Note: Descriptions are shown in the official language in which they were submitted.
WO 96/07428 PCI/US94/10262
8 8 9
PEPTIDE COMPOSITIONS CAPABLE OF DOWN REGULATING AN ANTIGEN SPECIFIC IMMUNE
RESPONSE.
Back~round of the Invention
Allergies have been the bane of human existence since the beginning of human
20 history. Those individuals afflicted with allergies suffer with continual or repeated
bouts of sinusitis, rhinitis, itchy watery eyes, nose and mouth, and allergic asthma
which may be so severe as to cause hospitalization. The most dangerous human
allergic response is anaphylaxis, which will cause death without medical intervention.
Pharmacellticals to alleviate many of the less severe symptoms have been
25 developed such as oral ~dmini~tration of antihistamines, inhalers for ~thm~ and
portable injection kits co~ adrenaline to suppress anaphylactic shock until
mr~ir,~l help can be reached. However, these pharm~ceuti~ do nothing more than
mask the symptoms caused by hi~mine release, the end result of the human immnne
response which begins when the offending allergen enters the body. Furth~rmore,
30 many of the more powerful ~ntihi~t~min~ drugs have undesirable side effects such as
excessive dryness of the mucosa or drowsiness.
Over the past several decades a therapy involving desPn~iti7~tion of hllm~n~ hasbeen developed. Desensitation therapy involves repeated injections with increasing
dosages of a crude allergen extract of the offending allergen. Although llc~ llk.ll with
35 allergen extracts has been proven somewhat effective in the clinic for alleviating
allergen-related symptoms, and is a common therapy used most widely in allergy
clinics today, the m~çh~ni~m of ~lesçn~iti7~tion remains unclear. Furthermore,
desen~iti7~tion therapy must be undertaken with e~ cllle caution as the side effects rnay
be significant or even fatal (i.e. anaphylaxis). Thus, it is n~cess~ry for the patient to
40 undergo multiple injections of incre,~ tally larger dosages of the crude extract, and
remain under medical observation for one or more hours every time an injection is
W0 96/07428 ~ 8 ~ 9 PCT/US94110262
given. In addition, it is often n.ocess~ry for an allergic patient to receive L~ nt until
",~ nce dose is reached which usually requires L.edt~l,e~lt injection once a week for
six to twelve months and at intervals of four to six weeks thel~drlel. Thus, this
tlCdllllClll is very time intensive, inconvenient and not without side effects or danger for
5 the patient.
In an attempt to make desen~iti7~tion immunotherapy more effective and safer,
several investiE~tors have dllell~ted to develop modified allergens with the intention of
effecting the immunological events which occur during immunotherapy (i.e. to increase
blocking IgG antibodies or decrease the number of allergen specific IgE antibodies).
10 Such modifications include pl~,p~ing and testing: urea-denatured allergens, T~hi7~k~ et
al, E.J. Immunol. 114:100-115 (1975); allergoids created by formalin treatment of
allergens; Norman et al., JAllergy Clin Immunol, 70:248-260 (1982); allergens bound
to tolerogens such as D-glutamic acid-D-lysine, Katz D., Immunology, 41:1-24
(1980);or polyet~yleneglycol, Sehon et al, JAllergy Clin Immunol 64:242-250 (197n~;
and glutaraldehyde-polymerized allergens, Metzger et al. N Engl J Med 295: 1160- 1164
(1976).
Michael et al., US Pat. No. 4,338,297, disclose a polypeptide active pollen
immuno~upplc;ssallt prepared by proteolytic enzyme digestion of pollen antigen and
purified by reaction with antipollen antibody potentially for use as a therapeutic in
20 desen~iti7~tion. Later, the same group published experiments indicating that it was
possible to ~Uppl~;Ss the immune response in mice to protein antigens such as bovine
serum and honey bee venom phospholipase A2 by Lle~ with fragments derived by
enzymatic digestion (Michael et al., J Clin Immunol, 75:200(abstr) (1985), and
Ferguson et al., Cell Immunol, 78:1-12 (1983)), and in addition found that fr~EmPnting
25 allergenic proteins of short ragweed pollen by peptic digestion and arlmini~t~ring
digestion products in the molecular weight range of 5-15 kD either prior to, or after
il,l,d~eliloneal i~.. i7~lion with a ragweed l"~p~dlion resulted in ~u~p~Gssion of the
immune response (Michael et al., Clin Exp Allergy, 20:669-674 (1990))~ According to
Michael et al., it was believed that the ~u~ ,ssion of the immune response to the
30 ragweed pl~ dtion involved T cells, and that the fragments of the peptic digest were
capable of stim~ ting T cells that regulate immunosuppression.
In addition, the same group of investig~tors tested a colllposilion comprising
peptic fr~Em~ntc of short ragweed for clinical effecti~ncss in hnm~n~, (Litwin et al.
Clin Exp Allergy 21:457-465 (1991)). In this study, three groups of ragweed hay fever
35 patients were placed on pre-seasonal i~llmunolherapy. One group received a
conventional ragweed pl~p~dlion that had been enriched for ragweed antigen (Amb a I)
~ie~i n~t~cl Pool 2. The proteins in Pool 2 contailled about 26% Amb a I. Pool 2 was
enzymatically digested and a fraction con~illillg proteins in the 10kD range or less was
W096/07428 ~ 8 8 ~ 9 PCT/US94tlO262
used as the peptic fragment colllposilion and ~e~ t~ fSRW. The third group was
given a placebo. Although the results indicated that the groups given either Pool 2 or
fSRW had significantly reduced symptom-medication scores co.llpa~ed with the
placebo-llc~ t group, the dirrclcllces bclwcen the fSRW l~cat~lælll and the Pool 2
5 tle~l . "~ were not ~i~ific~nt Thus, the fSRW peptic digest composition did not
totally achieve its goal of providing significant efficacy or convenience over aconventional immunotherapy composition comprising a crude extract of the allergen to
which the individual is sensitive, and has not been thus far approved for use in the
United States or is such a peptic composition presently the subject of a clinical study in
10 the United States.
Furthermore, the use of a composition comprising peptic fragments of a protein
allergen such as that described by Litwin et al. has serious drawbacks. For example the
protein extract (Pool 2) which was digested to produce the fSRW peptide composition
was erlliched for the desired Amb a I protein to only - 6% and 3r.y nurnber of other
15 proteins and con~min~nt.c were likely present in the Pool 2 composition and were
carried over to the fSRW digest. According to Litwin et al., the precise Amb a I content
in fSRW could not be dc~lllulled because components of the fSRW did not remain in
solution. Thus the fSRW may have contained undesirable proteins which may have
adverse effects on the patients being treated, and therefore, provides no advantage over
20 the use of a conventional crude extract, and moreover, may have many of the same
disadvantages associated with classic immunotherapy. In addition, the enzymatic
digest of a crude protein does not con~ tently produce the same composition of
peptides every time. Thelcfolc, it would be almost impossible to produce consistent,
precisely-defined, highly purified compositions of peptides for use as a pharm~-eutical
25 for human ll~all,-llll as is l~u~,d by most regulatory agencies throughout the world
using a composition of enzymatically digested proteins, similar to that of fSRW
described by Litwin et al.
In addition, similar to allergy, autoi ~ . " ~ .e diseases such as Type I diabetes,
multiple sclerosis, and rheumatoid arLhritis are generally accepted as being the result of
30 an antigen specific T cell mP~ ted response against an antigen which in the case of
autoi~""u.,~. di~e~e, is the body's own tissue. Therefore, it is believed an approach to
treating autoi~"~"~.,.o disease which is conceptually similar to that for treating allergies
would be app~pliate. For example, WO 88tlO 120, WO 91/08760, WO 92/06704, WO
93/21222, and WO 94/07520, (ies~rihe oral or enteral ~-lmini~tration of whole
35 ~uto~rltigens, or fr~gm~-nt~ thereof such as mylein basic protein, (MBP a pl~_sulllcd
~uto~nti~en in mnltiple sclerosis), insulin for the L~ of ~ betes, or collagen for
the L.~~ of rh~..",7.loid ~uilis. For many of the reasons described above, thereare limits to the clinical applicability of oral, enteral or aerosol ~imini~tration of
w0 96/07428 ~ 9 ~ 94,l0262
~nto~ntigene such as an inability to cl~ t,~ - ;7~ the active colll~nent of a lhe~ Lic
collll)osilion once introduced in the stomach due to ~llbselluent enzymatic dc~,làdation
in the stomach. Thus, predictable and reproducible th.,la~Gulic effects may be difficult
to achieve using these methods, not to mention the potential for adverse side effects as
5 a result of the body's further processing of the therapeutic which may not be
predictable.
