U.S. patent application number 10/474521 was filed with the patent office on 2004-10-14 for methods and materials for cancer treatment.
This patent application is currently assigned to Mayo Foundation for Medical Education & Research. Invention is credited to Celis, Esteban.
Application Number | 20040202654 10/474521 |
Document ID | / |
Family ID | 23082398 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040202654 |
Kind Code |
A1 |
Celis, Esteban |
October 14, 2004 |
Methods and materials for cancer treatment
Abstract
Methods and materials for inducing anti-tumor responses in
melanoma patients are disclosed. These methods and materials
involve gp100-derived polypeptides that contain both a helper
T-cell epitope and a cytotoxic T-cell epitope.
Inventors: |
Celis, Esteban; (Rochester,
MN) |
Correspondence
Address: |
Mark S Ellinger
Fish & Richardson
3300 Dain Rauscher Plaza
60 South Sixth Street
Minneapolis
MN
55402
US
|
Assignee: |
Mayo Foundation for Medical
Education & Research
200 First Street S.W.
Rochester
MN
55905
|
Family ID: |
23082398 |
Appl. No.: |
10/474521 |
Filed: |
May 12, 2004 |
PCT Filed: |
April 5, 2002 |
PCT NO: |
PCT/US02/10583 |
Current U.S.
Class: |
424/143.1 ;
530/388.22 |
Current CPC
Class: |
C07K 14/4748 20130101;
A61K 38/00 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/143.1 ;
530/388.22 |
International
Class: |
A61K 039/395; C07K
016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 2001 |
US |
60282633 |
Claims
What is claimed is:
1. A polypeptide comprising an HTL epitope and a CTL epitope,
wherein said polypeptide is 13 to 30 amino acids long, and wherein
the amino acid sequence of said polypeptide is at least 90%
identical to a gp100 amino acid sequence.
2. The polypeptide of claim 1, wherein said polypeptide comprises
an amino acid sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.
3. The polypeptide of claim 1, wherein said polypeptide binds more
than one MHC class II allele.
4. The polypeptide of claim 1, wherein said polypeptide binds
HLA-DR-53 or HLA-DQw6.
5. The polypeptide of claim 1, wherein said CTL epitope comprises
MLGTHTMEV.
6. The polypeptide of claim 1, wherein said CTL epitope comprises
VSNDGPTLI.
7. The polypeptide of claim 1, wherein said CTL epitope comprises
ALNFPGSQK.
8. A method for inducing an anti-tumor response in a mammal, said
method comprising administering a polypeptide to said mammal,
wherein said polypeptide comprises an HTL epitope and a CTL
epitope, wherein said polypeptide is 13 to 30 amino acids long, and
wherein the amino acid sequence of said polypeptide is at least 90%
identical to a gp100 amino acid sequence.
9. The method of claim 8, wherein said polypeptide comprises an
amino acid sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, or 16.
10. The method of claim 8, wherein said polypeptide binds more than
one MHC class II allele.
11. The method of claim 8, wherein said polypeptide binds HLA-DR-53
or HLA-DQw6.
12. The method of claim 8, wherein said CTL epitope comprises
MLGTHTMEV.
13. The method of claim 8, wherein said CTL epitope comprises
VSNDGPTLI.
14. The method of claim 8, wherein said CTL epitope comprises
ALNFPGSQK.
Description
BACKGROUND
[0001] 1. Technical Field
[0002] The invention relates to polypeptides. More particularly,
the invention relates to gp100-derived polypeptides that contain
both a helper T-cell epitope and a cytotoxic T-cell epitope.
[0003] 2. Background Information
[0004] For some time many groups have been actively identifying
cytotoxic T lymphocyte (CTL) epitopes for various tumor-associated
antigens (TAA) and synthetic polypeptides representing these
epitopes have been used to prepare therapeutic vaccines to treat
cancer patients (Marchand M P et al., Int J of Cancer 63:883
(1995); Jager E et al., Int J of Cancer 67:54 (1996); Weber J et
al., J Immunother 22:431 (1999); Rosenberg S A et al., Nat Med
4:321 (1998); and Zalcs T Z et al., Cancer Res 58:4902 (1998)).
However, therapeutic effects of these vaccines are far from
optimal.
[0005] Several TAA are possible targets for developing T-cell
immunotherapy against malignant melanoma (Boon, T et al., J Exp Med
183:725 (1996) and Wang R F et al., Immunol Rev 170:85 (1999)).
Amongst these, the melanocyte-related antigen gp100 is currently
being evaluated in the clinic (Rosenberg S A et al., Nat Med 4:321
(1998) and Kawakami Y et al., J Immunol 154:3961 (1995)). Numerous
CTL epitopes restricted by the MHC class I alleles HLA-A2, -A3,
-A11, -A24 and Cw8 have been reported (Kawakami Y et al., J Immunol
154:3961 (1995); Cox A L et al., Science 264:716 (1994); Tsai V. et
al., J Immunol 158:1796(1997); Kawashima I et al., 1998. Int J
Cancer 78:518 (1998); Skipper J C et al., J Immunol 157:5027
(1996); Kawakami Y et al., J Immunol 161:6985 (1998); Robbins P F
et al., J Immunol 159:303 (1997); and Castelli C et al., J Immunol
162:1739 (1999)). On the other hand, only one HTL epitope, which is
restricted by the HLA-DR*0401 MHC class II allele, expressed in
approximately one fourth of the population, has been described (Li
K, et al., Cancer Immunol Immunother 47:32 (1998) and Toulouldan C
E et al., J Immunol 164:3535 (2000)).
SUMMARY
[0006] The invention provides gp100-derived polypeptides that
contain a CTL epitope and an HTL epitope. Such gp100-derived
polypeptides can be used to induce anti-tumor responses in melanoma
patients. Because a concurrently induced HTL response can
facilitate the induction and persistence of a CTL response, the
materials and methods provided herein offer advantages over current
approaches. In some embodiments of the invention, gp100-derived
polypeptides bind more than one MHC class II allele (e.g.,
HLA-DR-53 or HLA-DQw6). Such polypeptides can be used more broadly
to induce anti-tumor responses in melanoma patients.
[0007] In general, the invention features a polypeptide containing
an HTL epitope and a CTL epitope, where the polypeptide is 13 to 30
amino acids long, and where the amino acid sequence of the
polypeptide is at least 90% identical to a gp100 amino acid
sequence. The polypeptide can contain an amino acid sequence as set
forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, or 16. The polypeptide can bind more than one MHC class II
allele (e.g., HLA-DR-53 or HLA-DQw6). The CTL epitope can contain
MLGTHTMEV, VSNDGPTLI, or ALNFPGSQK.
[0008] In another embodiment, the invention features a method for
inducing an anti-tumor response in a mammal. The method includes
administering a polypeptide to the mammal, where the polypeptide
contains an HTL epitope and a CTL epitope, where the polypeptide is
13 to 30 amino acids long, and where the amino acid sequence of the
polypeptide is at least 90% identical to a gp100 amino acid
sequence. The polypeptide can contain an amino acid sequence as set
forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, or 16. The polypeptide can bind more than one MHC class II
allele (e.g., HLA-DR-53 or HLA-DQw6). The CTL epitope can contain
MLGTHTMEV, VSNDGPTLI, or ALNFPGSQK.
