:386-390;
Houghten, R.A. et al. (1991) Nature 354:84-86; Houghten, R.A. et al. (1992) BioTechniques 13:412-421; Pinilla, C. et al. (1992) PioTechni~ues 13:901-905; and Oldenburg, K.R. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:5393-5397).
Antibody-binding peptides selected from the library can be sequenced by standardtechniques and the amino acid sequences of the selected peptides then compared to generate a consensus motif that represents a preferred, or optimal, amino actd sequence recognized by the antibody. The consensus motif of an epitope bound by an antibody of the invention can further be used to design other molecules expressing the epitope. For example, a human or non-human CD4 protein, or peptide fragment thereof, can be sy~ ti~,~lly mutated (e.g., by site-directed ~ l. c;~) to alter the protein or peptide such that it constitutively expresses an epitope bound by the antibody of the invention (i.e., the CD4 protein is altered to express the epitope in the absence of gpl 20 treatment).
The molecules of the invention expressing at least one epitope exposed on a r~ y altered form of cell-surface cD4 are useful for generating antibodies of the invention by ;- " ", ~ ~ l; ,.;, .g a mammal with the molecule. Accordingly, the invention provides a method for producing an antibody that bind5 an epitope expo5ed on a ~ rl ~ llyaltered form of a human CD4 molecule involving;.. : ,.;.. g a mammal with a molecule of the invention described herein. Polyclonal antibodies can be induced by standard techniques and mnnnrl-~ns~l antibodies can be prepared and selected as described previously.
For in vivo ~.1. . .; . .: ~1 ., 1 ;nn the molecules of the invention expressing at least one epitope exposed on the cnnformotinn:~lly altered form of CD4 can be hlCUIu~ ' ~ into p l .~ J~ Such ~ typically include at least the epitope-~ expressing molecule and a ~ lly acceptable carrier. The forml~lotinn and ~,l,.,:..:~l.~;nn of such cnnnrocitinnc is similarto that described above forantibody 35 c~mrocitinnC of the invention. Preferably, the molecule is included in the ~nmrnCilinn in an amoumt effective for inducing an immume response against the molecule (e.g., an antibody response against the molecule) in a subject. Preferably the l.;UlllUU~l;tiUII is adl.l;l.;,,t.l~id y via one or~ more preferably~ several Al l ~

Wo 96/02647 ? I iq 5 2 3 8 ~1/. C a ..IJ~

To induce an immune (e.g., antibody) response against the molecule, it may be desirable to also include a r,l - ,,,~c~l;..,.lly acceptable adjuvant in the ~,VIII~)O~;IiUll.
Adjuvant is used in its broadest sense and is intended to include any immune stimulating compound. A typical adjuvant used in the art is Freund's incomplete adjuvant. An5 illustrative example of a suitable adjuvant and canier ~ r.~; l i,), . is a muramyl dipeptide derivative and a carrier which includes a detergent and a .., ., ..1 ,i ., -~ ;. in of free fatty acids.
Other suitable adjuvants and carriers for use in the compositions include, for example, ion eY~honger.c~ alumina, aluminun stearate, lecithin, serum proteins (such as human serum albumin), buffer substances, such as i ' , ' , glycine, sorbic acid, potassium sorbate, 10 partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine, sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,, . ,~ ., . trisilicate, polyvinyl pyrrolidone, cellulose-based subst_nces and polyethylene glycol. Adjuvants for topical or gel base forms may be selected from sodium ~,albuAyl~l.,alylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene block polymers, 15 polyethylene glycol and wood wax alcohols.
The ability of the molecules of the invention to induce an antibody response in a subject directed against one or more epitopes exposed on a cl~nfnrmoti~nolly altered form of cell-surface CD4 may be exploited in therapeutic methods for inhibiting infection of cells by HIV in a subject. While not intending to be limited by mf-nhonicnn it is thought that these 20 molecules may have therapeutic utility by inducing an antibody response that has anti-viral activity in a subject, e.g., the induced antibodies may inhibit a requisite int ~ ~ 1r step necessary for viral entry into cells and thereby inhibit infection of the cell by HIV.
Accordingly, in another . " ~l~od; ., ....1 the invention provides a method for inhibiting infection of a cell by HIV in a subject involving ~- h .: .' .s .; . .g to the subject a ll .., ~ lly effective 25 amount of a molecule that expresses at least one epitope exposed on a conformationally altered form of human CD4, such that an antibody response against the epitope(s) expressed by the molecule is induced in the subject. Preferably, the subject is immunized with a molecule that expresses the 3-47 epitope andlor the 3-51 epitope.
The molecule, preferably in a 1 ,~ -- . ., =~. . .1;. .~1 Cr~n~r~cition as described above (e.g., 30 including an adjuvant), is ~l~llhl;a~ d at a dosage and by a route sufficient to induce an antibody response in the subject. The molecule is typically ddlll;ll;~t~ d i.. l,,.. ~ "I_~ly~ but may be adl";-,;..t~ d by another suitable route, e.g., ~~.~ .. v~ly, intrAA~rrnolly~
hlllav~,lwu~ly etc. The molecule may be a.h.lill;,t.lc d at one time or, more preferably, is adlll;.l;i,t~ d over a series of treatments. The most effective mode of o..h ~ and 35 dosage regimen will depend upon the particular composition and/or adjuvant used for treatment, the severity and course of infection, previous therapy, the patient's health status and response to treatment and the judgement of the treating physician. It will be c.l,~l . ' ' ' that a more highly ;. "" "",~ r form of compound may require a lower dosage or treatment time, e.g., when an adjuvant is used.

, 21~9 5238 W0 96/02647 r~ 'os~l4 . t: -~27 -In a non-limiting illustratiYe ~?mho~iinn~nt a daily dosc of about 0.1 to 1.0 mg of active compound per kilogram of body weight is ad~ l; .t~ d to the subject once a day for about 30 days. rhe subject may require i~ boosters (e.g., about 0.1 to 1.0 mg/lcg ~ body weight on a weekly to monthiy basis).
.
~ Oth, - F,,.l".,l;,", ";~
It should be c~ , ' that antibodies that bind . . .,, r, ., ~ y aitered forms ofnon-human CD4 molecules induced on the surface of non-human cells upon binding of other CD4-tropic vinuses to the cells, e.g., altered fomms of simian CD4 molecules induced upon binding of simian immlmndrfirirnry vinus (SIV), can also be produced using the teachings of the invention and are within the scope of the invention. For example, an SlV-infected rhesus monkey can be immnni7~ with soluble rhesus monkey or human CD4, and antibodies which bindarnnfnrm~tinnAllyalteredformofrhesusmonkeyCD4inducedonthesurfaceofcells upon SIV binding can be selected as described herein.
Additionally, while the cnnfnrm~ltinn ~lly altered form of CD4 described herein (e.g., the form of CD4 that expresses the 3-47 and 3-51 epitopes) is induced upon binding of HIV, or an envelope protein thereof, it is possible that the CD4 neo-epitope(s) expressed upon HIV
binding are naturally-occurring cryptic epitopes of the CD4 molecuie. Accordingly, it may be possible to induce this altered form of CD4 by other ", ~ " 1~ in addition to HlV
binding (e.g., through other proteins with which CD4 naturally interacts).

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent ;. .llc cited throughout this application are hereby h~ ' by reference.
T1'~x~MpI~T1~ Elicitation of rsCD4-specific Antibodies by T
of HIV-infected Humans with Human rsCD4 In this example, HlV-infected humans were immunized with recnmhinAnt soluble human CD4 (rsCD4) in an adjuvant and the polyclonal anti-CD4 antibody titer was measured to detenmine whether an anti-CD4 response was elicited by ;.. ~ .;, l ;

~ Hnm~n ~ .eolllhh- (~n4 immlmi7~tinn of h~m~n ;mmnnn~ f ri~nny vinlc (HIV)-;nfrrtrd in-livi.1n~1c Four asymptomatic HlV-infected humans with absolute CD4 counts greater than 500 were immnni7~-d and boosted; 1 .1 . ,.1, .. ,~ . ,1~. Iy f ve times with I mg human recombinant soluble CD4 (rsCD4) (Biogen, Inc., ('~mhridge, MA) in incomplete Freund's adjuvant.

WO 96/0264~ 2 1 q ~ ~ 3 8 ~ .114~

D~ ;. ,, . of rs(~n4-spP~if c s3ntihodv titPr.c jn the serum of rscD4-immlmi7p~l HIV-jnfeotP~l hnm~nc Human rsCD4 was coated overnight at 4 degrees C onto Nunc Maxisorp Immunoplates at a final rnnrPntrAti~n of 0.3 ~g/mL. The wells were washed three times with S PBS and blocked for two hours at room L~ UI C with 0.5 % nonfat dry milk/PBS. The wells were then washed three times with 0.5 % nonfat dry milW0.05 % Tween 20 PBS. One ml of patient plasma was heat inactivated at 56 degrees C for thirty minutes, and diluted I :60, 1: 180, 1 :540, and 1: 1620 with 0.5 % nonfat dry milk/0.05 % Tween 20/PBS. F;fly microljters of diluted plasma was then added to the appropriate wells'and incubated for two hours at room Lt~ Lulc. After washing the wells three times with 0.5 % nonfat dry milk/0.05 % Tween 20/PBS, 50 111 1 :50,000 diluted HRP-conjugated F(ab')2 goat anti-human IgG (H+L) was added. After one hour at room L~ lLuuc, the wells were washed three times with 0.5 % nonfat dry milk/0.05 % Tween 20/PBS. Color was developed by adding 100 111 TMB One Component Substrate Solution (KPL, G~iLhclabul, MD). This reaction was stopped by adding 100 IlM N H2SO4. Plates were read on a Dynatech plate reader at an OD=450 nm.