The present invention overcollles the drawbacks described above and provides
methods for treating allergies and other ~iiee~ees associated with an i~ -c response
by the human to a protein antigen (i.e. aulo;.. ~ e diseases), using precisely-defined,
10 highly purified, plepala~ions comprising at least one peptide having a defined sequence
of amino acid residues which comprises at least one T cell epitope of the protein
antigen, ~dminietered in non-immunogenic form. Preferred, compositions and methods
of treating humans in accordance with the present invention have been tested in the
clinic and have been shcwr. to 'ce successfu!. The ramifications of these successful
15 clinical trials are expected to change the face of allergy immunotherapy forever, as well
as change the course of tre~tm~-nt of autoi.u...l....q ~lice~eee and other areas relating to
human disease conditions associated with the immlme response.
Summary of the Invention:
The present invention provides theldpeulic colllposilions and methods for
treating disease conditions in hllm~n~ associated with an antigen specific immnne
response by the human to an antigen such as a protein antigen (i.e. allergy and
autoi.. ~ ~iiee~ee,e). Th~ldpeulic compositions of the invention are precisely-
25 defined, highly purified reproducible plep~dlions which are ~ui~le for human
therapy. Preferred colllposilions of the invention colll~,lise at least one ieo! ~
purified peptide, free from all other polypeptides or col.t~ nte, the peptide having a
defined sequence of amino acid residues which comprise at least one T cell epitope of
an antigen of interest. A lh~;l~eulic composition of the invention is capable of down
30 regulating an antigen specific immnn~ Jollse to an antigen of interest in a population
of humans subject to the antigen specific immnne lesponse such that disease symptoms
are reduced or elimin~ted, and/or the onset or progression of disease ~ylllptOIllS is
prevented or slowed.
Colllposilions and methods of the invention may be used to treat sensitivity to
35 protein allergens in hnm~ne such as allergies to ragweed, grasses, trees, house dust mite
(dust), cats, dogs and other ~nim~le, and any other airborne or contact allergens.
Compositions and methods of the invention may also be used to treat dulu;.. ~.e
W096107428 ~ 8 ~ 6 ~ PCT/US94/10262
disease such as rh~ oid arthritis, diabetes, my~cth~ni~ gravis, Grave's ~lice~ce, Good
Pasture's syndrome, thyroiditis and multiple sclerosis.
Brief Description of the Drawin~s
s
Fig. 1 is a graphic rcplesentdtion of catroom symptom ag~avalion (nasal, lung, and
total allergy) at one and six weeks post tlG~t.ll~ll with pairwise co,l,p~isons versus
placebo (PBO) for total allergy symptoms at 75 ~g and 750 tlg at six weeks, trend tests
for dose response at one and six weeks for total allergy symptoms are indicated (see
10 starred data).
Detailed Description of the Invention
The patent ~nd scientific lil~.d~ul~ referred to herein establishes the knowlcdge
15 that is available to those with skill in the art. The issued U.S. patents, PCT
publications, and other publications cited herein are hereby incorporated by reference.
The present invention provides the,d~ ic compositions and methods for
treating disease conditions in hum~nc associated with an immlln~ response by thehuman to an antigen. Theld~Gulic compositions of the invention comprise at least one
20 isolated, purified peptide, free from all other proteins or co..~ ntC, and having a
defined sequence of amino acid residues which comprise at least one T cell epitope of
the antigen. As used herein, the term "isolated" refers to a peptide which is free of all
other polypeptides, co~.t;..~,in~l~t~, starting reagents or other m~t~ , and which is
unconjugated to any other molecule. The composition or col"posilions of the invention
25 when ~minictered to a patient in a th~ldp~.~lic ,~h,æl in non-immnnogenic form, are
capable of down regulating an antigen specific immnne response in a population of
hnm~nc subject to the antigen specific i~ --..r response.
Col"po~.ilions and methods of the invention may be used to treat disease
conditions relating to antigens, such as protein allergens, in hnm~nc i.e. allergies to
30 ragweed, grasses, trees, house dust mite (dust), cats, dogs and other ~nim~lc, and any
other airborne or contact allergens. Human clinical trials described in the Examples
discuss the successful use of compositions and methods of the invention in the
IIGdtlll~lll of hum~nc allergic to cats. This invention may also be used to treat
autoi-..--..--~to disease such as rh~llm~toid arthritis, ~ betes~ myasthenia gravis, Grave's
35 lice~ce~ Good Pasture's syndrome, psoriasis, thyroiditis and multiple sclerosis wherein
the antigen l~,i,po,lsible for the disease is a protein ;~ o -~1igen.
In accord~ce with this invention, a "peptide" refers to a defined sequence of
amino acid residues pl~;felably comprising no more than about 50 amino acid residues
W096/07428 ~ 8 ~ ~ ~ PCT/US94/10262
and co,..~ g at least approxim~tely seven amino acid residues in length, and
plcfeldbly at least about 12~0 amino acid residues in length, and more plefi,ldbly at
least 13-30 arnino acid residues in length and which, when derived from a protein
antigen, contains less than the amino acids of the entire protein antigen and pref~ably
5 no more than about 75% of the amino acid residues of the entire protein antigen.
Peptides used in accordance with the invention comprise at least one T cell epitope of
an ~ntigen A T cell epitope is the basic element or smallest unit of recognition by a T
cell receptor, where the epitope collll~lises arnino acids essenti~l to receptor recognition.
T cell epitopes are believed to be involved in the initiation and perpetuation of the
l0 immllne response to an antigen such as a protein allergen which is responsible for the
clinical symptoms of allergy. These T cell epitopes are thought to trigger early immune
response events at the level of the T helper cell by binding to an appropriate HLA
molecule on the surface of an antigen presenting cell and stimulating the relevant T cell
subpopulation. These events lead to T cell prolife;ation, lymphok~nc ,ecretiGrl, local
lS infl~mm~tory reactions, recruitment of additional immune cells to the site, and
activation of the B cell cascade leading to production of antibodies. One isotype of
these antibodies, IgE is fundamentally hllpo~ t to the development of allergic
symptoms and its production is influenced early in the cascade of events, at the level of
the T helper cell by the nature of the lymphokines secreted. In the case of an
20 autoimmune ~ e~e7 the antibodies produced are al-to~ntibodies against an al~to~ntigen
resulting in the clinical symptoms of an autoimm--ne disease i.e. ~uto~ntihodies directed
against myelin basic protein, a presumed ~uto~ntigen in multiple sclerosis.
A therapeutic/prophylactic tl~ia~ ellt regimen in accordance with the invention
(which results in prevention of, or delay in, the onset of disease SylllptulllS caused by an
25 offending antigen or results in reduction, progression, or alleviation of ~ylllptollls
caused by an offen~iing antigen i.e. down regulation of an antigen specific immnn.o
response) comprises ~tlmini~tration, in non-irnmunogenic form (e.g. without adjuvant)
of a theldpeulic composition of the invention comprising at least one isolated peptide
which may be derived from a protein antigen l~sllollsible for the disease condition
30 being treated (or a peptide derived from an immunologically cross-reactive protein
antigen). While not intending to be limited to any theory, it is believed that
~-lmini~tration of a theld~;ulic composition of the invention may: a) cause T cell non
ponsiveness of appropliate T cell subpopulations such that they become
unresponsive to the offending antigen and do not participate in stimlll~ting an imm--n.o
35 l~,s~onse upon eA~osul~, to the offenllin~ protein antigen (i.e. via anergy or apoptosis);
b) modify the lymphokine secretion profile as cOlll~ ,d with eAl,o~ul~ to the naturally
occu~ing offending antigen (e.g. result in a decrease of ~ ~ and/or an increase in IL-
2); c) cause T cell subpopulations which normally participate in the response to the
w096/07428 ~ 8 ~ 9 PCr/USg4/10262
off~ntling antigen to be drawn away from the sites of normal eA~o~ule (e.g. nasal
m.lcQs~ skin and lung for allergy) towards the sites of ~rlminictration of the
co~ osition (this redistribution of T cell subpopulations may ameliorate or reduce the
ability of an individual's immnnlo system to stim~ t~ the usual immnn~ response at the
5 site of normal exposure to the offending antigen, res~lting in rliminl~tion in allergic
symptoms); or d) cause induction of T ~u~plc;ssor cells.