[0009] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0010] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DESCRIPTION OF DRAWINGS
[0011] FIG. 1. HLA-DR7-resricted T-cell proliferative responses to
gp100 polypeptides. HTL lines were selected by weeldy stimulation
using polypeptides gp100.sub.74-89 (a and c) and gp100.sub.576-590
(b and d). Polypeptide dose curve responses were performed to
estimate the overall avidity of the HTL for their respective
antigens (a and b). Dashed lines represent the polypeptide
concentration required to obtain 50% of the maximal proliferation
(.about.0.6 .mu.g/ml for polypeptide gp100.sub.74-89 and
.about.0.04 .mu.g/ml for polypeptide gp100.sub.576-590). MHC
restriction analyses demonstrate that these HTL recognize their
respective polypeptides in the context of the HLA-DR7 allele (c and
d). In both cases the T cell proliferative responses to polypeptide
presented by autologous PBMC were significantly inhibited by
monoclonal anti-HLA-DR, ".alpha.DR" (but not anti-HLA-DQ,
".alpha.DQ") antibodies *=numbers in parentheses represent the %
inhibition of proliferation induced by the antibodies. Mouse
fibroblast lines (L-cells) transfected with human HLA-DR7 (but no
those transfected with HLA-DR53) were efficient in presenting
polypeptide to both HTL lines. Values shown are the means of
triplicate determinations; bars, SD.
[0012] FIG. 2. Polypeptide gp100.sub.175-189 can be recognized by
HTL in the context of HLA-DR53 (a and c) or HLA-DQw6 (b and d).
Polypeptide dose curve responses were performed to estimate the
overall avidity of these HTL for polypeptide gp100.sub.175-189 (a
and b). Dashed lines represent the polypeptide concentration
required to obtain 50% of the maximal proliferation (.about.2
.mu.g/ml for both HTL lines). MHC restriction analyses demonstrate
that one of the HTL recognize polypeptide gp100.sub.175-189 in the
context of the HLA-DR53 allele (panel c) and the other one in the
context of HLA-DQw6 (panel d). In both cases the T cell
proliferative responses to polypeptide presented by autologous PBMC
were significantly inhibited by monoclonal anti-HLA-DR (c) or
-HLA-DQ (d) antibodies * numbers in parentheses represent the %
inhibition of proliferation. Mouse fibroblast lines (L-cells)
transfected with human HLA-DR53 but not with HLA-DR7 were efficient
in presenting polypeptide gp100.sub.175-189 to one of the HTL lines
(c). In addition an allogeneic lymphoblastoid cell line (LCL) only
sharing HLA-DR53 with the T cells was also efficient in stimulating
this cell line (c). Allogeneic LCL that only share HLA-DQw6 can
present polypeptide gp100.sub.175-189 to the second HTL line (d).
Values shown are the means of triplicate determinations; bars,
SD.
[0013] FIG. 3. HLA-DR7-restricted HTL specific for polypeptide
gp10074-89 can recognize naturally processed antigen. Panel a:
Autologous (DR7.sup.+) PBMC are efficient in stimulating HTL
responses (assessed by the production of GM-CSF, measured by ELISA)
by presenting polypeptide gp100.sub.74-89 or a lysate from
gp100.sup.+ melanoma cells (697mel, DR7 negative) but not a lysate
from Jurkat cells. Panel b: Production of GM-CSF by HTL is also
observed with polypeptide-pulsed APC (autologous PBMC or L-DR7
cells) or with intact DR7 positive melanoma cells (HT-144), but not
with a DR7 negative melanoma (888mel). These responses are
inhibited by anti-DR monoclonal antibodies (aDR). Values shown are
the means of triplicate determinations; bars, SD. These experiments
were repeated at least three times with similar results.
[0014] FIG. 4. Recognition of naturally processed antigen by
gp100.sub.175-189-specific, HLA-DR53-restricted HTL. A T-cell clone
derived from 5D10 was tested for its capacity IFN-.gamma. when
challenged with intact DR53.sup.+ melanomas (697mel and 624mel).
Production of IFN-.gamma. was inhibited by anti-DR antibodies and
could not be detected using DR53 negative melanoma cells (888mel).
Controls shown are the production of IFN-.gamma. by HTL stimulated
with DR53 positive lymphoblastoid cells (M35-EBV) in the presence
and absence of polypeptide gp100.sub.175-189. Values are the means
of triplicate determinations; bars, SD.
[0015] FIG. 5. gp100.sub.175-189-specific CD4.sup.+ T-cells
restricted by HLA-DQw6 can also recognize naturally processed
antigen. Panel a: autologous PBMC present synthetic polypeptide or
melanoma (gp100.sup.+) lysates to HTL resulting in the production
of GM-CSF. HTL response to a lysate from a gp100 negative cell line
(Jurkat) was significantly lower. Panel b: Intact HLA-DQw6 positive
melanomas can induce the production of GM-CSF by antigen-specific
HTL. Results demonstrate that this response is inhibited by anti-DQ
(aDQ) monoclonal antibodies, but not by anti-DR antibodies. Values
shown are the means of triplicate determinations; bars, SD.
[0016] FIG. 6. Cytolytic activity against melanoma cells of
gp100.sub.175-189-specific CD4.sup.+ T-cells. The
HLA-DQw6-restricted HTL clone was tested for its ability to kill
melanoma cells using the JAM assay as described in "Materials and
Methods". Target cells tested were: 1102mel (open circles) and M35
EBV-LCL (closed circles).
[0017] FIG. 7 is a table listing amino acid sequences.
DETAILED DESCRIPTION
[0018] The invention provides polypeptides such as gp100-derived
polypeptides that contain both an HTL epitope and a CTL epitope.
The invention also provides methods for using such gp100 derived
polypeptides to induce anti-tumor CTL and HTL responses in melanoma
patients. Some polypeptides of the invention can bind to several
MHC class II alleles, making them more broadly useful for inducing
HTL and CTL anti-tumor responses in melanoma patients.
[0019] Polypeptides, and polypeptide segments, are contiguous
sequences of 2 or more amino acids linked one to the other by
peptide bonds. Polypeptides suitable for the present invention are
between about 15 amino acids and about 30 amino acids long (e.g.,
about 15 to about 20 amino acids long, about 15 to about 25 amino
acids long, about 20 to 25 amino acids long, and about 25 to 30
amino acids long).
[0020] Polypeptides of the invention are substantially pure (i.e.,
substantially free of other naturally occurring viral, bacterial,
parasitic, tumor, or self polypeptides and fragments thereof).
Polypeptides of the invention can have a CTL epitope (i.e., a
polypeptide segment that can elicit a CTL response). Polypeptides
of the invention can also have an HTL epitope (i.e., a polypeptide
segment that can elicit an HTL response). In one embodiment, more
than one MHC class II allele recognizes the polypeptide.
[0021] HTL and CTL epitopes of the polypeptides can be present in
the configuration naturally found in a gp100 or gp100-derived
melanoma tumor associated antigen. HTL and CTL epitopes of a
polypeptide can also be present in a different configuration than
naturally found in a gp100 or gp100-derived melanoma tumor
associated antigen. HTL and CTL epitopes of a polypeptide can be
discrete, where no amino acid of the HTL epitope is part of the CTL
epitope. HTL and CTL epitopes of a polypeptide can also comprise
common amino acids, where one or more amino acids of an HTL epitope
is also part of a CTL epitope.
[0022] Polypeptides suitable for the compositions and methods of
the present invention need not be identical to specific
polypeptides disclosed. Thus, the polypeptides may be subject to
various changes, such as insertions, deletions, and substitutions,
either conservative or nonconservative, where such changes might
provide for certain advantages in their use. Conservative
substitutions result in the replacement of an amino acid residue
with another that is biologically and/or chemically similar, e.g.,
one hydrophobic residue for another, or one polar residue for Ala;
Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe,
Tyr. Typically, the portion of the sequence that is intended to act
as an CTL or HTL epitope will not differ by more than about 20%
from the corresponding sequence of a native gp100 or gp100-derived
antigen, except where additional amino acids may be added at either
terminus for the purpose of modifying the physical or chemical
properties of the polypeptide for, e.g., ease of linking or
coupling, and the like.