~'L
The anti-CD4 antibody titer of the four immunized individuals, measured by ELISA, is shown in Figure I . Antibodies in the serum of all four rsCD4-immuni~d individuals could be shown to bind to human rsCD4 by standard ELISA. However, these antibodies were present at a relatively low titer. The elicitation of these antibodies by;, . " ., l ~
with a self protein suggests that epitopes to wbich these individuals were not tolerant were presented to the immune system by the human rsCD4 molecule.
~ ~ _ ~,XAMPI 1~.2: P~.~, . ' of a Human C~ ;al T
Library from an rsCD 1 ~ ~ d, HlV-infected Individual In tbis example, a human: ' ' ' ' imml~nnglnb~lin library from the rsCD4-immnni7P~l HlV-infected subject HET, described in Example 1, was produced. This individual d ' ~ a higher anti-CD4 antibody titer post-immnni7~tion than did theotber subjects of this study (see Figure 1). The bone marrow cells used as a source of mRNA
for immllnn~lnbuljn gene ~."~ ", were obtained from this individual 200 days after the initial; " " " ~ , at the time of peak rsCD4-specific antibody titer. A comhin~tnrisll immlmn~loblllin library of 3 x I o6 members was expressed in the M13 bal,il,l;vlJlla~c vector pCOMB3. In this cloning system, Fabs are expressed on the surface of It~ vlllblllculL M13 tl d ~IIIh~ particles through the expression of the heavy chain as a fusion proteia with the M13 coat protein, gene III. Fab clones of a given antigen specificity can then be selected by ~ w096/02647 2 1 q 5 2 3 8 P~ C 114 panningthesel~c~""l,;.,- .l b~~tPril~hAg~overtheantigenofinterest. Thefollowing m~tho~ gy was used to produce the c~-m' ' immunoglobulin library:

HA--YP~ti~ An(~ St~--A,P~ of bonr mArroW 5Arr,rl~
Twenty ml of 1.. " ; ";, ~ d bone marrow cells were harvested from rsCD4 immlmi7.~
- HlV-infected individuals at the time of peak .t . . ,~ " ,.h IP rsCD4-specific antibody titer (200 days after the initial ;~ ) (see Figure l ). Lyl~ u~,~ t.,;. were isolated from these samples by Ficoll-diatrizoate density gradient ~. .,l. ir,.~,.l;..,. rapidly frozen on dry ice, amd stored at -70 degrees C prior to RNA extraction.
Amrlifi~Ation of immlmo.~l-blllin DNA
rnRNA was isolated from 2 x 107 bone marrow cells using the Quickprep Micro-mRNA Purification Kit (Pharmacia, Milwaukee, Wisconsin). T~.""""~,lnl,..l;., heavy chain cDNA was then ~ylllllc~i~d using primers specific for the third constant domain of human 15 heavy chains. Tmmlmf.glr~bulin light chain cDNA was similarly sy.~ i~d using primers .iullc~Julldillg to the constant domains of human kappa and lambda light chains. The nucleotide sequences of primers used for cDNA synthesis and PCR Amrlifif Ati~n of immllm-gll~lt Illin heavy and light chain DNA are shown below (restriction sites hl~ul~)ulalcd into primers for cloning purposes are underlined):
H~AVY (~h~in VAri~hlP l~ n prim~ rc VHA aggtgcagctg~ tctgg (SEQIDNO: l) VHB ag gtg cag ctg ctc eag tcg gg = (SEQ ID ~TO: 2) VHC ag gtg caa ttg ctc gag tct gg (SEQ ID NO: 3) VHD ag gtg caa ctg ~a3~ tcg gg (SEQ ID NO: 4) VHE ag gtg cag cta ctc gag tcg gg = (SEQ ID NO: 5) VHF ag gta cag ctg ç~ tca gg (SEQ ID NO: 6) (Xho 1) 25 I~A~pa L~ht ChAin VAriAhl~o R.o.~ n Primrr~

VKI gt gcc aga tgt ~a~ gtg atg acc cag tct cca (SEQ ID NO: 7) VK2 gt gcc aga tgt ga~ gtg ttg acg cag tct cca (SEQ ID NO: 8) (Sac I) W0 96/02647 ~ 9 5 2 ~

I ~mhr::l T ~ht Ch~in Vari:~hlp RP~;Or Pr;mPrC

VLI c tgc aca ggg tcc tgg gcc ~ gtg ttg acg ca (SEQ ID NO: 9) VL2 c tgc aca ggg tcc tgg gcc ga~ ata ctg acg ca (SEQ ID NO: 10) (Sac I) T~P~VY (~hAin Conct~nt RP~inn primPrc CHI agc atc ~L~ aca aga ttt ggg ctc (SEQ ID NO: I l) (Spe I) CH3 c tca gta tgg tgg ttg tgc (SEQ ID NO: 12) K~pa T.i,~ht Ch~in Conchlnt RP~inr~ PrimPr CLK t cct ~Lag_ tta cta aca ctc tcc cct gtt gaa gct ctt tgt gac ggg cga act c (SEQ ID N0: 13) (Xba I) T.slmhrl:~ T.irht Ch~lin CnnctS~nt RP~inn Primrr:

CLL g cat tct aga cta tta tga aca ttc tgt agg ggc = (SEQ ID NO: 14) (Xoa I) .
For cDNA synthesis, 400 ng of the appropriate constant region primer was added to 30 ~LI isolated mRNA (CUII~a~UUlldillg to one third of the total mRNA isolated from 2 x 107 bone marrow Iylll~/Lol"y h,a)7 heated tû 65 degrees C for five minutes, and cooled slowly to room Lc~llu~ Lul~: in a water bath. Reverse trAncrrirtion was initiated by adding reverse buffer (Gibco BRL, 6'J~ithPrcb~r~ MD), 80 U rRNAsin (Promega, Madison, WI), 0.8 mM dNTPs (Promega, Madison, WI), 200 U M-MuLV reverse ~ r (Gibco BRL, G~ithPrcb..rg MD) and 16.7 mM DTT (Gibco BRL, Gaithersburg, MD). After allowing this reaction to proceed for two hours at 37 degrees C, the reverse ~ was inactivated by heating the reaction to 65 degrees C for twenty minutes. The resulting cDNA
was stored at -20 degrees C prior to PCR ~mrlifir:lti~ln Two rounds of PCR ~mrlifir~tinn were employed to obtain sufficient quantities ofimm~.no~lohlllin heavy chain material for cloning. The first round PCR reaction contained 20 111 heavy chain specific cDNA, 2.5 U Pfu polymerase (Stratagene, La Jolla, CA) Pfu buffer II, 0.2 mM dNTPs (Promega, Madison, Wisconsin), 10 ng heavy chain CH3 constant ~-' c~1 95238 WOsClo2C47 ~ 114 regionprimer(SEQlDNO: 12;0pcron,Alameda.CA), 10ngofoneofsixvariableregion primers .,u~ uull.lhlg to six heavy chain families (SEQ ID NOs: 1-6; Operon~ Alameda, CA), and RNase-free water (United States Bio~hPmi~ Cleveland, Ohio) to make a final volume of 100 ,ul. Twenty-five rnnrlifirAtil~n cycles, utilizing the hot start technique, 5 followed by a 72 degrees C 10 minute final extension, were performed, using the following conditions: 94 degrees C for 1.5 minutes, 52 degrees C for 2.5 minutes, 72 degrees C for 3 minutes.
One microliter of the first round PCR product was then exposed to twenty-five identical cycles of ~rnrlifi~ ~ti~n under the same conditions except ~llh~l;llll;.,g the heavy 10 chain CH I constant region primer (SEQ ID NO: I l ) for the CH3 constant region primer (SEQ ID NO: 12).
I-",."",.-~,ln~"-l;,. Iight chain DNA was amplified by 35 cycles identical to those described above, except using primers CUll~ .I.ulldillg to the first constant domain of kappa or lambda light chains (SEQ ID NO: 13 or 14), as well as one of four variable region primers 15 specific for different kappa and lambda light chain families (SEQ ID NOs: 7-10).