While not int~nrling to be limited to any theory, it is believed that down
regulation of an antigen specific immnn~ response is in~u~ed as a result of not
providing an a~ ,pliate costim~ tory signal som~tim~s referred to as a "second
10 signal" Briefly, it is believed that stimulation of T cells requires two types of signals,
the first is the recognition by the T cell via the T cell receptor of applupliate MHC-
associated processed antigens on antigen presenting cells (APCs) and the second type
of signal is referred to as a costim~ tory signal(s) or "second signal" which may be
provided by certain compet.ent APCs. U.~hcri a composi;ion of the invention is
15 ~Amini~tered without in non-immunogenic form (e.g. without adjuvant), it is believed
that competent APCs which are capable of producing the second signal or
costim~ tQry signal are not engaged in the ctim~ tion of applûpliate T cells therefore
resulting in T cell non responsiveness or reduced T cell responsiveness. In addition,
there are a number of antibodies or other reagents capable of blocking the delivery of
20 costimul~tory signals such as the "second signal" which include, but are not limited to
B7 (including B7-1, B7-2, and BB-1), CD28, CTLA4, CD40 CD40L CD54 and
CD 11all 8 (Jenkins and Johnson, Current Opinion in Immunolo~y. 5:361-367 (1993),
and Clark and T ~ , Nature, 367:425428 (1994)) Thus, in accordance with the
invention a colllposilion may be ~rlminictPred in no~ -ogenic form as liccucse
25 above, in conjunction with a reagent capable of blocking co~ tQry signals such
that the level of T cell non responsiveness is enhanced.
Compositions and m~tho~s of the invention are useful for ~ ing humans for
allergies to any number of protein allergens such as: a protein allergen of the genus
Dermatophagoides; a protein allergen of the genus Felis; a protein allergen of the
30 genus Ambrosia; a protein allergen of the genus Lolium; a protein allergen of the genus
C~yptomeria; a protein allergen of the genus Alternaria; a protein allergen of the genus
Alder; a protein allergen of the genus Betula; a protein allergen of the genus Quercus; a
protein allergen of the genus Olea; a protein allergen of the genus Artemisia; a protein
allergen of the genus Plantago; a protein allergen of the genus Parietaria; a protein
35 allergen of the genus Canine; a protein allergen of the genus Blatt~l~a: a protein
allergen of the genus Apis; a protein allergen of the genus Cupressus; a protein allergen
of the genus Juniperus; a protein allergen of the genus Thuya; a protein allergen of the
genus Chamaecyparis; a protein allergen of the genus Periplaneta; a protein allergen of
W096/07428 0 2 ~ ~ 8 8 6 9 PCT/US94tlO262
the genus Agr~lJy~J,.; a protein allergen of the genus Secale; a protein allergen of the
genus Triticum; a protein allergen of the genus Dactylis; a protein allergen of the genus
Festuca; a protein allergen of the genus Poa; a protein allergen of the genus Avena; a
protein allergen of the genus Holcus; a protein allergen of the genus Anthoxanthum; a
5 protein allergen of the genus Arrhenatherum; a protein allergen of the genus Agrostis; a
protein allergen of the genus Phleum; a protein allergen of the genus Phalaris; a protein
allergen of the genus Paspalum; and a protein allergen of the genus Sorghum.
Examples of various known protein allergens derived from some of the above-
identified genus include: Dermatophagoides (pteronyssinus orfarinae) Der p I; Der p
10 II; Der p I~; Der p VII; Derf I; Derf II; Derf III; Derf VII; Felis (domesticus) Fel d I;
Ambrosia (artemiisfolia) Amb a I.l; Amb a I.2; Amb a I.3; Amb a I.4; Amb a II; Lollium
(perenne) Lol p I; Lol p II; Lol p III; Lol p rv; Lol p IX (Lol p V or Lol p Ib);
Cryptomeria (japonica) Cry j I; Cry j II; Can f I; Can f II; Juniperus (sabinoides or
virginia.n~7) r~n s I; lun v I; Da~t~lis (glomerata) Dac g I; Poa (pretensis) Poa p I; nhl
15 p I; and Sorghum (halepensis) Sor h I.
Peptides having a defined sequence of amino acid residues comprising at least
one T cell epitope and which induce T cell non responsiveness or reduced T cell
responsiveness, have been identified and isolated for many of the above named protein
allergens and are useful in compositions and methods of the present invention. For
20 example, peptides comprising T cell epitopes derived from Der p I; Der p ~, Der f I;
and Derf II are disclosed in USSN 08/227,772 and USSN 07/963,381 incol~olalGd
herein by reference, and published in W093/08279. Peptides comprising T cell
epilo~es derived from Fel d I are disclosed in USSN 07/662,276, 07/884,718, and
08/006,116 all incol~lated herein by ~ef~ ce. Peptides comprising T cell epilo?es
25 derived from Amb a I.l; Amb a I.2; Amb a I.3; Amb a I.4; and Amb a II are disclosed in
USSN 07/866,679 and W093/21321 incol~olatGd herein by çGf~ ,nce. Peptides
comprising T cell epitopes derived from Lol p I, Dac g I, Poa p I, and Phl p I are
disclosed in USSN 08/031,001 and peptides compri~ing T cell epitopes derived from
Lol p IX (Lol p V or Lol p Ib) are disclosed in W094/04564, and 08/106,016
30 incorporated herein by reference. Peptides compri~ing T cell e~)ilu~Gs derived from Cry
j I and Cry j J~, and Jun s I and Jun v I are disclosed in USSN 08/226,248 incorporated
herein by ~f~,lcnce. Peptides co...~ g T cell epitopes derived from Can f I and Can
f II are disclosed in USSN 08/156,549 incol~oldted herein by rGÇ~,ç~ce. Peptidescomprising T cell epilupes derived from Sor h I are disclosed in AU93/00559.
A nulllb~,l of antigens (i.e. ~lto~nti~n~) have been found to cause disease
symptoms in ~ntoimmlln~ e~çs ( i.e. ~lto~ntigen~ such as insulin; myelin basic
protein; rh factor; acetylcholine l~ceptol~; thyroid cell recel>tol~; b~ ,ellll"~e
proteins; thyroid proteins; ICA-69 (PM-l); glutamic acid decarboxylase (64K or 65 K);
W096/07428 ~ 8 8 6 9 PCT/US94110~62
proteolipid protein (PLP), myelin associated glyco~r~ (MAG), Collagen (Type II),Heat Shock Protein and carboxypeptidase H) in autoimmnn~ ce~ces such as diabetes,
rh~ oid arthritis, and multiple sclerosis. For example, peptides which may
comprise T cell epitopes derived from myelin oligodendrocyte protein MOG, a protein
5 which is believed to be one of the ~llto~nti~ens involved in multiple sclerosis are
disclosed in USSN 08/116,824 incol~o~aled herein by reference. Peptides which are
believed to be able to down regulate the antigen specific response to MBP (myelin
basic protein), a protein believed to be an ~uto~ntigen in multiple sclerosis have been
identified in WO 93/21222, EP 0 304 279, WO 91/15225, Ota et al, Letters to Nature,
346:183-187 (1990), Wucherpfennig et al., J. E~. Med., 170:279-290 (1994). Peptides
which are believed to be able to down regulate the antigen specific response to soluble
Type II collagen, a protein antigen believed to be an ~llto~ntigen in rheumatoid arthritis,
have been identified in WO 94/07520. WO 92/06704 describes methods for identifying
peptides of insulin, which are believed to be effect~ve and the ~reatmcrlt and prevcntior
15 of Type I diabetes.
In addition, peptides having defined amino acid compositions and which
comprise T cell epitopes, may be identified for any protein antigen or ~ o~ntigen.