[0023] Exemplary polypeptides include, without limitation:
LAEMSTPEATGMTPA (SEQ ID NO:1), TEVISTAPVQMPTAE (SEQ ID NO:2),
SLAVVSTQLIMPGQE (SEQ ID NO:3), GILLVLMAVVLASLI (SEQ ID NO:4),
PVQMPTAESTGMTPE (SEQ ID NO:5), GRAMLGTHTMEVTVY (SEQ ID NO:6),
GPTLIGANASFSIALN (SEQ ID NO:7), QVVLQAAIPLTSCGS (SEQ ID NO:8),
PGPVTAQVVLQAAIP (SEQ ID NO:9), KHFLRNQPLTFALQL (SEQ ID NO:10),
SLADTNSLAVVSTQLIMPGQE (SEQ ID NO:11), GILLVLMAVVLASLIYRRRLMK (SEQ
ID NO:12), GILLVLMAVVLASLIYRRRLMKQDFSV (SEQ ID NO:13),
VSNDGPTLIGANASFSIALN (SEQ ID NO:14), GPTLIGANASFSIALNFPGSQK (SEQ ID
NO:15), AND LEPGPVTAQVVLQAAIPLTSCGSS (SEQ ID NO:16).
[0024] The polypeptides of the invention can be prepared in a wide
variety of ways. Because of their relatively short size, the
polypeptides can be synthesized in solution or on a solid automatic
synthesizer in accordance with known protocols. See, for example,
Stewart and Young, Solid Phase Polypeptide Synthesis, 2d. ed.,
Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc., 105:6442
(1983); Merrifield, The Polypeptides, Gross and Meienhofer, ed.,
academic Press, New York, pp. 1-284 (1979).
[0025] Alternatively, recombinant DNA technology can be used
wherein a nucleotide sequence which encodes a polypeptide of
interest is inserted into an expression vector, introduced (e.g.,
by transformation or transfection) into an appropriate host cell,
and cultivated under conditions suitable for expression. These
procedures are generally known in the art, as described generally
in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1982), and Ausubel
et al., (ed.) Current Protocols in Molecular Biology, John Wiley
and Sons, Inc., New York (1987), and U.S. Pat. Nos. 4,237,224;
4,273,875; 4,431,739; 4,363,877; and 4,428,941, for example.
[0026] As the coding sequence for polypeptides of the length
contemplated herein can be synthesized by chemical techniques, for
example the phosphotriester method of Matteucci et al., J. Am.
Chem. Soc. 103:3185 (1981), modification can be made simply by
substituting the appropriate base(s) for those encoding the native
polypeptide sequence. The coding sequence can then be provided with
appropriate linkers and ligated into expression vectors commonly
available in the art, and the vectors used to transform suitable
hosts to produce the desired fusion polypeptide. A number of such
vectors and suitable host systems are now available. For expression
of the polypeptides, the coding sequence will typically be provided
with operably linked start and stop codons, promoter and terminator
regions and a replication system to provide an expression vector
for expression in a suitable cellular host (e.g., bacterial, yeast
or mammalian cells).
[0027] Polypeptides can be administered to mammals, particularly
humans, to induce melanoma-specific anti-tumor CTL and HTL
responses. Polypeptides can be administered intravenously,
subcutaneously, intradermally, or intramuscularly, topically,
orally or locally. Compositions for parenteral (i.e., intravenous,
subcutaneous, intradermal, or intramuscular) administration
comprise an HTL and CTL stimulatory polypeptide dissolved or
suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., water, buffered
water, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like.
These compositions can be sterilized by conventional sterilization
techniques. The resulting aqueous solutions can be packaged for use
as is or lyophilized--the lyophilized preparation being combined
with a sterile solution prior to administration. The compositions
can contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, methanol, and dissolving
agents such as DMSO. The amount HTL and CTL stimulatory polypeptide
in a pharmaceutical formulations can vary widely, i.e., from less
than about 1%, usually at or at least about 10% to as much as 20 to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected. Actual methods for preparing
parenterally administrable compounds will be known or apparent to
those skilled in the art and are described in more detail in for
example, Remington's Pharmaceutical Sciences, 17th ed., Mack
Publishing Company, Easton, Pa. (1985), which is incorporated
herein by reference.
[0028] Polypeptides can also be administered via liposomes, which
can target the polypeptides to a particular tissue, such as
lymphoid tissue, or targeted selectively to tumor cells, as well as
increase the half-life of the polypeptide composition. Liposomes
can include, without limitation, emulsions, foams, micelles,
isoluble monolayers, liquid crystals, phospholipid dispersions,
lamellar layers, and the like. In these preparations, the
polypeptide to be delivered can be incorporated as part of a
liposome, alone or in conjunction with a molecule which bonds to,
e.g., a receptor prevalent among lymphoid cells, such as monoclonal
antibodies which bind to the CD45 antigen, or with other
therapeutic or immunogenic compositions. Thus, liposomes filled
with a desired polypeptide can be directed to the site of lymphoid
cells, where the liposomes then deliver the selected
therapeutic/immunogenic polypeptide compositions. Liposomes for use
in the invention can be formed from standard vesicle-forming
lipids, which generally include neutral and negatively charged
phospholipids and a sterol, such as cholesterol. The selection of
lipids is generally guided by consideration of, e.g., liposome size
and stability of the liposomes in the blood stream. A variety of
methods are available for preparing liposomes, as described in
e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.
Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369. For
targeting to the immune cells, a ligand to be incorporated into the
lipsome can include, e.g., antibodies or fragments thereof specific
for cell surface determinants of the desired immune system cells. A
liposome suspension containing a polypeptide can be administered,
for example, intravenously, locally, topically, in a dose that can
vary according to, among other things, the manner of
administration, the polypeptide being delivered, and the
progression of the disease.
[0029] For solid compositions, conventional nontoxic solid carriers
can be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of the active ingredient, that is, one or more
polypeptide compositions of the invention, and more preferably at a
concentration of 25%-75%.
[0030] For aerosol administration, an HTL and CTL stimulatory
polypeptide can be provided in finely divided form along with a
surfactant and propellant. Typical percentages of polypeptides are
0.01%-20% by weight, preferably 1%-10%. The surfactant is typically
nontoxic and is typically soluble in the propellant. Representative
surfactants include esters or partial esters of fatty acids
containing from 6 to 22 carbon atoms, such as caproic, octanoic,
lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic
acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters, such as mixed or natural glycerides can be employed.
Surfactant can constitute 0.1%-20% by weight of the composition,
preferably 0.25-5%. The balance of the composition is ordinarily
propellant. A carrier such as lecithin, for one example, can also
be included for intranasal delivery.
[0031] An HTL and CTL stimulatory polypeptide can be introduced
into a mammalian host, including humans, linked to its own carrier
or as a homopolymer or heteropolymer of active polypeptide units.
Such a polymer can elicit increase immunological reaction and,
where different polypeptides are used to make up the polymer, the
additional ability to induce antibodies and/or T cells that react
with different antigenic determinants of the melanoma tumor. Useful
carriers known in the art include, for example, thyroglobulin,
albumins such as human serum albumin, tetanus toxoid, polyamino
acids such as poly(D-lysine:D-glutamic acid), influenza
polypeptide, and the like. Adjuvants such as incomplete Freunds
adjuvant, GM-CSF, aluminum phosphate, aluminum hydroxide, alum, or
montanide can also be used in the administration of an HTL and CTL
stimulatory polypeptide.
[0032] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Synthetic Polypeptides and Algorithm Analysis
[0033] Potential HLA-DR promiscuous CD4.sup.+ T-cell epitopes
capable of binding to more than one MHC class II allele were
selected from the amino acid sequence of the gp100 antigen using
the algorithm tables for three HLA-DR alleles (DRB1* 0101,
DRB1*0401, and DRB1*0701) published by Southwood, et al (J Immunol
160:3363 (1998). A computer program based on Microsoft Excel was
created for each of the HLA-DR alleles. For each position of a
9-mer core, a value was assigned for each possible specific amino
acid taking the tables published by Southwood, et al (J Immunol
160:3363 (1998). The computer program calculated the average
binding (ARB) values for each possible 9mer of the gp100 sequence.