Clonin~ of i~ h-bUl;l l hP~Vy :~n~ ht ch~lin DNAs intf~ thP Ml 3 rhs~Pmi~l vP~t~r pCOMB3 PCR amplified heavy and light chain DNAs were purified from a 1.5 % agarose gel 20 using the Sephaglas BandPrep kit (Pharmacia, Piscataway, NJ). Equal amounts of material from each light chain family were then pooled and digested with the enzymes Xba I and Sac I (Boehringer M~nnhrim In~lirn~rnli~ IN). The digested material was gel purified as above and ligated with Xba I/Sac I digested pCOMB3 vector (TSI, La Jolla, CA) (illustrated in Figure 2) overnight at 16 degrees C at a 2: 1 hl.~,l L. vc~,Lul molar ratio in a 150 ,ul reaction 25 containing T4 DNA ligase buffer and 10 U T4 DNA ligase (Gibco BRL, GGiLLcl~l/uly, MD).
Theligationproductswerell,.,.~r~.,.l..dintocle~,LIu.,ulll~,t~,.lLXLl-bluecells (Stratagene, La Jolla, CA) by clc~,LIulJol~Liull. Specifically, phenol/chloroform extracted, ethanol ulc~,iu;LaLcd ligation products were added to 300 111 cl.,~,LIu~,ul~ t~,llL XLI-blue cells in prechilled 0.2 cm gene pulser cuvettes (Bio-Rad, Melville, NY). These cells were pulsed in a Bio-Rad gene pulser apparatus set at 25 uF, 2.5 kV, and 200 ohms. Three milliliters SOC buffer was then added, and the cells were grown at 37 degrees C for one hour in a shaking incubator. Tl ,., .~ r ~, ~ d cells were selected for one hour at 37 degrees C in a 10 ml volume of superbroth, 20 ~Lg/ml carbenicillin, 10 llg/ml l~Ll~,.y.,ihle. Finally, l, ,,. I~r(,. " ,. .1 cells were amplified overnight in a 37 degree C shaker in 100 ml superbroth, 50 ,ug/ml carbenicillin, 10 llglml tetracycline.
R~c..,..l,;.,""l plasmid DNA was isolated from this overnight culture using the Wizard Magic Miniprep Kit (Promega, Madison, WI). 3~ 11 DNA was then digested with the restriction enzymes Xho I and Spe I (Boehringer Mannheim, lndiallcl.oli~, IN), gel W0 96/o2647 ' 2 l 9 5 2 3 8 r~ s ~ 4~

purified, and ligated overnight at 16 degrees C with pooled Xho l/Spe I digested, gel purified light chain PCR products using a 1.6:1 h~ ..L~ll ratio.
The cr/ml lrirl ligation products were then cl~.i. uuu....d into XL l-blue cells, as described abûve. A restrictiûn analysis of the resulting clones was performed to insure that 5 this library consisted of clones with bûth light and heavy chain inserts.

~onvercinn ûfthe romhins~tori6~1ihr~ry to ~n F~h-PyrrPccin,~ M13 1.~. t~ lh~r formrt The initiaH,o. ' ' library was amplified at 37 degrees C in a shaking incubator for one hour in a 100 ml vûlume ûf superbroth, 50 ~Lg/ml carbenicillin, 10 ,ug/ml Lci.a."yulil.e.
TheM13helperphageVCSM13(Stratagene,LaJûlla,CA)(10l2pfu)wasthenaddedto direct the assembly of M13 ~ . .,.,1,;,~ ~t~ H~ ;u~)llage After two hours growth at 37 degrees C, 70 ,ug/ml kanamycin was added, and helper phage infected cells were grown overnight at 37 degrees C.

15 Prpriritatir~n of Fah-Pl~,nreccin,~ M13 1,~- Ir d ~,ruh~e Recomhin:~nt M13 I;a~t~l;uluL~6~ were 1~ . ' on ice for one hour in the presenceof four percent polyethylene glycol-8000 (Sigma, St. Louis, MO) and 3 % sodiurn chloride (Sigma, St. Louis, MO). P~cc;~u;ldLcd phage were pelleted at 9000 rpm for twenty minutes at 4 degrees C. Finally, the phage pellet was lc~uau.lldcd in 2 ml PBS and stored at -20 20 degrees C.

TitPrir~ M13 ~,u~l.h,,.l,.d~l h~~tPrinphr~e Various dilutions of l;a~t~l;u~ulld~c were incubated for fifteen minutes at roomt~lll,U.l~liUlC with 50 111 of an XL I -blue culture grown to an OD6001ml of 1Ø The infected 25 cells were then plated onto LB, 50 llg/ml carbenicillin agar plates.

li X~ IPT ,li 3: Screening of a Human 12~ ~ b,l 1 Library from an rsCD1 1, HIV-infected Individual To .1,~ r,;~.~ theantibodieselicitedbyrsCD4 ;,",." ,;, I;.,.. ofanHIV-infected individual, the human c, I I ;.".,....,o~lob.~l;.. library, produced as described in Example 2, was screened for CD4-specific antibodies. Two panning strategies wereemployed to select CD4-specific Fabs from this immuni7ed individual. In the first strategy, the library was panned against human rsCD4. In the second strategy, the library was panned against preformed CD41gpl20 complexes captured onto ELISA wells with the anti-CD4 monorlonrl antibody 5D4. Clûnes ûf interest selected by each of the panning procedures were then converted to sûluble Fab expressing clones to facilitate the eh ~-~rtPri7~tion of their binding specificity by ELISA. Clones that bound to CD4 but not gpl20 by ELISA were then selected for further ..1. ~ Lrl ;~,.lion The following methodology was used:

2! q5238 Wo 96t02647 . ~ '. 1 .1 14 A. PZInnins s~inct hllm:m rqCD4 Four wells of a Maxisorp 96 well plate (Nunc, Newbury Park, CA) were coated overnight at 4 degrees C with 2 llg of the CD4-specific antibody SD4 (Hasunuma, T. et al., (1992) J. Immunol. 148:1841-1846) in a 100 1l1 volume PBS. Unbound antibody was removed by washing three times with 350 ~LI TBS. The wells were then blocked with 350 ~11 2 % nonfat dry milk/TBS for thirty minutes at room t~ J.,IaL~ . After shaking out the block solution, 1.25 llg human rsCD4 was added to each well in a 100 ~I volume, and allowed to bind for two hours at room t~ .,laLul~. Unbound rsCD4 was removed by performing three 350 111 washes with sterile double-distilled water. The wells were again blocked at 37 degrees C for one hour with 3501113% BSAtPBS. After shaking out the block solution, 100 ~11 Ml 3 combinatorial phage ( I ol 2 pfu) were added to each well and incubated for two hours at 37 degrees C. Nol~.llLI~ phage were removed and each well was washed one time with sterile double-distilled water. Each well was then washed ten times over a period of one hour at room Ltlll~laL~c~ with 350111 TBSI0.5 % Tween-20. Detergent was removed by washing one time with sterile double-distilled water, and phage were eluted by adding 100 111 phage elution buffer (0.1 M HCI, I mg/ml BSA-pH 2.2 with glycine). The elution was allowed to proceed for ten minutes at room LtllllJ.,IaLul~. The solution was then pipetted up and down several times, transferred to a sterile tube, and neutralized with 6 111 2 M Tris base per 100 111 eluted phage. The panned phage were titered and stored at -20 degrees C in preparation for ~mrlifi~film and further rounds of panning. The panning procedure was repeated until enrichment for antigen-specific clones was achieved, as determined by the percent yield phage (the number of phage eluted divided by the number of phage applied multiplied by 100) after each amplification/panning cycle.

B. P~nnir~ ~inqt hnm~n r~CD4/1~nl 20 r~mri~Yrq This procedure was identical to that for rsCD4 panning described above except that SD4 coated wells, after blocking, were incubated with preformed rsCD41gpl20 complexes.
The complexes were formed by incubating gpl 20 and human rsCD4 in a 1.4:1 molar ratio at 37 degrees C for 1.5 hours.

.Arr~,nlifi~tinnofr nnedb ~~trrin~nh~e Elutedphagewereincubatedwith2mlofanOD6oo/ml=l.O~DLl-blueculturefor fifteen minutes at room t~ ,laLLIl~. The cells were then grown in a 37 degree C shaking incubator for one hour in ten ml superbroth, 20 llg/ml carbenicillin, 10 ,ug/ml tetracycline.
These cells were then grown for an additional hour in a 100 ml volume of superbroth, WO 96/02647 2 1 9 5 2 3 8 s ~ 114~

50 llg/ml carbenicillin, 10 llg/ml t~LIa~y~li..e. At this time, the cells were infected with 1012 pfuoftheM13helperphageVCSM13todirecttheassemblyofFabcAu~ lgM13 I,a~,t.fiuullage. Finally, cells which had been infected with the helper phage were selected by growing the culture overnight in the presence of 70 llg/ml kanamycin.
S = , .
(lonvercir~n ûf rlonrc to a sol-~hlr Fah ~ ~n,reccinn form ~
In order to obtain soluble Fab, it was necessary to remove gene III, the proteinresponsible for anchoring the Fab molecules on the phage surface, from the ~" L~plasmids. Plasmid DNA was isolated from 3 mL overnight cultures of individual parned clones using the Wizard Magic Miniprep Kit (Promega, Madison, Wl). This DNA was digested with Spe I and Nhe I to remove gene III (see Figure 2). The remaining 4.7 Kb vector fragment was then gel purified. Because the Spe I and Nhe I restriction sites are romp~tihlr, ~ h~ ulo.~ d plasmid was made by ligating this vector overnight at 16 degrees C. The ligation product was ",."~r~" Illrd into competent XLI-blue cells in preparation for the induction of Fab expression.