One method includes dividing the protein antigen into non-overlapping, or
ovellappillg peptides of desired lengths and synthesi7ing, purifying and testing those
20 peptides to determine whether the peptides comprise at least one T cell epitope using
any number of assays (i.e. T cell proliferation assays, lymphokine secretion assays,
and T cell non-responsiveness studies). In another method an algolillllll is used for
predicting those peptides which are likely to comprise T cell epitopes and then
synth~ci7ing, ~ulirying and testing the peptides predicted by the algolilhlll in T cell
25 assays to determine if such predicted peptides cause T cell proliferation or lymphokine
secretion, or T cell non-responsiveness and are therefore likely to contain T cell
epitopes. As ~iccllsse~ in many of the ~o.;u...~ c cited above, human T cell
ctimlll~ting activity can be tested by cnltllring T cells obtained from an individual
sensitive to a predetermined protein antigen (i.e. an allergen or an ~nto~ntigen) with a
30 peptide derived from the antigen and ~et~ ....i..il~g whether proliferation of T cells
occurs in response to the peptide as measured, e.g., by cellular uptake of triti~t~d
thymidine. Stimnl~tion indices for responses by T cells to peptides can be calculated
as the m~ximl~m counts per minute (CPM) in response to a peptide divided by the
control CPM. A T cell stimnl~tion index (S.I.) equal to or greater than two times the
35 background level is considered "posilive". Positive results are used to c~l~ul~te the
mean stimlll~tion index for each peptide for the group of peptides tested. ~,Çell.,d
peptides useful in accol.lallce with this invention comprise at least one T cell epitope
and preferably at least two or more T cell epitopes and have a mean T cell stimlll~tion
W096/07428 q) ~ 8 6 ~ PCT/US94/10262
index of greater than or equal to 2Ø A peptide having a T cell stimnlq-tion index of
greater than or equal to 2.0 is considered useful as a thel~peulic agent. E~cÇ~ ,d
peptides have a mean T cell stimnlqtion index of at least 2.5, more preferably at least
3.5, even more pl~r~ldbly at least 4.0, and most plcfeldbly at least 5Ø
S In addition, pl~f.,ll~,d peptides have a positivity index (P.I.) of at least about
100, more preferably at least 150, even more preferably at least about 200 and most
preferably at least about 250. The posilivily index for a peptide is determined by
multiplying the mean T cell stim~ tion index by the percent of individuals, in apopulation of individuals tested sensitive to the antigen being tested (e.g., plc;feldbly at
least 9 individuals, more preferably at least 16 individuals or more, more preferably at
least 20 individuals or more, or even more preferably at least 30 individuals or more),
who have T cells that respond to the peptide. Thus, the positivity index represents both
the strength of a T cell response to a peptide (S.I.) and the frequency of a T cell
response to a peptide in a popu!atior. of irdividu31s sensitive tc the, ntigen being tested.
One algorithm for predicting peptides having T cell stimulating activity which
has been used with success is reported in Rothbard, 1st Forum in Virology, Annals of
~he Pasteur Institute, pp 518-526 (December, 1986), Rothbard and Taylor, Embo, 7:93-
100 (1988), and EP 0 304 279. These do~;u...~nt~ report defining a general T cell
pattern (algofilllll.), its stq-ti~ticql significance and its correlation with known epitopes
20 as well as its successful use in predicting previously unidentified T cell epitopes of
various protein antigens and ~lltoqntigens. The general pattern for a peptide known to
bind Class II MHC well as reported in the above-mentioned documents appears to
contain a linear pattern composed of a charged amino acid residue or glycine followed
by two hydroi)hobic residues. After d~ ll uling if a peptide conforms to the general
25 pattern, the peptide can then be tested for T cell reactivity. Other algolil~lllls that have
been used to predict T cell epitopes of previously undefined proteins include analgorithm reported by Margalit et al., J. Immunol., 138:2213-2229 (1987), which is
based on an amphipathic helix model.
Additionally, peptides comprising "cryptic epit-~pes" may be det~rmined and
30 are also useful in accordance with the methods of this invention. Cryptic epitopes are
those det_l..-i--qnt~ in a protein antigen or protein ~qaltoqntigen which, due to proces~ing
and plGselltdtion of the native protein antigen to the appr~pliate MHC molecule, are
not normally revealed to the immnne system. However, a peptide comprising a cryptic
epitope is capable of c~ncing T cells to become non-responsive, and when a subject is
35 primed with the peptide, T cells obtained from the subject will proliferate in vitro in
response to the peptide or the protein antigen from which the peptide is derived.
Peptides which comprise at least one cryptic epitope derived from a protein antigen or
tQ~ntigen are referred to herein as "cryptic peptides". To confirm the presence of
W096/07428 d ~ 69 PCT/US94/10262
cryptic epilopes a T cell proliferation assay may be used as is known in the art and is
described above. In this assay, antigen-primed T cells are cultured in vitro in the
presence of each peptide sepal~tely to establish peptide-reactive T cell lines. A
peptide is considered to comprise at least one cryptic epitope if a T cell line can be
5 established with a given peptide and T cells are capable of proliferation uponchallenge with the peptide and the protein antigen from which the peptide is derived.
It is also possible to modify the structure of any of the above-described peptides
for use in accordance with the present invention for such purposes as increasingsolubility (particularly desirable if the composition is to be injected), enhancing
10 therapeutic or preventive efficacy, or stability (e.g., shelf life ex vivo, and resistance to
proteolytic degradation in vivo). A modified peptide can be produced in which the
amino acid sequence has been altered as compared to the native protein sequence from
which it is derived, or as compared to the peptide to be modified such as by amino acid
sl~bstitutdon, de!etion, or additicr., to modify immunogenicity, or to which a component
has been added for the same purpose. For example, WO 94/06828 describes substituted
peptides in which essentially every amino acid residue may be substituted with aconservative amino acid, an amino acid not found in nature, or alanine, and yet the
~ub~ u~d peptide is still capable of down regulating an antigen specific immnn~
response In addition, many of the documents cited above disclose various
20 modifications to peptides with defined amino acid compositions which have been
shown to induce T cell non-responsiveness.
In addition, it is not nece~ry that a peptide used in accordance with the methodof this invention be derived from a known antgenic protein. Any peptide comprising a
defined sequence of amino acid residues or capable of down-regulating an antigen25 specific imml~nP response to an antigen or ~u~o~ntigen may be used in accordance with
the method of the present invention. For example, peptides may be synth~-si7P~
comprising a defined amino acid sequence not based on a known protein antigen
sequence, and yet are capable of down regulating an antigen specific immnnP response
e.g. the peptide mimics a T cell epitope of the protein antigen and causes down
30 regulation of the imm--nP response to that protein antigen, or causes down regulation of
the immnn~ response for another reason, such as it is derived from a bystander antigen.
Without being limited to any theory, it is believed that bystander antigens, which are
also tissue specific (but are not the target of immnn.o or autoi.. ~ .. o attack) possess the
ability to elicit ~u~ ,ssor T cells at the site of i~ ----r. attack which may in turn result
in down regulating the i.. ~.e responses in the locality of the immnn~ attack (e.g.
afflicted "self" tissue in the case of autoi,.. ~ disease or nasal mucosa, skin and lung
in the case of allergy). Bystander antigens include but are not limited to portions of the
w096/07428 0 2 ~ ~ 8 8 6 9 ~ 54/10262
antigen which are not th.c...~elvG5 the target of immnnr attack, and which possess
~up~ ssive activity at the site of immnn~ attack.
In addition, any compound that mimics a peptide capable of down regulating an
antigen specific immlm~ 1c~onse to an antigen or ~u~o~tigen may be used in
5 accordance with the invention. Such a co111pou1ld may not be composed entirely of
subunits joined by peptide bonds, but joined by other link~s (e.g. thiolester bonds),
providing that the non-peptide compound mimics a peptide capable of down regulating
an antigen specific immnne response to the antigen of interest as indicated by effective
therapeutic/prophylactic llc~ ent of sy111~lo111s.