The higher the ARB value, the more likely the polypeptide will bind
to the corresponding HLA-DR allele. The data in FIG. 7 demonstrate
that the sequences, ARB values for each allele, ranks and rank sum
values for the top 10 candidate polypeptides. Polypeptides that
displayed high ARB values and were proximal with known CTL epitopes
were synthesized and purified as described. The purity (>95%)
and identity of polypeptides were determined by mass
spectrometry.
Example 2
In Vitro Induction of Antigen-Specific HTL Using Synthetic
Polypeptides
[0034] Polypeptides gp100.sub.74-89, gp100.sub.175-189 and
gp100.sub.576-590 were tested for their capacity to stimulate
CD4.sup.+ T cells obtained from four healthy, MHC-typed individuals
in primary in vitro cultures using polypeptide-pulsed autologous
dendritic cells (DC). The method used to generate tumor
antigen-reactive HTL lines and clones using polypeptide-stimulated
peripheral blood mononuclear cells (PBMC) was recently described in
detail (Kobayashi H et al., Cancer Res 60:5228 (2000)). Briefly, DC
were generated in tissue culture from adherent monocytes that were
cultured for 7 days in the presence of GM-CSF and IL-4. A total of
(1.times.10.sup.4) polypeptide-pulsed DC (10 .mu.g/ml for 2 hr)
were irradiated (4,200 rad) and co-cultured with 3.times.10.sup.4
autologous purified CD4.sup.+ T cells (using magnetic microbeads
from Miltenyi Biotech) in each well of a 96-round bottom well
culture plate. Seven days later, the cultures were restimulated
with polypeptide-pulsed irradiated autologous PBMC and two days
later, human rIL-2 was added at a final concentration of 10 IU/ml.
After 7 days, the microcultures were tested for their proliferative
responses to polypeptide as described below. Those micro-wells
showing a proliferative response to polypeptide (at least 2.5 fold
over background) were expanded in 24 or 48-well plates by weeldy
restimulation with polypeptides and irradiated autologous PBMC. In
some instances T-cell lines were cloned by limiting dilution and
used for further studies. Culture medium for all procedures
consisted of RPMI-1640 supplemented with 5% human male AB serum,
0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 2 mM
L-glutamine, and 50 .mu.g/ml gentamicin.
[0035] Polypeptides gp100.sub.74-89 and gp100.sub.576-590 induced
proliferative responses in CD4.sup.+ T-cells that were restricted
by the HLA-DR7 allele (FIG. 1). In both cases, the T-cells
responded to polypeptide presented by autologous PBMC in a dose
dependent manner (FIGS. 1a and 1b). The affinity of T-cell response
to polypeptide gp100.sub.576-590, however, appeared to be higher
(.about.15 fold) than the response to polypeptide gp100.sub.74-89
as determined by the amount of polypeptide required to achieve 50%
of maximal proliferative response. The response of both T-cell
lines were restricted by HLA-DR7 molecules as determined by the
capacity of DR7-transfected L-cells to present polypeptide to the
T-cells and by the capacity of anti-DR antibodies (but not anti-DQ)
to block the T-cell proliferative responses (FIGS. 1c and 1a).
Thus, gp100.sub.74-89 and gp100.sub.576-590 trigger HTL restricted
by the HLA-DR7 allele, which is expressed in approximately 26% of
Caucasians. Although both of these polypeptides were selected as
potential "promiscuous" epitopes, they were not able to trigger HTL
restricted by other alleles such as HLA-DR1 or -DR4. Nevertheless,
given the limited number of attempts to induce these responses (2
experiments with cells from 2 donors), these epitopes may indeed
have some degree of promiscuity.
[0036] Two separate T-cell clones reactive with polypeptide
gp100.sub.175-189 were isolated from two normal individuals and
studied in more detail for their proliferative response to antigen.
The data presented in FIG. 2 demonstrates that both of the T-cell
clones recognized polypeptide in a dose dependent way with similar
levels of affinity (FIGS. 2a and 2b). Studies performed to
determine the HLA restriction elements for these clones indicated
that in one case the response to polypeptide gp100.sub.175-189 was
restricted by HLA-DR53 (FIG. 2c) and in the other case by HLA-DQw6
(FIG. 2d). Thus, gp100.sub.175-189 represents a promiscuous HTL
epitope capable of eliciting HTL responses restricted by HLA-DR53
and -DQw6. Since approximately one half of the population expresses
one or both of these MHC alleles, this polypeptide could be usefull
for eliciting anti-tumor HTL responses to melanoma in a large
number of tumor patients.
Example 3
Antigen-Specific Proliferative Response of T Cells
[0037] Recognition of naturally processed antigen by
polypeptide-reactive T-cells can be an important parameter to
determine whether a predicted T-cell epitope can be used to elicit
an immune response against tumor cells. HTL may recognize
polypeptide/MHC class II complexes directly on tumor cells that are
MHC class II positive and as a consequence the T-cells will become
activated and produce cytokines that provide help to CTL or
cytokines that may inhibit tumor cell growth.
[0038] T cells (3.times.10.sup.4/well) were mixed with irradiated
APC in the presence of various concentrations of antigen
(polypeptides, tumors or tumor lysates), in 96-well culture plates.
APC consisted of either PBMC (1.times.10.sup.5/well),
HLA-DR-expressing L-cells (3.times.10.sup.4/well), EBV-LCL or
melanoma tumor cells (3.times.10.sup.4/well), pretreated with
IFN-.gamma. (500 U/ml for 48 h) to enhance MHC expression.
Proliferation assays were incubated at 37.degree. C. in a 5%
CO.sub.2 incubator for 72 h and during the last 16 h, each well was
pulsed with 0.5 .mu.Ci/well of [.sup.3H]-thymidine (Amersham
Pharmacia Biotech, Piscataway, N.J.). In some cases, culture
supernatants were collected before the addition of
[.sup.3H]-thymidine for the determination of lympholine production
using ELISA kits (Pharmingen, San Diego, Calif.). The radioactivity
incorporated into DNA, which correlates with cell proliferation,
was measured in a liquid scintillation counter after harvesting the
cell cultures onto glass fiber filters. To determine MHC
restriction molecules involved in antigen presentation, blocking of
the antigen-induced proliferative response was investigated by
adding anti-HLA-DR mAb L243 (IgG2a, prepared from hybridoma
supernatants obtained from the (American Type Culture Collection)
or anti-HLA-DQ mAb SPVL3 (IgG2a, Beclcman Coulter, Inc., Fullerton,
Calif.). Both antibodies were used at a final concentration of 10
.mu.g/ml throughout the 72 h assay. The specificity of these
antibodies and their capacity to specifically inhibit T helper
responses has been determined in our laboratory in numerous
occasions. All assessments of proliferative responses were carried
out at least in triplicate and results correspond to the means. The
stimulation index (SI) was calculated by dividing the mean
radioactivity (cpm) obtained in the presence of antigen by the mean
radioactivity (cpm) obtained in the absence of antigen but in the
presence of APC.
[0039] FIG. 3a demonstrates that the HLA-DR7-restricted T-cell line
(10B1) induced with polypeptide gp100.sub.74-89 was efficient at
recognizing tumor cell lysates from a gp100.sup.+ melanoma tumor
(697mel), but not control tumor lysates (Jurkat), presented by
autologous APC. For these experiments, the lysates were prepared
using a DR7 negative melanoma (697mel) to ensure that the
autologous APC were indeed capturing the gp100 antigen from the
lysate to process and present the epitope to the helper T-cells.
Furthermore, these HTL were also effective in recognizing antigen
presented directly by HLA-DR7.sup.+ melanoma tumors (HT-144), and
this recognition could be inhibited with anti-HLA-DR antibodies
(FIG. 3b). These results indicate that the T-cell epitope
represented by polypeptide gp100.sub.74-89 is not only processed by
APC from tumor cell lysates but is also expressed on MHC class II
molecules on melanoma cells. A similar analysis was performed using
the HTL line induced with polypeptide gp100.sub.576-590, but in
this case T-cell responses could not be detected using tumor
lysates or intact melanoma cells.