Indnrtion ûf sFAh e ~reccion Cllllll/;l.rll."~1 clones were inoculated into 500 ml superbroth, 50 ~Lg/ml carbenicillin, 20 mM MgC12 and grown at 37 degrees C in a shaking incubator to an OD600/ml 1ØSoluble Fab expression was then induced by growing these cultures at 30 degrees C overnight in the presence of I mM IPTG (Stratagene, La Jolla, CA) and 4 nM dibutyryl cAMP~ (Sigma, St. Louis, MO).

Tcols~tion of sF~h fr~.m in~lnr~ h~rl~ori~l rllltllres Since the pelB leader sequence of the pComb3 vector directs Fab molecules to theperiplasm of induced bacterial cells, an osmotic shock procedure was performed to obtain a p~,l;ula~ , extract from these cells. Specifically, induced bacterial cells were pelleted at 4000 rpm for thirty minutes at 4 degrees C. After discarding the ~ the induced bacterial cells were 1~ d on ice in 40 ml osmotic shock solution A (100 mM Tris-HCI, pH .6, 500 mM sucrose, 0.5 mM EDTA). The cell wall was Iysed by adding 2 ml4 mg/ml Iysûzyme, ;------- I 'y follûwed by 160 ml osmotic shock solution B (50 mM Tris-HCI, pH 8.6, 250 mM sucrose, 0.25 mM EDTA, 2.5 mM MgC12). After a ten minute incubation on ice, bacterial debris was pelleted by rrntrifilgin,o the Iysate in 35 ml Oakridge tubes at 12,500 rpm for five minutes at 4 degrees C. After "r,.~r~, . ;"g the ~ to a new Oakridge tube, the protease inhibitor AEBSF ((~lhionhrm San Diego7 CA) was added to a final ~ . of I mM. Remaining bacterial debris was removed by r~ntrifiloing the extract again at 12,500 rpm for fifteen m;nutes at 4 degrees C. Finally. the 7u,u.lllalallL
was filtered through a 0.2 lam acrodisc (Gelman Sciences, Ann Arbor, Michigan).

21 952~38 W096/02647 ~ ,.14 Purifi~tion of s~hlhle Ff~h In order to obtain a pure source of Fab fo m l,," ... ~ ion, the ~ )la ~lll;C extracts of induced cells were applied to goat anti-human F(ab')2 affinity columns. The affinity columms were prepared by incubating 4 mg goat anti-human F(ab')2 (Jackson Immunoresearch, West S Grove, PA) with 2 ml ~'r~mm lhinrl G Sepharose beads (Pharmacia, Piscataway, N.J.) for one hour at room tl,lll~ ltUI~ on a rocking platfomm. After washing the conjugated beads four times with ten ml 0.2 M sodium borate, pH 9.0, the beads were incubated on a rocker for 1.5 hours with the coupling reagent DMP at a r/~n~Pntr~tion of 20 mM in 0.2 M sodium borate, pH 9Ø The coupling reaction was terminated by washing one time with 0.2 M
eth~nnl~rninP, pH 8.0, and incubating the beads in eth:~n~iRrnine for two hours at room t~ ltUlCi on a rocker. The beads were washed three times in 0.2 M sodiurn borate, pH 9.0, . ."1Pd in 10 ml PBS/0.05 % sodium azide, and stored at 4 degrees C.
The coupled beads were loaded onto Econo-Pac columns (Bio-Rad, Hercules, CA), and sodium azide was removed by washing the columul several times with PBS. The columm was then P~ ilihr:~tPd with 10 column volumes elution buffer (3.5 M sodium thiocyanate) to remove uncoupled IgG. After washing the columns several tirnes with PBS, rPriplslenni~
extract isolated from a 500 ml induced culture was applied to the column at 4 degrees C.
N.~nere~ifi( llly bound proteins were washed off with 200 ml PBS/I mM AEBSF
(Calbiochem, San Diego, CA). Finally, bound Fab was eluted with eight column volurnes 3.5 M NaSCN, and the samples were desalted using Centriprep 30 nlfr~filtr~ti~n devices (Arnicon, Beverly, MA).

ScrPPnir~ s~hlhle F~hs for ~nti~en 5pPeifitity Wells of a 96 well Maxisorp plate were coated overnight at 4 degrees C with I llg of the antigen of interest in a 150 ,ul volume. After washing eight times with TBS, the wells were blocked for one hour at 37 degrees C with 0.5 % nonfat dry milW0.05 % Tween-20/TBS. Blocking solution was then removed, and the Fabs were incubated in the ~.I",lul wells for one hour at 37 degrees C. Unbound Fab was removed by washing eight times with TBS. Bound Fab was then detected by incubating the wells for one hour at 37 degreee C with a 1:50,000 dilution of horse radish peroxidase-conjugated F(ab')2 goat anti-human F(ab')2 (Jackson lullllullu~ ,ll, West Grove, PA). After eight TBS washes, TMB
one component substrate (KPL, ('.";~ , MD) was added. The reactions were terminated after twenty minutes by the addition of 2/3 N H2SO4, amd the absorbance was read on a Dynatech MR5000 ELISA reader at an ODr-450 nm (Dynatech Lab., Inc., Chantilly, VA).

D~ ~ 1,,;~.,.1;.~" ûfsFAh ef~nrPntr~tion - ~,~ntif~tiye FT T~
Wells were coated overnight at 4 degrees C with I llg F(ab')2 goat anti-human F(ab')2 (Jackson l~ uuu-~ u~ , West Grove, PA). ARer eight TBS washes, ~2e wells were WO96t02647 i~ 2~ ~ , "- s 114~

blocked as described previously. Various dilutions of each Fab preparation were then loaded.
as well as known cnnrrntratinnc of a purified human Fab standard (Biodesign Kennebunk, ME). The detection of bound Fab was performed as described above.

S E~
After two rounds of rsCD4 panning, the library was enriched three-fold for rsCD4-specific clones, as determined by the percent yield of phage (the number of phage eluted divided by the number of phage applied, multiplied by 100). Fifty five clones from this enriched library were converted to soluble Fab expressing clones to facilitate the 10 ~llala~,ttl;~LiOn of their specificity by ELISA. Nine of these clones flrm~ ' strong reactivity with human rsCD4, but not with l~c.""l,;,.~ .l gpl20, by ELISA. The ELISA
results for a IclJlca~llLalive clone, 2-6, are shown below in Table 1.
Second, to gain access to antibodies which might be specific for epitopes of CD4 that are exposed only upon HIV binding to the CD4 receptor, the library was panned agamst 15 preformed CD4/gpl20 complexes captured onto ELISA wells with the anti-CD4 l,.,.,~
antibody 5D4. After three rounds of panning, the library was enriched two-fold for CD41 gpl20-specific clones. Thirty-five clones from this enriched library were converted to soluble Fab expressing clones. Nine of these clones were shown to react with human rsCD4, but not with rgpl20, by ELISA. The ELISA results for two Ic~..c:,c..La~ive clones, 3-47 and 20 3-51, are shown below in Table I .

Table I

Clone 2-6 Clon~ 3~7 Clone 3-51 humanrsCD4 2.411 2.368 2.108 rgpl20 0.284 0.253 O.lg4 To determine whether all of the rsCD4-specific Fab clones identif ed were unique, conventional DNA sequencing was performed in the heavy and light chain variable regions of these clones. Fourteen of the eighteen rsCD4-specific Fabs identified were shown to have unique roml~in:ltinn~ of heavy and light chains, including clones 3-47 and 3-51. The partial nucleotide sequences of the nucleic acid molecules encoding the light and heavy chain variable regions of clone 3-47 are shown in SEQ ID NO: 15 and 16, lc~Jc~ ly . The partial nucleotide sequences of the nucleic acid molecules encoding the light and heavy chain variable regions of clone 3-51 are shown in SEQ ID NO: 17 and 18~ lc~ 1y. The full-length sequences of these molecules can be determined by standard DNA sequencingtechniques (e.g., dideoxy sequencing) using olignm.rlroti~lr primers designed based upon the sequences disclosed herein.

- 2~1 952~38 Wos6/02647 r~ C'1.114 ~X~MPl.F,4: Cl~. .t~ of rsCD4-specific Soluble Fab Clones In this example, the binding ~ .,.. I r~ of the fourteen CD4-specific antibodiesS described in Example 3 were examined. In a first series of .,AI.~fi~ lL~, the ability of the ~ rsCD4-specific Fab fragments to bind to native CD4 expressed on the surface of human peripheral blood Iymphocytes (PBLs), or CD4 on the surface of PBLs pulsed with gpl 20, was investigated. Fab binding to CD4 on the surface of PBLs was assessed by incubating the cells with the antibody, followed by a fluorescein i ~vlhio~ y (FITC)-labelled goat anti-10 human F(ab')2 secondary antibody. Bound antibody was then detected by FACS analysis.