Additionally, peptide compositions ~llmini~tered in accordance with the
invention preferably comprise a sufficient percentage of the T cell epitopes of the
offending protein antigen (i.e. at least about 10% and more preferably about 20%, more
preferably about 30%, more preferably about 40%, and even more preferably about
50% or greater, of the T cell reactivity to an offendil;g ....tige;l of intercst) are included
15 in the composition such that a thc1~pculic regimen of ~rlrnini~tration of the composition
to an individual sensitive to a particular protein antigen in accordance with the
invention, results in T cells of the individual being rendered nom~spo1lsive to the
protein ~ntigen To determine whether a peptide (candidate peptide) or a combination
of candidate peptides are likely to contain a sufficient percentage of T cell epitopes of
20 the protein antigen of interest to induce T cell nonresponsiveness in a ~ub~llial
pe,ce1lt~ge of a population of individuals sensitive to the protein antigen, an algorithm
can be used. In accordallce with one such algo1iltll11, a human T cell stimnl~tion index
(rli~cu~secl above) for the peptide(s) in an in vitro T cell proliferation assay is calculated
for each individual tested in a population of individuals sensitive to the protein antigen
25 of interest. The 1,,...~ini..g peptides in the in vitro T cell proliferation assay are
overlapping peptides (overlapping by bGIween about 5 - 15 amino acid residues) which
cover the remainder of the protein not covered by the candidate peptide(s), which
~e~ ;ning peptides are at least about 12 amino acids long and which are preferably no
longer than 30 and more preferably no longer than 25 amino acid residues in length. A
30 human T cell ~tim~ tion index for each such ,~;,..~;..i..g peptide in the set of peptides
produced in the in vitro T-cell proliferation assay with T-cells obtained from each
individual in the population of individuals tested is calculated and added together. For
each individual, the human T cell stim~ tion index for the candidate peptide(s) is
divided by the sum of the human T cell stimnl~tion indices of the ,~ peptides in
the set of peptides tested to d~ t.. ;~-P- a percent. This percent is obtained for at least
twenty (20) and p1Gfe~ably at least thirty (30) individuals sensilive to the protein
antigen of interest and a mean percent is d~t. . ~ r~ A mean percent of about lO%,
preferably about 20%, preferably about 30% and even more preferably about 50% or
W096/07428 ~ 2 ~ ~ ~ 8 6 9 PCT/US94110262
greater for the c~n~liA~te peptide(s) together with a percent positive (defined as the
p~centage of posilive T cell l~sponses (S.I.s of greater than or equal to 2.0) in response
to the cAnAid~t~ peptide or coll~bindtion of cAndiA~te peptides) of at least about 60%,
preferably about 75% and more preferably about 90% indicates that the cAnAiA~te
S peptide(s) sele-cte~l is likely to contain a sufficient pel~;cllldge of T cell epitopes to
induce T cell non responsi~ .ess in a ~u~s~llial l,cl-;cllldge of a population of
individuals sensitive to the protein antigen of interest.
For the l1G~ of allergy in accordance with the methods of the invention, it
is preferred that a peptide used in conjunction therewith does not bind immunoglobulin
10 E (IgE) or binds IgE to a substantially lesser extent (i.e. at least 100-fold less binding
and more preferably, at least 1,000-fold less binding) than the protein allergen from
which the peptide is derived binds IgE. The major complications of standard
immunotherapy are IgE-m-oAiAte~ responses such as anaphylaxis. Immunoglobulin E is
a mediator of anaphylaetic reqctions .:hich result frorn thc bir.ding and cross-linking of
15 antigen to IgE on mast cells or basophils and the release of m~AiAtQrs (e.g., histamine,
serotonin, eosinophil chemotacic factors) in allergic ("atopic") patients. Thus,anaphylaxis in a substantial percentage of a population of individuals sensitive to the
allergen being treated could be avoided by the use in immunotherapy of a peptide or
peptides which do not bind IgE in a ~ul~ tial per~nldge (e.g., at least about 75%) of
20 a population of individuals sensitive to a given allergen, or if the peptide binds IgE,
such binding does not result in the release of m~Aiqtors from mast cells or basophils.
The risk of anaphylaxis could be reduced by the use in imrnunotherapy of a peptide or
peptides which have reduced IgE binding. Moreover, peptides which have minimql IgE
stimnl~ting activity are desirable for th~,ld~cu~ic effectiveness. Minimal IgE
25 stim~lqting activity refers to IgE production that is less than the amount of IgE
production and/or IL~ production stimnl~ted by the native protein allergen (e.g., Der p
I). If a peptide binds IgE, it is preferable that such binding does not result in the release
of m~Aiqtors (e.g. hi.ct~nnines) from mast cells or basophils. To detel.~ e whether a
peptide which binds IgE results in the release of m~Aiqtors, a hict~min~ release assay
30 can be pclÇolllled using standard reagents and protocols obtained for example, from
Amac, Inc. (Westbrook, ME). Briefly, a buffered solution of a peptide to be tested is
combined with an equal volume of whole h~p~ i7eA. blood from an allergic subject.
After mixing and incubation, the cells are pelleted and the supçrn~tqntc are processed
and analyzed using a radio immunoassay to determine the amount of hict~rnine
35 rele~ced
Highly purified peptides free from all other polypeptides and co~
having a defined sequence of amino acid residues compricing at least one T cell
epitope, used in lllclal~culic colll~osilions of this invention, may be produced
W 0 96/07428 ~ 8 6 9 PC~r~US94/10262
synth~oti~lly by chemical ~yl~tl~e~is using standard techniques. Various methods of
chemically synth~si7in~ peptides are known in the art such as solid phase ~ylllllesis
which has been fully or semi a~lOIlla~d on col""l~ ;ially available peptide
~yl~t~ 7çrs. Synth~ti~lly produced peptides may then be purified to homogeneity
5 (i.e. at least 90%, more prGf~bly at least 95% and even more prefelably at least 97%
purity), free from all other polypeptides and co,-~...in~-t~ using any number oftechniques known in the li~lalulG for protein purification.
In accordance with one procedure for producing highly purified homogenous
peptide compositions, a peptide produced by synthetic chemical means (either anchored
10 to a polymer support "solid phase synthesis" or by conventional homogenous chemical
reactions "solution synthesis") may be purified by preparative reverse phase
chromatography. In this method, the synthetically produced peptide in "crude" form is
dissolved in an app~o~,iate solvent (typically an aqueous buffer) and applied to a
sepæration co!wrn (t.ypically a reverse phase silica based media, in addition, polymer or
15 carbon based media may be used). Peptide is eluted from the column by increasing the
concentration of an organic component (typically acetonitrile or methanol) in anaqueous buffer (typically TFA, triethylamine phosphate, acetate or similar buffer).
Fractions of the eluate will be collected and analyzed by appropliate analytical methods
(typically reverse phase HPLC or CZE chrolll~tography). Those fractions having the
20 required homogeneity will be pooled. The counter ion present may be changed by
additional reverse phase chromatography in the salt of choice or by ion exchangeresins. The peptide may then be isolated as its acetate or other applo~ e salt. The
peptide is then filtered and the water removed (typically by lyophilization) to give a
homogenous peptide composition CO~ g at least 90%, more preferably at least 95%
25 and even more pl~,feldbly at least 97% of the required peptide colllponent. Optionally,
or in conjull.;lion with reverse phase HPLC as described above, purification may be
accomplished by affinity ch,ul"~ography, ion exchange, size exclusion, counter
current or normal phase separation sy~ms, or any combination of these-methods.
Peptide may additionally be concc;,,lldlt;d using ultra filtration, rotary evaporation,
30 precipitation, dialysis or other similar techniques.
The highly purified homogenous peptide composition is then characteri7çd by
any of the following techniques or combinations thereof: a) mass ~I,e~ oscopy todt;~t;lllfine molec~ r weight to check peptide identity; b) amino acid analysis to check
the identity of the peptide via amino acid composition; c) amino acid sequencing (using
35 an ~ulo~l~at~d protein sequencer or manually) to cOllr~ the defined sequence of amino
acid residues; d) HPLC (multiple s~t~ s if desired) used to check peptide identity and
purity (i.e. identifiPs peptide illl~uliliesj; e) water content to ~eterminç the water
concentration of the peptide compositions; f) ion content to d~lt;lllline the presence of
14
w096/07428 ~ 8 8 6 9 Pcr/US94~l0262
salts in the peptide co~ o~ilion; and g) residual organics to check for the p~se.lce of
residual organic rea~nt~ starting m~t~.ri~l~, and/or organic Co,~
Synthetically produced peptides of the invention comprising up to
approxim~t~ly forty-five amino acid residues in length, and most preferably up to
5 appro~ ely thirty amino acid residues in length are particularly desirable as
inclcases in length may result in difficulty in peptide synth~si~ Peptides of longer
length may be produced by lccolllbinalll DNA techniques as discussed below.
Peptides useful in the methods of the present invention may also be produced
using recombinant DNA techniques in a host cell transformed with a nucleic acid
l 0 sequence coding for such peptide. When produced by recombinant techniques, host
cells transformed with nucleic acid encoding the desired peptide are cultured in a
medium suitable for the cells and isolated peptides can be purified from cell culture
medium, host cells, or both using techniques known in the art for purifying peptides
ænd proteins including ion-exchange chromatography, ult;a filtrat,on, elcct;Gphoresis or
15 immunopurification with antibodies specific for the desired peptide. Peptides produced
recombinantly may be isolated and purified to homogeneity, free of cellular material,
other polypeptides or culture medium for use in accordallce with the methods
described above for synthetically produced peptides.