[0040] The two HTL lines reactive with polypeptide
gp100.sub.175-189 were also tested for their ability to recognize
naturally processed antigen. HTL clone 5D10 (HLA-DR53-restricted)
was unable to recognize melanoma cell lysates presented by
autologous APC. However, the HTL were very effective in recognizing
intact HLA-DR53 positive melanomas, but not DR53 negative melanomas
or DR53 positive lymphoblastoid cells that do not produce gp100
(M35-EBV), unless pulsed with polypeptide (FIG. 4). In addition,
the recognition of gp100.sup.+ melanoma tumor cells by these HTL
was inhibited with anti-HLA-DR antibodies (FIG. 4), confirming that
this interaction requires the presentation of polypeptide by MHC
class II molecules. A T-cell clone (4D16) derived from the
HLA-DQw6-restricted line was studied for its ability to recognize
processed antigen derived from tumor cell lysates presented by APC
or intact class II positive melanomas. The results shown in FIG. 5a
demonstrate that these HTL were capable of reacting with antigen
derived from melanoma lysates presented by autologous APC. In
addition, this HTL clone was also quite effective in recognizing
antigen directly on DQw6+melanomas (FIG. 5b). Thus, it appears that
the T-cell epitope represented by polypeptide gp100.sub.175-189 can
be found expressed on both HLA-DR53 and HLA-DQw6 molecules on MHC
class II melanomas.
[0041] Thus, HTL reactive to gp100.sub.175-189 and HTL reactive to
gp100.sub.74-89 were capable of recognizing naturally processed
antigen in MHC class II positive melanomas or in APC fed with tumor
lysates. On the other hand, HTL that were induced using polypeptide
gp100.sub.576-590 did not proliferate or produce lympholdnes when
interacting with MHC class II positive melanomas or tumor lysates
presented by APC.
Example 4
Antigen-Specific Cytotoxicity Assays
[0042] In some cases, HTL exhibit cytotoxicity towards antigenic
MHC class II tumors. See, Manici et al., J Exp Med 189:871(1999);
Fujita et al., Eur J Immunol 28:305 (1998); Zarour et al., Cancer
Res 60:4946 (2000); Touloukian et al., J Immunol 164:3535 (2000);
Zarour et al., Proc Natl Acad Sci USA 97:400 (2000); Yasukawaet
al., Blood 92:3355 (1998). Polypeptide gp100.sub.175-189, which
generated DR53- and DQw6-restricted HTL capable of recognizing
naturally processed antigen, includes a potent HLA-A2-restricted
CTL epitope (gp100.sub.177-186). Polypeptide gp100.sub.177-186 is
very effective in inducing anti-tumor CTL responses by in vitro
immunization of PBMC from normal individuals using
polypeptide-pulsed DC.
[0043] The capacity of HTL lines and clones to kill tumor cells
expressing the appropriate polypeptide/MHC complexes was evaluated
using the JAM assay (Matzinger P, J Immunol Methods 145:185
(1991)), which measures the DNA retained by target living cells
after incubation with effector T-cells. Briefly, target cells were
labeled with of [.sup.3-H]-thymidine for 18 hrs in tissue culture.
After washing and counting the cells, they were incubated at
various effectors to target ratio in 96-well round-bottomed plates
for 36 hours. The cultures were then harvested onto glass fiber
filters, and the amount of radioactivity-retained in DNA is
measured in a scintillation counter. The percent cytotoxicity was
calculated by the ratio of cpm obtained in the absence of T-cells
over the cpm obtained in the presence of different amounts of
T-cells X 100. All determinations were done in triplicates.
[0044] FIG. 6 demonstrates that HLA-DQw6-restricted HTL that react
with polypeptide gp100.sub.175-189 display high levels of
cytotoxicity against a DQw6 positive melanoma cell line. Thus,
polypeptide gp100.sub.175-189 can be used to elicit anti-tumor HTL
and CTL responses in patients expressing HLA-DR-53 or HLA-DQw6
class II alleles.
[0045] The other gp100-reactive HTL lines exhibited lower levels of
cytotoxicity against melanoma cell lines. Polypeptide
gp100.sub.74-89, which stimulated an HLA-DR7-restricted HTL
response, partially overlaps (in its carboxyl end) with another CTL
epitope, but this one is restricted by the HLA-A3 and -A11 alleles
(gp100.sub.87-95; ALNFPGSQK). Thus, a synthetic polypeptide of 22
residues (gp100.sub.74-95) can be used to elicit anti-tumor CTL and
HTL responses in patients expressing HLA-A3 (or -A11) and HLA-DR7.
Polypeptide gp100.sub.74-89 also overlaps in its amino terminal end
with another CTL epitope (gp100.sub.70-78), this one being
restricted by HLA-Cw8. Thus, a synthetic polypeptide of 20 residues
(gp100.sub.70-89) can be used to elicit anti-tumor CTL and HTL
responses in patients expressing HLA-Cw8 and HLA-DR7.
Example 5
Clinical Study
[0046] To assess polypeptides of the invention, the following study
is performed. Metastatic melanoma HLA-A2 positive patients are
immunized with an immunodominant peptide derived from gp-100. The
immunization is performed in the presence of GM-CSF and IFA as
immune-adjuvants and goals include quantification of specific CTL
responses and observation for therapeutic efficacy. Of particular
interest is the comparison of the efficacy of immunization in
patients that share the class II HLA antigens specific for the
peptide relative to those that do not. This will provide
preliminary data for the need of T helper cell participation with
shared epitopes recognized by CTLs.
[0047] In the proposed study, the efficacy of immunization measured
by the following is examined: specific cytotoxic T lymphocyte (CTL)
activity against melanoma tumor cells; frequency of
peptide-specific T cells determined by fluorescent tetramer
analysis; levels of activation of tetramer-specific T cells
measured by cellular cytokine production; in vivo delayed type
hypersensitivity reactions to peptide challenge after immunization;
and, whenever possible, measurement of numbers/function of tumor
related/infiltrating T cells. We will collect preliminary data
relating to the efficacy of treatment is collected by following
changes in tumor size for the duration of the clinical trial.
[0048] Goals
[0049] Determine the immunologic effects of immunization using a
HLA class I and II specific gp-100-derived peptide suspended in IFA
in the presence or absence of GM-CSF.
[0050] Define the safety and toxicity profile of the immunization
protocol.
[0051] Collect preliminary data on:
[0052] Therapeutic efficacy as it relates to parameters of immune
function in patients with stage IV malignant melanoma.
[0053] Compare immunization efficacy of patients that share the
HLA-DR53 and/or DQw6 haplotypes versus those who do not.
[0054] Patient Eligibility
[0055] Required Characteristics
[0056] Age .gtoreq.18 years.
[0057] HLA-A2 positive.
[0058] Histologic proof of measurable stage IV malignant
melanoma.
[0059] Melanoma positive for gp-100.
[0060] Laboratory values obtained .ltoreq.14 days prior to
registration:
[0061] Absolute Neutrophil Count (ANC)
.gtoreq.1500.times.10.sup.9/L
[0062] Platelets .gtoreq.100,000.times.10.sup.9/L
[0063] Hemoglobin >9.0 g/dL
[0064] AST .ltoreq.3.times.ULN
[0065] Alkaline phosphatase .ltoreq.3.times.ULN
[0066] Creatinine .ltoreq.2.5.times.ULN
[0067] Ability to provide informed consent.
[0068] Willingness to return to clinic for follow-up.
[0069] Life expectancy .gtoreq.12 weeks.
[0070] ECOG performance status 0, 1 or 2.
[0071] Contraindications
[0072] Uncontrolled or current infection
[0073] Known standard therapy for the patient's disease that is
potentially curative or proven capable of extending life
expectancy.