1. Cell Stz.inin~
Human peripheral blood IYIU~ YICS were isolated from 10 ml h. ~.,.. ;.,;,. .1 blood by Ficoll diatrizoate density gradient ~. .,I.;r"~ . The cells were l~lchlculJ~ILcd at 37 degrees C for one hour with either PBS or 35 llg/ml lC~' .. ,.l.;,.~.,l gpl20 (HIV-ISF2 strain) (kindly provided by Chiron, Emeryville, CA). After washing, the cells were incubated for twenty minutes on ice with the Fab or control antibody at a cnnl~Pnir~finn of 2 ~Lg/ml. 19thy5D7 is a CD4 domain one-specific antibody, whereas OKT4 recognizes CD4 domain three. L736523 is a gpl20 specific amtibody. After washing with PBS, the cells were stained at 4 degrees C
20 for twenty minutes with a 1 :50 dilution of either FlTC-conjugated F(ab')2 goat anti-human F(ab')2 (Jackson Llll~ lul~a~ ,ll, West Grove, PA) or FlTC-conjugated F(ab')2 goat anti-mouse F(ab')2(Jackson luu~ lu~ ,L, West Grove PA). After washing the samples were analyzed on an EPICS-.C flow cytometer (Coulter Corp., Hialeah, FL). As a negative control, am indirect stain, ~..l .~I;I~.I;,~o PBS for the primary antibody, was performed.
B~
R.~lcaclllalivc results of the surface staining CA~ are shown in the series of flow cytometric profiles depicted in Figure 3, panels A-J. In panels A-E, human peripheral blood Iymphocytes ~lc;ll~,ub~,~,J with PBS were stained with various antibodies. In panels F-30 J, human peripheral blood IylulJllo~"~t~,~ Ul~ ' ' with l~ ..h;l~ gpl20 were stainedwith various antibodies. Figure 3 depicts cell staining with the following antibodies: control FlTC-labelled goat anti-human secondary antibody (panels A and F), 19thy5D7 (specific for thegpl20bindingsiteofCD4)(panelsBandG),L736523(specificfortheV3100pdomain of gpl 20) (panels C and H), Fab clone 3-47 (panels D and 1) and Fab clone 3-51 (panels E
35 and J).
While the CD4-specific antibody 19thy5D7 bound to native cell-surface CD4 on PBS-treated PBLs (Figure 3, panel B), none of the Fab fragments examined bound to native cell-surface CD4 on PBLs (as PYPmplifiPd by clone 3-47; Figure 3, panel D). To determine whether the rsCD4-specific Fabs elicited by rsCD4 immllni7~finn of the HlV-infected W0 96/02647 z i 9 5 2 3 8 - 38 - ~ Jy ~

humans recognized CD4 epitopes potentially exposed upon HIV binding to the CD4 molecule, the ability of these cloned Fabs to bind to human PBLs l~lch~cub..t~d with HIV envelope protein gpl20 was assessed. To confirm that gpl20 was bound to CD4 on this cell population, decreased staining of gpl20-pretreated PBLs with a monoclonal antibody specific for the gpl 20 binding site of CD4 (I 9thy5D7) (Figure 3, panel G)? as compared to 19thy5D7-binding to untreated cells (Figure 3, panel B), was ~ lrl~
Moreover, the antibody L736523 (specific for the V3 loop domain of gpl20) bound to the gp 1 20-treated PBLs (Figure 3, panel H). Fab clone 3-47, which had been panned against CD4/gpl20 complexes, stained at least 90 % of the gpl20-bound human PBLs (Figure 3, panel I). Fab clone 3-51, which also had been panned against CD4/gpl20 complexes, stained at least 30 % of the gp 1 20-bound human PBLs (Figure 3, panel J). These results .1. . """~
that Fab clones 3-47 and 3-51 recognize previously unidentified epitopes of the CD4 receptor which are only exposed on the cell surf~e after gpl20 binding.

To provide further evidence for the CD4 specificity of Fab clone 3-47, a Western blot of a human PBL Iysate was probed with this purified Fab and ;IIIIIIUUIU,UICI,;,U;klliUII
c~,u~lihll~ were carried out, as described below.

II. Wpctpm Blots Peripheral blood Iyul,ullocytcs were isolated from seventy ml bPrqrini7pd human bloodbyFicolldiatrizoategradientc. ,I.ir"~,.l;~,,. AfterwashingwithPBS,thecellswere Iysed in Triton X-100 Iysis buffer (300 mM NaCI, 50 mM Tris-HCL, pH 7.6, 0.5 % Triton X100, 10 llg/ml leupeptin, 10 llg/ml aprotinin, I mM PMSF, 1.8 mg/ml io/1Oq~ PrqrniolP) on ice for forty-five minutes with occasional mixing. After pelleting cell debris at 12~500 rpm for ffteen minutes at 4 degrees C, the ~ t - ~ I was transferred to a 1.5 ml eppendorf tube.
Sodium dodecyl sulfate and sodium deoxycholate were added to a final ronrrntrqtior of 0.2 percent, and the Iysates were stored on ice ptior to loading the gel.
After adding nu.ucdu.,;.~g 5 X sample buffer (60 mM Tris-HCL, pH 6.8, 25 %
glycerol, 2 % SDS, 0.1% blulllu~ lul blue), the Iysate was heated to 95 degrees C for ten minutes and loaded onto a six percent Sc,ualal;ll~/'i ~/O stacking SDS polyacrylamide gel.
Human rsCD4 and lc~,ulllbillalli gpl20 samples (i5 llg/well) were also loaded onto the gel.
Tl,e gel was run at I OOV for a u,ulw~hl.,lt~ly six hours before llall~r~ g the proteins overnight at 4 degrees C in transfer buffer (50 mM Tris base. 380 mM glycine, 0.1 % SDS, (20 % methanol) to a nitrocellulose membrane (Stratagene, La Jolla, CA) in a Transphor Transfer cl~.,L~uuhulc~;s TE 42 unit (Hûefer Scientific Iu~l~uu~,ul~, San Francisco, CA).
After rinsing in Tris-buffered saline (TBS) three times, the membrane was air dried and blocked in TBST (100 mMTris-HCI, pH 7.5, 0.9 % NaCI, 0.1 % Tween 20) for twohours with gentle rocking. The membrane was cut into strips and probed for one hour at room t~,~llu~lalulc with gentle rocking with the appropriate antibody or Fab diluted to a final 21 9-523~
Wo 961~2C47 1 ~~ 14 ~,u~ alivll of 2 llg/ml in TBST. After performing five five-minute TBST washes, the stripswereprobedwithal:SOdilutionofl.."~..,,.1;~1.peroxidase(HRP)-conjugatedgoatanti-human F(ab')2 (Jackson Illullullul~a~ l,ll, West Point, PA) for one hour at room L~ .flLul~ with gentle rocking. The strips were then washed for two hours with generous 5 quantities of TBST. Bound Fab was detected using ECL nhPmih.,,.;~ . f .~1 Western blotting detection reagents (Amersham) according to the lllallural,Lul~ instruction.

~a~
The Western blotting results are depicted in Figure 4, which illustrates the reactivity of an irrelevant gpl 20-specific antibody ~L736523) with human rsCD4 (lane 1), a CD4-specific control antibody (humani~d SA8) with hurnan rsCD4 (lane 2), the ~,.. .,~ lm~l Fab 3-47 with rsCD4 (lane 3), the gpl20-specific antibody (L736523) with a human PBL Iysate (lane 4) and the ."."...~1..""1 Fab 3-47 with a human PBL Iysate (lane 5). The data d that clone 3-47 specifically recognizes denatured rsCD4 (lane 3). In addition this Fab clone recognizes a 60 kDa protein, CUlll,.~l~Ulldillg to the molecular weight of the CD4 molecule, from a human PBL Iysate (lane 5). These results confirm the CD4 specificity of Fab clone 3-47, and in r . " . ,1.;, Ir I ;~ 11 1 with the cell staining data described above, suggest that Fab clone 3-47 recognizes an epitope exposed on a confn-m~tinn~lly altered form ofthe CD4 molecule exposed upon gpl20 binding to CD4. Thus, Fab clone 3-47 provides direct evidence for an HIV-induced crnft)rm ~ n~l change in the CD4 receptor. The ability of the HIV envelope protein to induce this ~,u~lrul~ iul~al change in CD4 suggests that HIV entry involves not only envelope binding to the first domain of CD4, followed by fusion with the cell membrane, but a series of events involving multiple regions of the CD4 receptor.