In certain limited chcull~lces, peptides may also be produced by chemical or
20 enzymatic cleavage of a highly purified full length or native protein of which the sites
of chemical digest or enzymatic cleavage have been predetermined and the resulting
digest is reproducible. Peptides having defined amino acid sequences can be highly
purified and isolated free of any other poly peptides or co..1;t...i..~nt~ present in the
enzymatic or chemical digest by any of the procedures described above for highlypurified, and isolated synth~tic~lly or l~,colllbinantly produced peptides.
Highly purified and isolated peptides produced as discussed above may be
formnl~te~ into therapeutic compositions of the invention suitable for human therapy.
If a tlle~ ic composition of the invention is to be ~Amini~tered by injection (i.e.
subcutaneous injection), then it is preferable that the highly purified peptide be soluble
in an aqueous solution at a ph~rrn~cel1~ic~11y acceptable pH (i.e. pH range of about 4-9)
such that the composition is fluid and easy syringability exists. The composition also
plcft;lably includes a pharmaceutically acceptable carrier. As used herein
"ph~rm~.-eutir~11y acceptable carrier" includes any and all excipients, solvents,
per~ion media, co~tingS~ ~ntih~cteri~1 and antifungal agents, toxicity agents,
b.lrf~,,L[~g agents, absorption delaying or enh~ncing agents, s~ t~nt~ and miclle
forming agents, lipids, lipoSollRS, and liquid complex forming agents, stabilizing
agents, and the like. The use of such media and agents for ph~....~ceutically active
substance is known in the art. Except insofar as any conventional media or agent is
W096/07428 ~ 9 8 8 6 ~ PCI/US94110262
hlcGIllpdtible with the active compound, use thereof in the th~dpeulic compositions is
contemplated. Supplelllellt~y active compounds can also be incol~oldted into thecompositions.
The~dpeulic compositions of the invention suitable for injectable use are
5 preferably sterile aqueous solutions prepared by inc~ u~ting active compound (i.e.,
one or more highly purified and isolated peptide as described above) in the required
amount in an a~pl~Jplidt~ vehicle with one or a cGIl.billation of ingredients enullleldled
above and below, as required, followed by filtered sterilization. Preferred
pharm~ceutically acceptable carriers include at least one excipient such as sterile water,
10 sodium phosphate, mannitol, sorbitol, or sodium cloride or any combination thereof.
Other pharmareutically acceptable carriers which may be suitable include solvents or
dispersion medium containing, for example, water, ethanol, polyol (for example
glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The p;opcr fiuidity can be m,.intained for example
15 by the use of coating such as lecithin, by the Ill~nt~,.lance of the required particle size in
the case of dispersions and by the use of surfactants. Prevention of the action of
microorg~nicmc can be achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid, Ih. Ill~r~ sol and the like.
Prolonged absorption of the injectable compositions can be brought about by including
20 in the composition, an agent which delays absorption, for example, aluminum
monostearate and gelatin.
A th~l~eulic composition of the invention should be sterile, stable under
conditions of manufacture, storage, distribution and use and should be preservedagainst the cont~ ;.-g action of microorg~ni~lllc such as bacteria and fungi. A
25 plc~llc;d means for man--factnring a Ill~,.~)eUliC compositions of the invention in order
to ~ai~ in the integrity of the composition (i.e. prevent con~..in~;on, prolong
storage, etc.) is to prepare the formlllation of peptide and ph~rm~rentically accelJIdble
carrier(s) such that the composition may be in the form of a lyophili7~d powder which
is recon~itutecl in a pharmaceutically acceptable carrier, such as sterile water, just prior
to use. In the case of sterile powders for the prep~dtion of sterile injectable solutions,
the preferred methods of pl.,pa~dlion are vacuum drying, freeze-drying or spin drying
which yields a powder of the active ingredient plus any additional desired ingredient
from a previously sterile-filtered solution thereof. Specific formulations of therapeutic
compositions of the invention are described below and in the Examples.
In many cases, a ~ ~.lliC composition of the invention co,llplises more than
one isolated peptide. A ~hPI~ ;C coml,o~ilion colll~lisillg a multipeptide formulation
suitable for pharrnar,eutical ~rlmini~tration to hnman.~ may be desirable for
~Iminictration of several active peptides. The multipeptide f~rrnlllation includes at
16
wo 96,07428 ~ 6 9 PCT/US94/10262
least two or more isolated pepti~es having a defined amino acid sequence and is
capable of down regulating an antigen specific i~ .c response. Special
considerations when p~palulg a multipeptide form~ tion include IllA;ll~ ;llg thesolubility, and stability of all peptides in the formnlAtiQn in an aqueous solution at a
S physiologically ~çc~pl~hle pH. This l~uks choosing one or more pharm~e~ltically
acceptable solvents and excipients which are coln~ible with all the peptides in the
multipeptide formulation. For example, suitable excipients include sterile water,
sodium phosphate, m~nnitol or both sodium phosphate and m~nnitol. An additional
consideration in a multipeptide formulation is the prevention of dimerization of the
10 peptides if necessary. Agents may be included in the multipeptide formulation which
prevent dimerization such as EDTA or any other material or procedures known in the
art to prevent dimerization. The following is an example of the multipeptide
formulation used in a Phase II human clinical trial as a therapeutic for the treatment of
allergies to cat. Two active peptides, peptide X (SEQ ID NO 1) and peptide Y (SEQ ID
15 NO 2), each having a defined composition of amino acid residues, and derived from the
protein allergen Fel d I, were synthesiæd via chemical synthesis and purified tohomogeneity as described above for use in a multipeptide formulation for treating
hllm~nc allergic to cats in accordance with the present invention. For this embodiment,
Peptide X (SEQ ID NO 1 ) and Peptide Y (SEQ ID NO 2) were in the form of a
20 lyophilized powder which was reconstituted in sterile water, prior to use. Peptide X
(SEQ ID NO 1) and Peptide Y (SEQ ID NO 2) were combined during mannfA~ ring
using known techniques to produce a vial col.lAi~ g a sterile, pyrogen free, lyophilized
powder havin~ the following composition:
Active: 0.75 mg peptide X (SEQ ID NO 1 ) and 0.75 mg peptide Y (SEQ
ID NO 4)
Inactives: 0.05 M Sodium Pl,osph~le, pH 6.2
5% w/v Mannitol, U.S.P.
Diluent: Sterile Water for Injection, U.S.P. (initial reconstitution)
0.9% Sodium Chloride for Injection
(dilution beyond initial recor~ )l ion)
~lc?alalion of this multipeptide formnl~tion requires reconstitution of the vials with
sterile water for injection. In another embodiment, the multipeptide formulation may
further include three (see Example 2), four, five or more additional peptides suitable for
human therapy.
A-lmini~tration of the th~la~ulic compositions as described above to an
individual, in a non-immunogenic form, can be carried out using known procedures at
dosages and for periods of time ef~cclivc to cause down regulation of the antigen
specific immnn~ response (i.e., reduce the disease ~ylllplollls of antigen specific
W096/07428 0 ;2 li ~ ~ 8 6 ~ PCT/US94/10262
".~.~r ~ onse caused by the off~n~ling antigen) of the individual. Down regulation
of an antigen specific i " ""~ response to an antigen assoc,d~ed with a disease
condition in hllm~nc may be dclclllfilled clinically whenever possible depending the
disease condition being treated, or may be clete~minPd subjectively (i.e. the patient feels
as if some or all of the ~y~ lol~s related to the disease condition being treated have
been alleviated).
Effective amounts of the lhcldpculic co"~posilions of the invention will vary
according to factors such as the degree of sensitivity of the individual to the antigen, the
age, sex, and weight of the individual, and the ability of peptide to cause downregulation of the antigen specific immune response in the individual. A therapeutic
composition of the invention may be ~lminict~red in non-immunogenic form, in a
convenient manner such as by injection (subcutaneous, intravenous, etc.), oral
~riminictration, subligual, inhalation, transdermal application, rectal ~dminictration. or
~Qy other route of ~lminictration known in the art for :l~m.ini~tering thcrapcutic agents
It may be desirable to ~dminicter simultaneously or sequentially a therapeutically
effective amount of one or more of the therapeutic compositions of the invention to an
individual. Each of such compositions for ~Aminictration simultaneously or
sequentially, may comprise only one peptide or may comprise a multipeptide
formulation as described above.