[0074] Known allergy to vaccine or adjuvant components.
[0075] Any of the following prior therapies with interval since
most recent treatment:
[0076] Chemotherapy .ltoreq.4 weeks
[0077] Mitomycin C/nitrosoureas .ltoreq.6 weeks
[0078] Biologic therapy .ltoreq.4 weeks
[0079] Radiation therapy .ltoreq.4 weeks
[0080] Radiation to >25% of bone marrow
[0081] Failure to fully recover from effects of prior chemotherapy
regardless of interval since last treatment.
[0082] Other concurrent chemotherapy, immunotherapy, or
radiotherapy.
[0083] New York Heart Association classification III or IV (Table
II).
[0084] Seizure disorder.
[0085] Any of the following as this regimen may be harmful to a
developing fetus or nursing child:
[0086] Pregnant women
[0087] Nursing women
[0088] Women of childbearing potential or their sexual partners who
are unwilling to employ adequate contraception (condoms, diaphragm,
birth control pills, injections, intrauterine device [IUD],
surgical sterilization, subcutaneous implants, or abstinence,
etc.)
[0089] Known immune deficiency, as patients with known immune
deficiencies will likely not be able to mount an immune response to
the study vaccine.
[0090] Active psychiatric disorder requiring anti-psychotic
medications.
[0091] Known central nervous system metastases or carcinomatous
meningitis.
[0092] History of other malignancy in last 5 years with the
exception of basal cell or squamous cell carcinoma of the skin
treated with local resection only (it is impossible to predict the
effect of study treatment on other, potentially dormant malignant
diseases)
1TABLE II NEW YORK HEART ASSOCIATION CLASSIFICATIONS Clinical
Evaluation of Functional Capacity of Patients with Heart Disease in
Relation to Ordinary Physical Activity Physical Cardiac Need for
Ability Class Symptoms Limitations Additional Rest* to work** I
None None None Full time II Only moderate Slight Usually only
Usually full slight or time occasional III Defined, with Marked
Usually moderate Usually part less than time ordinary activity IV
May be present Extreme Marked Unable to even at rest, work and any
activity increases discomfort *To control or relieve symptoms, as
determined by the patient, rather than as advised by the physician.
**At accustomed occupation or usual tasks. Reference: Bruce, R. A.:
Mod. Concepts Cardiovasc. Dis. 25: 321, 1956. (Modified from New
York Heart Association, 1953).
[0093]
2 Test Schedule Long term follow-up q.sup.3 .ltoreq.14 days prior
to Prior to months until prior to each cycle 7 Week month 24 post
Tests and procedures registration cycle treatment 24 registration
History and exam, X X X X X weight, performance status Height X
Tumor measurement/ X .sup. X.sup.4 X X X evaluation Hematology
group X X X X X WBC, ANC, Hgb, PLT Chemistry group X X X X X AST,
LDH, Alk Phos, Creat, K, Na) Injection site skin .sup. X.sup.6
.sup. X.sup.6 reaction DTH anergy panel R R DTH tumor antigens R R
Immunology studies.sup.1 R R HLA typing .sup. R.sup.5 Gp-100 tumor
stain .sup. R.sup.5 Lymph node aspiration.sup.2 R Serum pregnancy
test.sup.3 R Picture of all injection R R sites documenting local
skin reactions.sup.7 .sup.1100 cc of peripheral blood will be
collected for immunologic studies. .sup.2Only if immunization site
regional lymph node is clinically enlarged .sup.3For women of
childbearing potential only. Must be done .ltoreq.7 days prior to
registration .sup.4Before treatments for cycles # 3 and 6.
.sup.5May be done up to 6 months before registration. .sup.6Measure
vaccine injection sites maximum diameter of erythema and induration
(i.e. gp-100 (Feb. 12, 2001): E = 5.3 cm, I = 2.0 cm). Code each
vaccine site in sequence of injection (site of 1.sup.st injection,
etc.) and record erythyma/induration for each site at each time
point. See Appendix IV for documentation form. .sup.7Optional. R
Research funded.
[0094] Stratification Factor:
[0095] Dominant disease: Visceral vs. Non-visceral.
[0096] HLA-DR53 and/or DQw6 positive vs negative.
[0097] Registration/Randomization Procedures
[0098] Prior to discussing protocol entry with the
patient,determine for dose level.
[0099] Register patient.
[0100] Obtain signed consent form.
[0101] Confirm patient eligibility and the existence of a signed
consent form.
[0102] Treatment assigmnent is calculated using a dynamic
allocation procedure that balances the marginal distributions of
the stratification factor among the treatment groups. The
stratification factor is defined in Section 5.0.
[0103] Treatment on this protocol commences at the clinic under the
supervision of a medical oncologist.
[0104] Treatment begins prior to registration and begins within 3
days of registration.
[0105] Pretreatment tests are completed within the guidelines
specified on the test schedule.
[0106] All baseline symptoms are documented and graded on the
oncology record.
[0107] Protocol Treatment
3 Treatment groups: Peptide (.mu.g) Group in IFA GM-CSF (.mu.g)
Retreat A 1000 0 Day 1 of wk 0, 3, 6, 9, 12, 15, 18 and 24 B 1000
62.5 Day 1 of wk 0, 3, 6, 9, 12, 15, 18 and 24 C 1000 100 Days 1-5
of wk 0, 3, 6, 9, 12, 15, 18 and 24
[0108] Treatment Schedule:
4 Group A: (1 treatment cycle = 3 weeks) Day Agent Dose (.mu.g)
Route Re-treat 0 peptide 1000 s.q. Repeat entire cycle every 3
weeks (see table 7.1)
[0109]
5 Group B: (1 treatment cycle = 3 weeks) Day Agent Dose (.mu.g)
Route Re-treat 1 GM-CSF 62.5 s.q. Repeat entire cycle every 3 weeks
peptide 1000 (see table 7.1)
[0110]
6 Group C: (1 treatment cycle = 3 weeks) Day Agent Dose (.mu.g)
Route Re-treat 1 GM-CSF 100 s.q. Repeat entire cycle every 3 weeks
peptide 1000 .mu.g s.q. (see table 7.1)
[0111] Ten patients are randomly assigned to receive one of the
three treatment combinations.
[0112] Doses are not escalated in any individual patient.
7 Dosage Modification Based on Toxicity Use Common Toxicity
Criteria (CTC) Version 2.0 unless otherwise specified. CTC Category
Toxicity peptide GM-CSF BASED ON INTERVAL TOXICITY Blood/
Hematologic: Bone Marrow ANC <1,500 or PLT <100,000 .dwnarw.
by 50% No change All Non-hematologic: Grade 3 .dwnarw. by 50%
.dwnarw. by 50% Grade 4 Stop Rx Stop Rx Pain Bone pain No change
.dwnarw. by 50% (grade 3, 4) Myalgia/arthralgia .dwnarw. by 50%
.dwnarw. by 50% (grade 3,4) Skin Grade 3 .dwnarw. by 50% .dwnarw.
by 50% Grade 4 Stop Rx Stop Rx AT SCHEDULED RETREATMENT Blood/
Hematologic: Bone Marrow ANC <1,500 or Hold and .dwnarw. by
50%.sup.1 PLT <100,000 No change All Non-hematologic: Grade 3
Hold and .dwnarw. by 50%.sup.1 Hold and .dwnarw. by Grade 4 Stop Rx
50%.sup.1 Stop Rx Pain Bone pain Hold and .dwnarw. by 50%.sup.1
Hold and .dwnarw. by (grade 3, 4) 50%.sup.1 Myalgia/arthralgia Hold
and .dwnarw. by 50%.sup.1 Hold and .dwnarw. by (grade 3, 4)
50%.sup.1 Skin Grade 3 Hold and .dwnarw. by 50%.sup.1 Hold and
.dwnarw. by Grade 4 Stop Rx 50%.sup.1 Stop Rx
[0113] 1. Hold treatment until level of toxicity is grade 2 or less
before proceeding with therapy at 50% decreased dose. If patient
does not recover to grade 2 toxicity or less after a 4-week delay
in therapy, remove from study.