To further ~1f m( that Fab 3-47 recognizes the CD4 molecule, immuno-precipitation CA~J~.I;III.~III:; were conducted with b;ul;ll~l.lt~,d H9 cells. To biotinylate the cells, 3 x 107 H9 cells were washed three times with PBS-Plus (PBS/O.I mM CaC12/0.1 mM
MgC12) and 1~ .1 in 3 ml of Sulfo-NHS-Biotin (obtained from Pierce; at I mg/ml in PBS-Plus). After incubating for I hour at 4 ~C with gentle agitation, the cells were washed once v~ith RPMI-1640 media, followed by three washes with PBS-Plus.
The b;u~ H9 cell pellet was Iysed in Triton X-100 Iysis buffer (300 mM NaCI, 50 mM Tris-HCI, pH 7.6, 0.5 % Triton X-100, 100 llg/ml leupeptin, 10 lag/ml aprotinin, I mM PMSF, 1.8 mg/ml i-l~r.~rf~lrmi~lf ) on ice for 45 minutes with occasional mixing. After pelleting cell debris at 12,500 rpm for lS minutes at 4 ~C in a l..iC.u~. .,a ir~lsf, the was transferred to a 1.5 ml eppendorf tube. To preclear the cell Iysate, the Iysate was incubated for I hour at 4 ~C with 300 111 of a 50 % suspension of ~'J~mm~hin~ G beads (Pharrnacia) in PBS with gentle agitation. These samples were cPntrifilgPd at 12, 5000 rpm :2t 95238 Wo s6/02647 P~ 4~

for I minute in a ~l~;c~vccllLlirugc The precleared Iysate was then incubated overnight with Fab 3-47 or a negative control antibody (the gpl20-specific .". .",.~J~",~I antibody L736523) at a cor rrntrption of 15 ~Lg/ml. Next, the samples were incubated with 200 111 of a 75 %
suspension of goat anti-human IgG, FSab')2-conjugated ('T~mmPhind G beads in PBS for 5 I hour at 4 ~C with shaking. The beads were then pelleted as described above, washed three times with high salt wash buffer (0.5 M NaCI, 20 mM Tris-HCI, I mM EDTA, I %
Na-Deoxycholate, 0.5 % NP-4, 30 ~/O Sucrose), followed by two washes with low sa~t wash buffer (10 mM NaCI, 10 mM Tris-HCI, pH 7.6).
l~uu~llvlJlc~ ..t~,d proteins were eluted from the beads in IIOIICdUC;IIg samplebuffer (60 mM Tris-HCI, pH 6.8, 25 ~/O glycerol, 2 % SDS, 0.1 % blVIIIU,UII~,.IOI blue? at 95 ~C
for 10 minutes and loaded onto a 10 ~/c SDS-~Jvlya.,lylalll;dc gel. After ck,.,LIu,ullulc~;i, these proteins were transferred overnight to nitrocellulose. The membrane was blocked with 5 ~/O
bovine serum albumin (BSA)/TBST (100 mM Tris-HCI, pH 7.5, 0.9 % NaCI, 0.1 ~/O Tween-20) and probed with 2 ~Lg/ml horse radish peroxidase-conjugated avidin (Pierce)/0.3 %
15 BSA/TBST to detect biotinylated proteins. After extensive washing with TBST, HRP-avidin-bound, b;uLilly' ' ' proteins were visualized suing the ECL detection system (Amersham).
The results, illustrated in Figure 5"1. .".."~1, m that Fab 3-47, but not the negative control anti-gpl20 l~lulloclvl~al antibody, ;IIIIIIUIIU,UICI ', " ' a 55 kD protein (i.e., cullca~ulld;llg to the molecular weight of CD4) from the biul;~y' ' H9 cell Iysate.

F.XAMPI .F 5: Mapping the Domain Specificity of CD4-Specific Fabs To determine the location within the CD4 molecule at which Fab 3-47 binds, full-length and truncated CD4 molecules were used in enzyme linked iu~uu~osvlb~,.lL assays (ELISAs) with Fab 3-47 and control antibodies. One ,ug of full-length humam rsCD4 (comprising amino acids 1-371) or truncated human rsCD4 (comprising amino acids 1-183, UUII l,i~)Ulld;llg to the V I and V2 domains) was absorbed onto the wells of a Maxisorp plate overnight at 4 ~C. After washing eight times with Tris-buffered saline (TBS), the wells were blocked for one hour at 37 ~C with 0.5 % nonfat dry milk/0.05 ~/O Tween-20/TBS. Blocking solution was then removed and the Fab fragments were incubated in the appropriate wells for one hour at 37 ~C. Unbound Fab was removed by washing eight times with TBS. Bound Fab was then detected by incubating the wells for one hour at 37 ~C with a 1:50,000 dilution of horse radish peroxidase-conjugated goaf anti-human IgG F(ab')2 (Jackson Ill~ unu~e~ucll, West Grove, PA). Afler eight TBS washes, TMP one component substrate solution (KPL, Ca;Lll~,lah~, MD) was added. The reactions were terminated after twenty minutes by the addition of 2/3 N H2SO4. The absorbance was read on a Dynatech MR5000 ELISA reader (Dynatech Lab, Inc. Chantilly, VA) at OD=450 nrn.
Rel,lc~...L~L;v~ results for Fab 3-47 are ~ d below:

. _ ., ., _, , . , ,, .. ,,,,, , ,, _ , _ , _ _ : . . .. .. . . ... . ..

Wo 96/02647 r~ 4 Human rsCD4 Human rsCD4 DnmAin~ 4 DnmAin~ Z
Fab 3-47 1.224 1.193 These results .1. ."..,.~1l A Ir that Fab 3-47 binds to the truncated form of rsCD4 (containing only domains I and 2) equally well as to the full-length rsCD4 (containing domains 1-4), S indicating that the binding site for Fab 3-47 is contained within domains I and 2 (i.e., V I and V2) of human CD4.

F,~MP~ .~.6: Conditions for Formation of a C~ Altered CD4 Molecule R ~ by CD4-Specihc Fabs To further examine the conditions that resulted in the formation of the rnnfnrrnAtinnAIly altered CD4 molecule recognized by specific anti-CD antibodies of the invention, such as Fab 3-47, additional cell stuining studies similar to those described in Example 4, Section I, above, were performed. In a first series of r~ , Fab 3-47 or 15 various control antibodies were incubated with human peripheral blood Iymphocytes (PBLs) that had been ~l chl~,ul~ l with HIV-I .~ gpl 20 (rgp 120) at either 4 ~C or 37 ~C.
The controls included a positive control anti-gpl20 ".",-n~ AI antibody (L736523), a negative control Fab (2-36) and a no antibody control (i.e., PBS alone). The cell staining fllll.,ll~ were performed as described in Example 4, Section 1, above. The flow 20 cytometric profiles are shown in Figure 6, panels A-H. Panels A-D represent human PBLs ~/IC;;II~.Ub..~,d with rgpl20 at 4 ~C. Panels E-H represent human PBLs ~l~h~ lJat~:i with rgpl20 at ~37 C. Cells in panels A and D were incubated with PBS alone. Cells in panels B
and F were incubated with the control anti-gpl 20 amtibody. Cells in panels C and G were incubated with the control Fab 2-36. Cells in panels D and H were incubated with Fab 3-47.
25 Theresultsd,",~ rthec.",r.,",.Al;~".AllyalteredformofCD4thatisrecognizedbyFab3-47 is formed when human PBLs are ~l~ ' ' with rgpl20 at 37 ~C, but not when human PBLs are ~ ' ' with rgp 120 at 4 ~C (i.e., binding of Fab 3-47 to human PBLs was observed when the pr~inAnhqtinn was carried out at 37 ~C but not when the A~ I I was carried out at 4 ~C).
In a second series of ~AIJ~,I;llI~ll~:i, the ability of Fab 3-47 to bind to H9 cells h ' ~ with live HIV-I virus was examined. In the virus binding ~A~ ;lhll~ , H9 cells were incubated overnight at 37 C with infectious lr~ llh;ll ~ HIV-IMN (20 ng p24 per 1.5 x 106 cells) in the presence of either Fab 3-47, a positive control anti-CD4 u l- .. ,-,~1. ~, ~AI
antibody (Hu5A8, a humanized antibody that recognizes domain 2 of CD4) or a negative control Fab (2-36), each at a .. ,.. , .n ,.1 ;. ", of 2 llg/ml. After washing, bound antibodies were detected by incubating these cells at 4 C for 20 minutes with a I :50 dilution of a goat anti-~ 1 95238 W0 96102647 - 42 - P~ 0..114 human IgG F(ab')2 (Jackson l~ --u~ul ,~lel., West Grove, PA). After washing. the cell samples were analyzed on an EPICS-C flow cytometer (Coulter Corp.. Hialeah. FL). As a negative control, an indirect stain, ~ ,g PBS for the primary antibody, was performed.
The results are shown in Figure 7, panels A-D. Panel A represents the PBS control. Panel B
5 represents the cell staining with the positive control anti-CD4 antibody (HuSA8). Panel C
represents cell staining with the negative control Fab 2-36. Panel D represents cell staining with Fab 3-47. The results ,1~ ."..,.~a. . that Fab 3-47 binds to H9 cells ~l~hl~ uba~d with liveHIV-l,indicatingthel,.~;l....l.AI;r~l~ofH9cellswithliveHlV-l leadstotheformationof the confnnnAlir nAlly altered form of CD4 that is recognized by Fab 3-47.

EQTJIVAT .F~TS ~ : : E
Those skilled in the art will recognize, or be able to ascertain using no more than 15 routine ~,A~ A I ~ , many equivalents to the specif c embodiments of the invention described herein. Such equivalents are intended to be ~ d by the following claims.