For subcutaneous injection of one or more therapeutic compositions of the
invention, preferably about 1 ~lg- 3 mg and more p~cfcldbly from about 20~g-1.5 mg,
and even more preferably about 50 ~g- 750 ~lg of each active component (peptide) per
dosage unit may be ~minict~red. It is especially advantageous to formnl~te p~u~ dl
compositions in unit dosage form for ease of ~minictration and uniformity of dosage.
Unit dosage form as used herein refers to physically discrete units suited as unitary
dosages for human subjects to be treated; each unit co..l~h~ g a predctcl,l~ined quantity
of active compound calculated to produce the desired theldpculic effect in association
with the desired ph~rm~ tic~l carrier. The specification for the novel unit dosage
forms of the invention are dictated by and directly dependent on (a) the unique
30 characteristics of the active compound and the particular theld~ lic effect to be
achieved, and (b) the limitations inherent in the art of co",pou"ding such an active
col"poulld for the tlC;;~'IIr.nl of human subjects.
Dosage regimen may be adjusted to provide the O~)lilllUIII ~ 1dPCUIiC response.
For eY~mrl~, several divided doses may be ~rlmini~ored over the course of days,
35 weeks, months or years, or the dose may be proportionally increased or reduced with
each ~ul se-luent injection as in~ at~o-d by the exigencies of the thela?culic situation. In
one p,ef~,l,cd th.,l~peL~lic rcgim~n, ~ullcu~neous injections of th~ peulic col"posilions
are given once a week for 3-6 weeks. The dosage may remain constant for each
WO 96/07428 PCT/US94110262
8 8 6 9
injection or may increase or decrease with each subsequent injection. A booster
injection may be Arlmini~tered at intervals of about three months to about one year after
initial LI~AI ".. ht and may involve only a single injection or may involve another series
of injections similar to that of the initial treatment.
S To ~rimini~tçr a composition of the invention by other than ~ ,nt~
~mini.~trationt (i.e. oral ~mini~tration) it may be necessAry to coat the composition
with, or co-~lmini~ter the composition with, a material to prevent its inactivation or
enhance its absorption and bioavailability. For example, a peptide formulation may be
co-~flmini~tered with enzyme inhibitors or in liposomes. Enzyme inhibitors include
pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
Liposomes include water-in-oil-in-water CGF emulsions as well as conventional
liposomes (Strejan et al., (1984) J. Neuroimmunol.. 7:27). When a peptide is suitably
protected, the peptide may be orally ~mini~tered, for example, with an inert diluent or
an assimilable edible carrier. Th.e peptide .a.-ld other ir.~,led~ents rr.ay also be enclosed in
a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly
into the individual's diet. For oral therapeutic ~rlminictration, the active compound may
be incorporated with excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, solutions, gels, suspensions, syrups, wafers, and the
like. Such compositions and preparations should contain at least 1% by weight ofactive compound. The percentage of the composition and pl~p~tions may, of course,
be varied and may conveniently be between about 5 to 80% of the weight of the unit.
The amount of active compound in such therapeutically useful compositions is such
that a suitable dosage will be obtained. In addition, the active compound may beincorporated into su~Ained-release or controlled release (steady state or pulsatile
release) plep~lions and formulations.
This invention is illustrated by the following non-limiting examples.
EXAMPLE 1: Administration of Peptides to ~llm~n Subjects for
Treatment of Allerw to Cat
A. Composition of Phase I Formulation
For Phase I clinical trials the drug product was comprised of two freeze-dried
peptide formulations, peptide X (SEQ ID NO 1) and peptide Y (SEQ ID NO 2).
35 Peptide X (SEQ ID NO 1) and peptide Y (SEQ ID NO 2) were manufactured
separately, and packaged into two distinct pyrogen free vials.
19
w096/07428 0 ;! ~ ~ 8 ~ 6 ~ PCT~sg4/l0262
1 ~l ll~E X (SEQ ID NO 1)
Active: 1.5 mg/vial
Inacti~es: 0.1 M Sodium Phosphate, pH 5.7
5% w/v Mannitol, U.S.P.
Diluent: Sterile Water for Injection, U.S.P.
'l ll)E Y (SEQ ID NO 2)
Active: 1.5 mg/vial
Inactives: 0.1 M Sodium Phosphate, pH 5.7
5% w/v Mannitol, U.S.P.
Diluent: Sterile Water for Injection, U.S.P.
In tllis study, the ~ug product was supplied in two separate vials as sterile,
pyrogen free, lyophilized powders cont~ining sodium phosphate and mannitol as
excipients, and the active component peptide X (SEQ ID NO 1 ) or peptide Y (SEQ ID
NO 2).
Preparation of the drug product in these studies required reconstitution of the
vials with sterile water for injection. Dilution of reconstituted vials was required to
deliver lower doses in the dose titration concentrations of 7.5 or 75~1g/mL, and also
accomplished using sterile 0.9% sodium chloride for injection. If desired, the vials
could have been plep~ed with only 7.5 or 75 ~lg per peptide without need for dilutions.
The lyophilized drug product was stored at or below -20C and the drug product
was admini~tPred imm~Ai~tely after recon!~l ;l ul ion. These storage conditions and use
conditions are not limiting.
B. Composition of Phase II Formulation
For Phase II clinical trials a single formulation cor.l~;..ing 750 ~lg/ml final
concentration of each peptide was developed. Peptide X (SEQ ID NO 1 ) and peptide Y
30 (SEQ ID NO 2) were combined during the fill finish process to produce a vial
co~ ;ning a sterile, pyrogen free, lyophilized powder.
W0 96/0~428 ~ PCT/US94tlO262
Active: 0.75 mg peptide X (SEQ ID NO 1) and 0.75 mg peptide Y (SEQ
ID NO 4)
Inactives: 0.05 M Sodium Phosphate, pH 6.2
5% w/v Mannitol, U.S.P.
Diluent: Sterile Water for Injection, U.S.P. (initial reconstitution)
0.9% Sodium Chloride for Injection
(dilution beyond initial reconstitution)
The freeæ-dried drug product was recon~ uled to 750 ~g/ml concentration
10 with sterile water for injection. Dilution of recon~titut~.d vials for lower doses, e.g., to
achieve concentrations of 7.5 or 75~g/mL, was accomplished using sterile 0.9% sodium
chloride for injection. Subjects were ~Amini~tered the drug product according to the
a~lmini~tration instructions contained in the clinical protocol.
The drug product was ~lmini~t~red immediately after recor.stitutiorl.
The lyophilized drug product was stored at or below -20C, however, this is not
limiting.
D. Hurnan Phase I and II Clinical Studies
The following Phase I and Phase II studies of peptides X and Y (SEQ ID NOS:
20 1 and 2) have been con-lucted in human cat-allergic subjects.
Protocol P92-01: Phase I Clinical Study of Safety and Activity of Peptides X and Y
This Phase I, open-label, dose-esc~l~ting safety study was conducted to
dct~line the safety of the ~tlmini~tration of the peptide X (SEQ ID NO 1) and peptide
25 Y (SEQ ID NO 2). The antigenicity of the co""lol-Gnt~ was also evaluated, as was the
activity of the peptides in altering skin test sensitivity.
Two centers, Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland
and New F.ngl~n-l Medical Center, Boston, Massachusetts, enrolled a total of nineteen
(19) cat-allergic individuals in this study. Peptide X (SEQ ID NO 1) and peptide Y
30 (SEQ ID NO 2) were ~tlmini~tered separately but conco",i~,lly by subcutaneousinjections in esc~l~ting doses ranging from 7.5 ~lg to 1500 ~lg over a five-week period.
Safety was ~sesse-1 by evaluations of clinical laboratory p~lllètel~, physical
eY~n~in~tion, antibody studies and adverse e~,icnces. Activity was ~ssesse~l by
analysis of skin testing. Prick and intr~.rm~l skin testing with both affinity purified
35 native Fel d I and the peptides were performed one week prior to, and two and six
weeks following the tre~tm~nt period. Sixteen patients were treated with peptides X
and Y during the study. The peptides were well tolerated at doses of up to 1500 llg.
There were no serious adverse experiences and the safety of the peptides to justify
wog6/07428 ~ ~ ~ Q ~ 8 6 Q PCI~/US94/10262
further clinical development was col.r.. ~1 Adverse events recorded with the trial
were typical of those seen in open label allergy studies. Patients were all cat allergic
individuals in this trial and tolerated dose orders of magnitude higher than can be
achieved with conventional immunotherapy.