[0114] Ancillary Treatment
[0115] Mild myalgia/artlralgia can be managed with acetaminophen or
non-steroidal anti-inflammatory agents.
[0116] Toxicity is Monitored and Adverse Reactions Reported
[0117] Treatment Evaluation
[0118] Schedule of Evaluations: For the purposes of this study,
patients are reevaluated every 6 weeks. In addition to a baseline
scan, confirmatory scans are also obtained 6 weeks following
initial documentation of objective response.
[0119] Guidelines for Evaluation of Measurable Disease
[0120] Minimum disease requirement is 1 cm.times.1 cm. All
measurements are recorded in metric notation using a ruler or
calipers. All baseline evaluations are performed as close as
possible to the treatment start and not more than 14 days before
the beginning of the treatment.
[0121] The same method of assessment and the same technique is used
to characterize each identified and reported lesion at baseline and
during follow-up. Imaging-based evaluation is preferred to
evaluation by clinical examination when both methods have been used
to assess the antitumor effect of a treatment.
[0122] Clinical lesions are considered measurable only when they
are superficial (e.g. skin nodules, palpable lymph nodes). In the
case of skin lesions, documentation by color photography including
a ruler to estimate the size of the lesions is recommended.
[0123] Lesions on chest x-ray are acceptable as measurable lesions
when they are clearly defined and surrounded by aerated lung.
However, CT is preferred.
[0124] Conventional CT and MRI is performed with cuts of 10 mm or
less in slice thickness contiguously. A single technique of imaging
is employed in all assessments of tumor response throughout the
study.
[0125] Cytology and Histology:
[0126] If the measurable disease is restricted to a solitary
lesion, its neoplastic nature is confirmed by
cytology/histology.
[0127] These techniques can be used to differentiate between
partial response and complete response in rare cases, for example,
residual lesions in tumor types such as germ cell tumors, where
known residual benign tumors can remain.
[0128] The cytological confirmation of the neoplastic origin of any
effusion that appears or worsens during treatment when the
measurable tumor has met criteria for response of stable disease is
mandatory to differentiate between response or stable disease (an
effusion may be a side effect of the treatment) and progressive
disease.
[0129] Measurement of Effect
[0130] Target Lesions
[0131] All measurable lesions up to a maximum of 10 lesions
representative of all involved organs are identified as target
lesions and recorded and measured at baseline. Target lesions are
selected on the basis of their size (lesions with the longest
diameter) and their suitability for accurate repetitive
measurements (either by imaging techniques or clinically).
[0132] The sum of the longest diameter (LD) for all target lesions
is calculated and reported as the baseline sum LD. The baseline LD
is used as reference to further characterize the objective tumor
response of the measurable dimension of the disease.
[0133] Non-Target Lesions: All other lesions (or sites of disease)
are identified as non-target lesions and are also recorded at
baseline. Measurements are not required, and these lesions are
followed as "present" or "absent".
[0134] Response Criteria
[0135] Evaluation of target lesions
[0136] Complete Response (CR): Disappearance of all target
lesions.
[0137] Partial Response (PR): At least a 30% decrease in the sum of
the longest diameter (LD) of target lesions taking as reference the
baseline sum LD.
[0138] Progression (PD): At least a 20% increase in the sum of LD
of target lesions taking as reference the smallest sum LD recorded
since the treatment started or the appearance of one or more new
lesions.
[0139] Stable Disease (SD): Neither sufficient shrinkage to qualify
for PR nor sufficient increase to qualify for PD talking as
references the smallest sum LD.
[0140] Evaluation of non-target lesions
[0141] Complete Response (CR): Disappearance of all non-target
lesions and normalization of tumor marker level.
[0142] Non-Complete Response (non-CR): Persistence of one or more
non-target lesions and/or maintenance of tumor marker level above
the normal limits.
[0143] Progression (PD): Appearance of one or more new lesions.
Unequivocal progression of existing non-target lesions.
[0144] Note: Although a clear progression of "non-target" lesions
only is exceptional, in such circumstances, the opinion of the
treating physician should prevail, and the progression status
should be confirmed at a later time by the review panel (or study
chair).
[0145] If tumor markers are initially above the upper normal limit,
they must normalize for a patient to be considered in complete
clinical response.
[0146] Confirmation
[0147] To be assigned a status of PR or CR, changes in tumor
measurements are confirmed by repeat studies that should be
performed 6 weeks after the criteria for response are first
met.
[0148] In the case of SD, follow-up measurements must have met the
SD criteria at least once after study entry at a minimum interval
of 6 weeks.
[0149] Evaluation of Best Overall Response
[0150] The best overall response is the best response recorded from
the start of the treatment until disease progression/recurrence
(taking as reference for progressive disease the smallest
measurements recorded since the treatment started). The patient's
best response assignment will depend on the achievement of both
measurement and confirmation criteria (see section 11.41).
8 Non-Target New Target Lesions Lesions Lesions Overall Response
Complete response Complete No Complete response response Complete
response Non-CR/Non-PD No Partial response Partial response Non-PD
No Partial response Stable disease Non-PD No Stable disease
Progressive disease Any Yes or No Progressive disease Any
Progressive Yes or No Progressive disease disease Any Any Yes
Progressive disease
[0151] Patients with global deterioration of health status
requiring discontinuation of treatment without objective evidence
of disease progression at that time are reported as "symptomatic
deterioration." Every effort is made to document the objective
progression even after discontinuation of treatment.
[0152] In some circumstances it may be difficult to distinguish
residual disease from normal tissue. When the evaluation of
complete response depends upon this determination, it is
recommended that the residual lesion be investigated (fine needle
aspirate/biopsy) before confirming the complete response
status.
[0153] Duration of Response
[0154] Duration of overall response is measured from the time
measurement criteria are met for CR/PR (whichever is first
recorded) until the first date that recurrent or progressive
disease is objectively documented (taking as reference for
progressive disease the smallest measurements recorded since the
treatment started).
[0155] Duration of overall complete response is measured from the
time measurement criteria are first met for CR until the first date
that recurrent disease is objectively documented.
[0156] Duration of stable disease is measured from the start of the
treatment until the criteria for progression are met, taking as
reference the smallest measurements recorded since the treatment
started.
[0157] Descriptive Factor Disease Status: None
[0158] Treatment/Follow-up Decision at Evaluation of Patient
Patients in whom disease has not progressed and who have
experienced acceptable toxicity are eligible for re-treatment at
the same dose level for the duration of the study until 24 weeks.
After 25 weeks, continual treatment every 3 months up to 18 months
post randomization is at the discretion of the clinician.
[0159] Patients in whom disease has not progressed and who have
experienced unacceptable toxicity are eligible for re-treatment at
a lower dose (see Section 8.0) until week 24 of the study. After 25
weeks, continued treatment every 3 months for up to 18 months post
randomization is at the discretion of the clinician. Re-treatment
of patients with grade 3 or 4 toxicities, other than those
identified in the dose modification table, is discussed with the
study chair before receiving further protocol therapy.
[0160] Those patients with progression of disease or who refuse
further treatment will go to the event-monitoring phase. Tumor
progression and death is monitored every 3 months until death or 3
years post randomization. No follow-up is required after 3 years
post randomization.
[0161] Re-treatment of patients: Decisions regarding re-treatment
of any patient is based upon the type, severity, duration, and
reversibility of the toxic reactions. In general, patients who show
improvement or stability of their disease continues to receive
treatment beyond the 24-week duration of the study. Immunizations
are performed at 3-month intervals. Patients having objective
progression of disease or clinical deterioration will not continue
to receive treatment on this study.