W096/02647 43 r~.,. ,114 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i~ APPLICANT
(A) NAME: BETH ISRAEL HOSPITAL
(B) STREET: 330 BROOKLINE AVEN~E
(C) CITY: BOSTON
(D) STATE: M~qq~TTTTqFTTS
i0 (E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02215 (G) TELEPHONE: (617) 667-4585 (ii) TITLE OF INVENTION: ~nt;hr~;~q that Bind a Confnrr-t;rnAlly Altered CD4 Molecule Induced Upon Binding of Human T - f;r; nry Virus (iii) NUP~3ER OF SEQUEN OE S: 18 ( iv ) ~KEb rU.J~N~ ADDRESS:
(A) ~nnT~Fq~qFF: L~HIVE h CO OE FIELD
(B) STREET: 60 State Street, Suite 510 (C) CITY: Boston (D) STATE: MAq~ q ( E) COUNTRY: USA
(F) ZIP: 02109-18?5 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC t;T l (C) OPERATING SYSTEM: PC DOS/MS-DOS
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(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NCMBER: US 08/277,080 (B) FILING DATE: 19-Jul-94 (C) CL~SSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NSMBER: US 08/305,903 (B) FILING DATE: 13-Sep-94 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: DeConti, Giulio A , Jr.
(B) REGISTRATION NCMBER: 31,503 - (C) ~NO~/DOCKET NU~3ER: BIZ-013CPPC
(ix) TEL~._.JNl~TION INFORMATION:
(A) TELEPHONE: (61i) 227-7400 (B) TELEFAX: (617) 227-5941 21 q5238 W096/02647 44 r~l,u~ 14 ~

(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE r~T~rTFDT.cTIcs:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) sTD~TrT.~NFc.c: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide I ~ ~.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
AGGTGCAGCT GCTCGAGTCT GG : ~ 22 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE r~D~rTrDrCTIcs (A) LENGTH: 22 base pairs (D) TYPE: nucleic acid (C) STD~NnT.~n~Tc.cc Gingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: n1;grn~1r1Pnti~r (xi) SEQUENCE ~ lON: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE r~D~rTrDTcTIcs:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) ~ NI]~IIN~ single ::
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: rl;r;rn11r1~rt;~r (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AGGTGCAATT GCTCGAGTCT GG ~ _ 22 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE r~D~rTFRT.cTIcs:
(A) LENGTH: 22 ba~e pairs (D) TYPE: nucleic acid (C) ~ N~:C::: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nl;Jrmlrl~rtide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
55 AGGTGCAACT GCTCGAGTCG GG ~ 22 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE rH~T~rTDDTcTIcs:

~ W 096/02647 2- 1 q 5 2 3 8 . ~ . ' .. 14 (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) 5T~NDFnNRCq: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: rl;g~nllrlPnt;~
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

~2) INFORMATION FOR SEQ ID NO:6:
~i) SEQUENCE r~v~rTF~TcTIcs:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STV~NDRnN~qc single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
~rrT~r~rrT GCTCGAGTCA GG ~ .22 25 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE r~DpcTFvTqTIcs:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) ST~ nNRCq: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: ~lirJ~nl~rl~rtide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GTGCCAG~TG TGAGCTCLiTG ATGACCCAGT CTCCA 35 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE rNLv~rT~vTqTIcs:
(A) LENGTH: 35 base pairs ~B) TYPE: nucleic acid ~C) ST~D~n~n~cq single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: oligonucleotide ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GTGCCAGATG TGAGCTCGTG TTGACGCAGT CTCCA 3s ~2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE r~v~rT~RTcTIcs:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) sT~ nN~qq single (D) TOPOLOGY: linear ~V096/02647 2 19 ~ ~ 3 8 - 46 ~ a,114~

~ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
s CTGCACAGGG l~L~ AGCTCGTGTT GACGCA ~ 36 ~2) INFORMATION FOR SEQ ID NO:10:
O (i) SEQUENCE r~R~rTERTsTIcs:
(A) LENGTP.: 36 base pairs (B) TYPE: nucleic acid (C) sTRANnpnN~cc single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nl;~n~ tide _ _ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
20 CTGCACAGGG IC~l~o~ AGCTCATACT GACGCA ~ 36 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CBARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STR~ nNEC,C: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE~ ~r--~l~tide . --(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AGCATCACTA rT~r~r~TT TGGGCTC ~ .27 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CBARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STR~T~EnNEcq single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CTCAGTATGG 1~l~VI~ 19 50 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTB: 56 base pairs (B) TYPE: nucleic acid (c) STR~E~ .cc: single ~
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide 21 95~38 W 096/02647 ~ 5'l.ll4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ~
TCCTTCTAGA TTACTAACAC l~lC~l TGA~GCTCTT TGT~ACGGGC GA~CTC 56 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE c~rTR~rcTIcs:
(A) LENGTH: 34 base pairs ~ (B) TYPE: nucleic acid (C) ST~Rn~TRC.C: single (D) TOPOLOGY: linear (ii) MOLECVLE TYPE: ~l;gnnl1nl~n~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:~
GCATTCTAGA CTATTATGAA CATTCTGTAG GGGC ~ ~ 34 (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE r~r~T~r~cTIcs:
(A) LENGTE: 44 base pairs (B) TYPE: nucleic acid (C) ST~R~NRc.~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

GTGTTGACTC AGCCTGCCTC ~~ ~b TCTGCTGGAC AGTC . 44 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE ~ rT~r~rcTIcs:
(A) LENGT~: 176 base pairs (B) TYPE: nucleic acid (C) ST~nRDNRc.5: single (D) TOPOLOGY: linear (ii) MOLECVLE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GAGTCAGGGG GAGGCTTGGT ~.~cr.~ G~IC~1~A GACTCTCCTG TACAACCTCT 60 GGATTCACCT TTAACACGTA TGCCATGAGT ~ ~ AGNCTCCAGG GAAGGGGCTG 120 GAATGGCTCT CAGGTATTA~ TAACAATGGT CGGACTGCAT TCTACGAGAC TCGTGA 17Ç
(2) INFORMATION FOR SEQ ID NO:17:
~ (i) SEQUENCE CEARACTERISTICS:
(A) LENGT~: 125 base pairs (B) TYPE: nucleic acid (C) STT~NDRr~CC: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

W 096/02647 2 1 9 5 2 3 8 48 ~

(xi) SEQUENCE DESCRIPTION: SEQ ID N~:17:- ~ -GTGTTGACTC ATGccTaTAA TrrrDrrDrT TTaGGAGGcc r~r~r~rrr~Grp GATCACGAGG 60 5 TcAçAAçATa TGGACCATCT GGTGAACACG TGTPP~rrrr CGCTCTCTAC TiA~PPTPrp 120 AA~AA 125 (2~ INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE r~D~PrT~TCTICS:
(A) LENGTE: 191 base pairc (B) TYPE: nucleic acid (C) sT~aNn~nN~qq ~ingle (D) TOPOLOGY: linear (ii) ~LECULE TYPE: cDNA ~ :
(xi) SEQUENCE DESCRIPTION: SEQ ~ID N0: 18:
rPrrTr~r TGCTCGAGTC Drrr.r~r~r GTGGTCCAÇC CTÇGGAÇGTC~CCTGAÇACTC 6Q
TCCTGTGCAÇ CCTCTGÇATT CACTTTCAÇT pr~rTprr~r~rp Tr~rDTTr~r~r~T rrr~rrDr~rT 120 25 rrPr.rr~Prr~ GGcTGaAGTG aGTGGcAGTT AT~TCAAATG Prr~ r~ TTAGATATAT 180 GcAçAcTcGa T

Claims (71)

1. An antibody, or fragment thereof, that binds a conformationally altered form of a CD4 molecule expressed on the surface of a CD4+ cell upon contact of the cell with human immunodeficiency virus (HIV), or an envelope protein thereof, wherein the antibody, or fragment thereof, does not substantially bind CD4 on the surface of the cell prior to contact of the cell with HIV or an envelope protein thereof.

2. The antibody of claim 1, which is a monoclonal antibody.

3. The antibody of claim 2, which is a human monoclonal antibody.

4. The antibody of claim 2, which binds a conformationally altered form of human CD4 that expresses an epitope bound by a monoclonal Fab 3-47, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69658 or an epitope bound by a monoclonal Fab 3-51, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69684.

5. The antibody of claim 4, which has an epitope binding specificity of a monoclonal Fab 3-47, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69658.

6. The antibody of claim 5, which is a full-length monoclonal antibody having immunoglobulin heavy and light chain variable regions of a monoclonal Fab 3-47.

7. The antibody of claim 4, which has an epitope binding specificity of a monoclonal Fab 3-51, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69684.

8. The antibody of claim 7, which is a full-length monoclonal antibody having immunoglobulin heavy and light chain variable regions of a monoclonal Fab 3-51.

9. The antibody of claim 1, which is a Fab fragment.

10. The antibody of claim 1, which is functionally linked to an agent selected from the group consisting of am antibody, an antibody mimetic agent, a detectable agent a cytotoxic agent and a pharmaceutical agent.