One patient discontinued the study due to an asthma attack which occurred
following skin testing with cat extract I and peptides on Week 1, and a second asthma
attack which occurred following the 7.5 llg dose of each peptide on Week 2. It is
unclear whether there is a direct relationship between treatment with the peptides and
the onset of asthma in this patient. It is noteworthy that the study was conducted during
one of the most severe pollen seasons of recent years and this patient, like many of
those enrolled, had seasonal allergies.
Antibody studies revealed that all evaluable patients had IgG and IgE antibodiesto Fel d I. In all but two patients, the concentrations did not change over the course of
the study. Two patients demonstr~ted IgE antibodies to peptide X (SEQ ID NO 1 )
and/or peptide Y (SEQ ID NO 2) at Weeks 8 and 12 which were not present at Week 1.
One of these patients had a substantial skin test reaction to peptide X (SEQ ID NO 1 ) at
Week 8 which was not present at Week 12. Five patients had endogenous IgG antibody
to peptide X (SEQ ID NO 1) and three patients had IgG antibody to peptide Y (SEQ ID
NO 2) prior to exposure to the peptides. Four patients with no pre-existing, anti-
20 peptide IgG antibodies to peptide X (SEQ ID NO 1) and/or peptide Y (SEQ ID NO 2)
developed increased concentrations of such antibodies during or after Llc~ Rnt. Two
patients developed a positive imm~ te skin test reaction to a treatment peptide during
the study but without clinical correlation. Another patient had a delayed local reaction
to skin testing with peptide Y (SEQ ID NO 2) at Week 8 which was related to the
25 intraderrnal dose of peptide Y (SEQ ID NO 2).
Skin test results to cat allergen showed a statistically-significant decrease inreactivity to cat extract col,t;.;.~i.,g Fel d I at two or three dilutions by prick test after 8
or 12 weeks, respectively. No statistically significant differences in reactivity were
detected by the intradermal method. There was a statistically-~ignific~nt decrease in
30 the late phase reactivity to Fel d I (~;E) at six and twenty-four hours at the Johns
Hopkins site when Week 8 was coll~pa~d to b~c~.linP (Week 1). The decrease was not
significant when Week 12 was col"paled to baseline.
Although this study was decignt.d to evaluate safety, the skin testing data
suggested that the peptides may be causing desenciti7~tion to Fel d I.
W O 96/07428 PC~rrUS94/10262
~ 2~ 98 869
Protocol P92-02: Phase II Clinir-~ Study of the Safety and Activity of Peptides X and
Y Usin~ a Cat Room Challen~e Model
This phase II safety and efficacy study was a double blind placebo-controlled
study of peptides X and Y given ~ub~;u~leously in four weekly doses of 7.5 ,ug, 75 ~g,
S or 750 llg per peptide. Patient's sensilivily to natural eApo~ule was ~csessecl by
mea~ur~ of symptom scores and pulmonary function during a 60-minute period in
a small room co~ g stuffed rulllilulc and two cats. This cat room challenge
occurred pleh~ ..t and one and six weeks po~Ulcatlllclll. In the cat room, patients
rated nose, eye and lung symptoms on a five point scale every five minutes. Pulmonary
10 function was tested every 15 minutes. Additional parameters ~sessed during the study
were skin test sensitivity to cat extract and peptides X and Y, specific IgE and IgG to
Fel d I and component peptides X and Y, and T cell responsiveness to cat antigen and
selected peptides.
Ninety-five patient~ were errolled at two cen~Rrs, Johns Hopkins Asthma and
15 Allergy Center, Baltimore, Maryland and New England Medical Center, Boston,
Massachusetts. Ninety-one patients completed the trial. Four patients discontinued,
two because of transient allergic symptoms associated with the therapy and two
because of sched-lling conflicts.
The therapy was generally well tolerated. Analysis of the primary efficacy data
20 for the study revealed a significant dose response relationship which was considered
ct~tictic~lly significant (Fig. 1) for control of allergic symptoms (nasal, lung, and total
allergy) in~uce~ by cat room exposure at one and six weeks po~ul.,~ t Statistically
significant pairwise coll.palisons versus placebo for nasal and total allergy symptoms at
75 ~lg and 750 ~lg was ~etPcte~ at six weeks (See Fig. 1). The 7.5 ~lg dose could not be
25 distinguished from placebo. Patients were prick test negative to study medication
pncLIcdlll~ but 13 p~tiPntc had positive prick and/or intradermal skin test to llc,.~ t
peptides. Two patients developed significant titres of IgE to hv.~ t peptide andwere skin test positive to study m~-lic~tion post-treatment. Only five patients with
positive skin tests to peptide had measurable IgE to peptide. A l~lupollion of patients
(placebo: 57.7%; 7.5 ~g: 60.9%; 75 ~lg: 63.6%; 750 llg: 83.3%) reported cat-allergic
Jllls such as rhinorrhea, nasal congestion, pllllilus, chest tightn.oss, and/or
wh~ing during this study. The study medication appealcd to induce some mild
transient ~yllll~lollls sugge~live of natural cat exposure. The incidence was dose-related
(placebo: 11.5-19.2%; 7.5 ~lg: 21.7-26.1%; 75 ,ug: 31.8-45.5%; and 750 llg: 50.0-
62.5%). Such symptoms were generally mild, self limited and required no h~dllllcllt or
were controlled with beta agonist and/or ~ntihi~t~min~. One patient was treated with
adrenaline, and six patients had ~ ic ~ylllptollls with drop in peak flow. All were
managed easily. The tre~tm~nt did not alter antibody responses to call allergies in the
w0 96/07428 ~ 8 ~ 6 9 PcTlus94llo262
time period s~ A did not effect skin teat l~,aclivily relative to placebo, but was
associated with h"l"u~e"~cnt of total allergy score in 80% of study patients. Additional
studies to better chaMcten7e the LIG~ effect are underway.
5 Example 2 Administration of Peptides to Humans for
Treatment of Aller~y to ~ ed
A. Phase I Formulation
For Phase I clinical trials, the drug product was a multipeptide formulation
comprising three freeze-dried peptides of Amb a I, the sequence of amino acid residues
of each comprising at least one T cell epitope of the ragweed protein allergen Amb a
I.1 (see, W093/21321, incorporated herein by reference). Each peptide was purified to
homogeneity (at least 97% pure) in accordance with the methods desc.,bed zbovc. The
15 multipeptide formulation was prepared in accordance with procedures described herein.
The multipeptide formulation used in this Phase I clinical Study was in the forrn of a
freeze-dried powder cake of each the of peptides in a single vial. The formulation was
reconstituted just prior to use with sterile water for injection and normal saline (0.9%)
was used for any dilutions beyond the initial reconstitution.
B. H~ n Phase I Clinical Studies
Preliminary data infli~ated that all doses were tolerated and the multipeptide
formulation appears to be safe. Fur~er analysis and Phase II Clinical Studies are
25 pending in the United States and C~
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than routine el-~filllc;lll~lion, numerous equivalents to the specific procedures
30 described herein. Such equivalents are considered to be within the scope of this
invention and are covered by the following claims.
24
wog6/07428 ~ 2 ~ ~ 8 8 6 9 Pcr/US94,l0262
S~QU~N~: LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: IMMULOGIC PHARMACEUTICAL CORPORATION
(ii) TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR
ADMINISTERING TO HUMANS, PEPTIDES CAPABLE OF DOWN REGULATING AN
ANTIGEN SP~l~lC IMMUNE RESPONSE
(iii) NUMBER OF ~u~ S: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: IMMULOGIC PHARMACEUTICAL CORPORATION
(B) STREET: 610 LINCOLN STREET
(C) CITY: WALTHAM
(D~ STATE: Massachusetts
( E) COUNTRY: USA
(F) ZIP: 02154
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatib1e
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII text
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) AllORN~Y/AGENT INFORMATION:
(A) NAME: DARLENE A. VANSTONE
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 092.0US
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 466-6000
(B) TELEFAX: (617) 466-6040
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Lys Arg Asp Val Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val
1 s lo 15
Glu Gln Val Ala Gln Tyr Lys Ala Leu Pro Val
W 096/07428 0 ~ 8 6 9 PCTnUS94110262
(2) INFORMATION FOR SEQ ID NO:2:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Lys Ala Leu Pro Val Val Leu Glu Asn Ala Arg Ile Leu Lys Asn Cys
l 5 l0 15
20 Val Asp Ala Lys Met Thr Glu Glu Asp Lys Glu
20 25
26