11. A monoclonal antibody, or fragment thereof, having an epitope binding specificity of a monoclonal Fab 3-47, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69658.

12. The monoclonal antibody of claim 11, which is the monoclonal Fab 3-47.

13. The monoclonal antibody of claim 11, which is functionally linked to an agent selected from the group consisting of an antibody, an antibody mimetic agent, a detectable agent, a cytotoxic agent and a pharmaceutical agent.

14. A monoclonal antibody, or fragment thereof, having an epitope binding specificity of a monoclonal Fab 3-51, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69684.

15. The monoclonal antibody of claim 14, which is the monoclonal Fab 3-51.

16. The monoclonal antibody of claim 14, which is functionally linked to an agent selected from the group consisting of an antibody, an antibody mimetic agent, a detectable agent, a cytotoxic agent and a pharmaceutical agent.

17. A composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising the monoclonal antibody of claim 11 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising the monoclonal antibody of claim 14 and a pharmaceutically acceptable carrier.

20. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the immunoglobulin light chain variable region of the monoclonal Fab 3-47 of claim 12.

21. The nucleic acid molecule of claim 20, further comprising a nucleotide sequence encoding an immunoglobulin light chain constant region.

22. An expression vector comprising the nucleic acid of claim 21.

23. A host cell into which the expression vector of claim 22 has been introduced.

24. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the immunoglobulin heavy chain variable region of the monoclonal Fab 3-47 of claim 12.

25. The nucleic acid molecule of claim 24, further comprising a nucleotide sequence encoding at least one immunoglobulin heavy chain constant region.

26. An expression vector comprising the nucleic acid of claim 25.

27. A host cell into which the expression vector of claim 26 has been introduced.

28. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the immunoglobulin light chain variable region of the monoclonal Fab 3-51 of claim 15.

29. The nucleic acid molecule of claim 28, further comprising a nucleotide sequence encoding an immunoglobulin light chain constant region.

30. An expression vector comprising the nucleic acid of claim 29.

31. A host cell into which the expression vector of claim 30 has been introduced.

32. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the immunoglobulin heavy chain variable region of the monoclonal Fab 3-51 of claim 15.

33. The nucleic acid molecule of claim 32, further comprising a nucleotide sequence encoding at least one immunoglobulin heavy chain constant region.

34. An expression vector comprising the nucleic acid of claim 33.

35. A host cell into which the expression vector of claim 34 has been introduced.

36. An antibody mimetic agent that binds a conformationally altered form of a CD4 molecule expressed on the surface of a CD4+ cell upon contact of the cell with human immunodeficiency virus (HIV), or an envelope protein thereof, wherein the antibody mimetic agent does not substantially bind CD4 on the surface of the cell prior to contact of the cell with HIV or an envelope protein thereof.

37. The antibody mimetic agent of claim 36, which has an epitope binding specificity of a monoclonal Fab 3-47, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69658.

38. The antibody mimetic agent of claim 36, which has an epitope binding specificity of a monoclonal Fab 3-51, encoded by a plasmid carried by a bacteria deposited with the American Type Culture Collection and assigned designation number 69684.

39. A method for detecting a CD4+ cell expressing on its surface a conformationally altered form of a CD4 molecule induced upon binding of human immunodeficiency virus, or an envelope protein thereof, to the cell, comprising:
contacting the CD4+ cell with a monoclonal Fab selected from 3-47 and 3-51; and detecting the monoclonal Fab bound to the cell surface to thereby detect a conformationally altered form of a CD4 molecule expressed on the cell surface.

40. A method for identifying an agent that inhibits formation of a conformationally altered form of a CD4 molecule expressed on the surface of a CD4+ cell upon binding of human immunodeficiency virus, or an envelope protein thereof, to the cell, comprising:
contacting the CD4+ cell with:
a gp120 composition; and an agent to be tested for the ability to inhibit formation of a conformationally altered form of CD4 on the cell surface;
further contacting the cell with a monoclonal Fab selected from 3-47 and 3-51; and determining the amount of monoclonal Fab bound to the cell, wherein a reduced amount of binding of the monoclonal Fab to the gp120-treated cell in the presence of the agent, as compared to the amount of binding of the monoclonal Fab to a gp120-treated cell in the absence of the agent, indicates that the agent inhibits formation of a conformationally altered form of CD4 on the cell surface.

41. A method for identifying an agent that induces formation of a conformationally altered form of a CD4 molecule on the surface of a CD4+ cell, comprising:
contacting the CD4+ cell with an agent to be tested for the ability to induce formation of a conformationally altered form of CD4 on the cell surface;
further contacting the cell with a monoclonal Fab selected from 3-47 and 3-51; and determining the amount of monoclonal Fab bound to the cell, wherein an increasedamount of binding of the monoclonal Fab to the CD4+ cell in the presence of the agent, as compared to the amount of binding of the monoclonal Fab to a CD4+ cell in the absence of the agent, indicates that the agent induces formation of a conformationally altered form of CD4 on the cell surface.

42. A method for inhibiting infection of a CD4+ cell by human immunodeficiency virus, comprising contacting the cell with an antibody, or fragment thereof, that binds a conformationally altered form of a CD4 molecule expressed on the surface of the cell upon contact of the cell with human immunodeficiency virus (HIV), or an envelope protein thereof, wherein the antibody, or fragment thereof, does not substantially bind CD4 on the surface of the cell prior to contact of the cell with HIV, or an envelope protein thereof.

43. The method of claim 42, wherein the antibody, or fragment thereof, is a monoclonal antibody, or fragment thereof.

44. The method of claim 43, wherein the monoclonal antibody, or fragment thereof, is a human monoclonal antibody.

45. The method of claim 44, wherein the antibody has an epitope binding specificity of a monoclonal Fab 3-47.

46. The method of claim 45, wherein the antibody is the monoclonal Fab 3-47.

47. The method of claim 44, wherein the antibody has an epitope binding specificity of a monoclonal Fab 3-51.

48. The method of claim 47, wherein the antibody is the monoclonal Fab 3-51.

49. The method of claim 42, wherein the antibody is administered to a subject.

50. An isolated molecule that expresses at least one epitope expressed on a conformationally altered form of CD4 induced on the surface of a CD4+ cell upon contact of the cell with human immunodeficiency virus, or an envelope protein thereof.

51. The molecule of claim 50, wherein the conformationally altered form of CD4 expresses am epitope bound by a monoclonal Fab 3-47.

52. The molecule of claim 51, which expresses the epitope bound by the monoclonal Fab 3-47.

53. The molecule of claim 50, wherein the conformationally altered form of CD4 expresses am epitope bound by a monoclonal Fab 3-51.

54. The molecule of claim 53, which expresses the epitope bound by the monoclonal Fab 3-51.

55. The molecule of claim 50, which is a protein or peptide.

56. The molecule of claim 55, which is a modified human CD4 protein, or peptide fragment thereof.

57. The molecule of claim 55, which is a non-human primate CD4 protein, or peptide fragment thereof.

58. The molecule of claim 57, which is a rhesus monkey or chimpanzee CD4 protein, or peptide fragment thereof.

59. The molecule of claim 55, which is an anti-idiotype antibody, or fragment thereof, that binds monoclonal Fab 3-47 or monoclonal Fab 3-51.

60. The molecule of claim 50, which is a peptide mimetic.

61. A pharmaceutical composition comprising the molecule of claim 50 and a pharmaceutically acceptable carrier.

62. The pharmaceutical composition of claim 61, further comprising a pharmaceutically acceptable adjuvant.

63. A method for producing an antibody that binds an epitope expressed on a conformationally altered form of a CD4 molecule induced on the surface of a CD4+ cell upon contact of the cell with human immunodeficiency virus, or an envelope protein thereof, comprising immunizing a mammal with the molecule of claim 50.

64. A method for inhibiting infection of a CD4+ cell by human immunodeficiency virus in a subject, comprising administering to the subject a therapeutically effective amount of a molecule that expresses at least one epitope expressed on a conformationally altered form of CD4 induced on the surface of a CD4+ cell upon contact of the cell with human immunodeficiency virus, or an envelope protein thereof, such that an antibody response against at least one epitope expressed by the molecule is induced in the subject.

65. The method of claim 64, wherein the conformationally altered form of CD4 expresses an epitope bound by a monoclonal Fab 3-47.

66. The method of claim 65, wherein the molecule expresses an epitope bound by amonoclonal Fab 3-47.

67. The method of claim 64, wherein the conformationally altered form of CD4 expresses an epitope bound by a monoclonal Fab 3-51.

68. The method of claim 67, wherein the molecule expresses an epitope bound by amonoclonal Fab 3-51.

69. The method of claim 64, wherein the molecule is a modified human CD4 protein, or peptide fragment thereof.

70. The method of claim 69, wherein the molecule is a non-human primate CD4 protein, or peptide fragment thereof.

71. The method of claim 70, wherein the non-human primate CD4 protein, or peptide fragment thereof, is from a rhesus monkey or chimpanzee.