[0162] If a patient fails to complete the initial cycle of therapy
(defined as drug administration and 3 weeks observation) for
reasons other than toxicity, the patient is regarded as treatment
intolerant and an additional patient is treated at the current dose
level; however, all toxicity information will be utilized in the
analysis. For these instances, a specific notation is made for
review by a study committee.
[0163] Pharmacologic/Ancillary Studies
[0164] Immlunological Assays: The principal objective of monitoring
the CTL response to gp-100 is to estimate the effect of in vivo
priming with peptides in conjunction with administration of GM-CSF
on the frequency of CTLs specific for these peptides. Such
estimates can not be accurately derived from standard assays of CTL
function, i.e. target lysis, but rather require methods that
actually estimate the frequency of CTLs. Two relatively new
approaches to estimating CTL frequency are promising for this
purpose: (1) staining with class I tetramers and (2) intracellular
staining for cytoline production. Class I tetramers are comprised
of monomeric class I heavy chains that have been folded with
.beta.2-microglobulin (.beta.2M) and specific immunogenic peptides;
these monomers are then biotinylated at sites on their carboxy ends
for conjugation into tetramers with PE-avidin. Such tetramers have
increased valency and can be mixed with FITC-labeled anti-CD8
antibody to stain peptide-specific CTLs for enumeration by flow
cytometry. Intracellular staining for cytolcines provides a
complementary approach in which CTLs are boosted in vitro with
peptide-sensitized stimulator cells in the presence of Brefeldin A
that blocks transport such that cytokines accumulate in the
responding cells. Stimulated cells are then permeabilized and
stained with anti-cytolcine and anti-CD8 antibodies to estimate the
frequency of specifically responding CD8.sup.+ T cells by flow
cytometry. Both approaches can be used to estimate the effect of in
vivo peptide priming on the frequency of specific CTLs.
[0165] One hundred milliliters of blood is drawn prior to
initiation of peptide immunization and at 7 d after each
immunization. Peripheral blood lymphocytes (PBLs) are enriched by
flotation in Ficoll gradients and frozen in 10% DMSO for storage.
PBLs from each blood draw are tested with a panel of monoclonal
antibodies to estimate the percentages of CD4.sup.+ and CD8.sup.+ T
cells as well as B cells, monocytes, and dendritic cells.
[0166] In addition, assays are performed to estimate T cell
responses to polyclonal stimulus (PHA), recall antigens (tetanus
toxoid), and HLA alloantigens. These two sets of experiments are
used for evaluation of overall T cell responsiveness.
[0167] PBLs are stimulated with the melanoma peptide to stimulate
and expand CTLs specific for the vaccine gp100 peptide and
influenza (GILGFVFTL) peptides. Briefly, PBLs are diluted to
2.5.times.10.sup.6 cells/ml and incubated at 37.degree. C./5%
CO.sub.2 in the presence of 100 .mu.g peptide in round bottom
microtiter wells. After 3 d of culture, recombinant IL-2 is added
to a concentration of 20U/ml. Responder T cells are harvested after
6-7 d of culture and mixed in a standard cell mediated lympholysis
(CML) assay at varying ratios with .sup.51Cr-labelled target cells
sensitized with titrated concentrations of peptide. Target cells
include T2 and 221.A2 cells and sensitizing peptides include the
specific target peptide as well as a negative control. These assays
are used to identify those PBL samples that include CTLs specific
for the melanoma peptide and provide a first-order comparison of
CTL function.
[0168] More definitive estimates of specific CTL frequency are
generated through tetramer analysis. The plasmid vectors for
synthesis of human .beta.2M and HLA-A2 heavy chains were obtained.
The laboratory successfully folded HLA-A2 heavy chain monomers that
include melanoma peptides. A2 heavy chain molecules are mixed with
b2M and gp100 and influenza peptides. Folded monomers are
biotinylated with BirA enzyme and conjugated with PE-avidin. Each
tetramer preparation is tested with specific CTLs to confirm
activity. The CTL lines were generated by repeated stimulation of
PBLs collected from normal HLA-A2.sup.+ donors with the specific
peptide.
[0169] Double staining with PE-labeled tetramers and FITC-labeled
anti-CD8 monoclonal antibody are performed in the presence of Fc
receptor-specific antibody to block non-specific staining. Stained
cells are analyzed by flow cytometry and estimates of the
percentages of doubly stained CTLs are obtained. Pre-immunization
PBLs are compared with PBLs collected after each of the successive
peptide immunizations to assess the efficiency of peptide
immunization on CTL frequency. Tetramers are also used to
specifically sort CD8.sup.+ CTLs for functional assays. Sorting is
performed to purify tetramer/CD8 double positive cells that are
expanded 10 d with ConA and IL-2; these expanded CTLs are tested
with peptide-sensitized targets in a standard CML assay to confirm
that the sorted CTLs exhibit lytic function.
[0170] Estimates of peptide-specific CTLs are also be obtained by
intracellular staining for cytokines following in vitro stimulation
with peptide-sensitized stimulator cells. Pre- and
post-immunization PBLs are stimulated with individual peptides as
outlined above. After 7 d of culture, cells are diluted and
stimulated with T2 cells pulsed with peptide for 5-6 hr in the
presence of Brefeldin A. Responder cells are then fixed,
permeabilized, and stained with anti-CD8 antibody in conjunction
with antibodies specific for IFN and GM-CSF for flow cytometiic
analysis. These assays yield the frequencies of CTLs that are
responsive to peptide stimulation through cytolidne release.
[0171] Estimates of peptide-specific, IFN-gamma-producing CTLs and
Th cells are obtained by Elispot assays following in vitro
stimulation with peptide-sensitized stimulator cells. CD8+ (CTLs)
and CD4+ (Th cells) are purified from pre- and post-immunization
PBLs by magnetic bead separation. These responder cells are
stimulated with autologous CD8-/CD4- PBLs pulsed with the gp100
peptide. After 7 d of culture, cells will be diluted, titrated and
stimulated with T2 cells pulsed with peptide for 16-24 hr in
microtiter wells coated with anti-IFN-gamma capture antibody. The
test peptides include the gp100 peptide and a negative control
peptide (influenza). The wells are then washed and treated with
ALP-conjugated detection antibody for estimation of the number of
spots observed for each responder cell titration. The difference
between the frequency of spots obtained with gp100 and influenza
peptide stimulation is the estimate of the frequency of gp100
peptide-specific, IFN-gamma-producing T cells with either the CD8+
or CD4+phenotype. In parallel, CD8+ and CD4.sup.+ T cells are
stimulated with T2 cells pulsed with gp100 and influenza peptide
and harvested after 6 hr for total RNA extraction. Quantitative,
competitive RT-PCR is performed to estimate the numbers of copies
of IFN-gamma message/ng total RNA. These three assays are used to
evaluate the effects of peptide immunization on (1) the frequency
of CTLs that recognize the gp100 peptide, (2) the frequencies of
CTLs and Th cells that produce IFN-ganuna in response to the gp100
peptide, and (3) the total amount of IFN-gamma message produced by
CTLs and Th cells in response to the gp100 peptide.
[0172] Delayed-type Hypersensitivity (DTH) Testing: General
cell-mediated immunity is functionally assessed by DTH testing
prior to the initiation at the conclusion of therapy. A standard
antigen series, including Candida, mumps and tetanus is applied to
the left or right volar forearm of each patient. 0.1 ml of a
standard, commercial preparation of each antigen is injected
intradermally. Forty-eight hours later, each injection site is
visually observed and the extent of erythema and induration
recorded on a scale of 0-4. The number of positive test sites is
compared before and after therapy. If the patient demonstrates
anergy (no positive test sites) prior to initiation of therapy,
he/she will still be eligible for enrollment.
[0173] Patients are also tested to the individual peptide antigens
at a dose of 10 .mu.g peptide saline solution. Initial testing is
performed prior to initiation of therapy and also prior to cycle 7.
Extent of erythema and induration is recorded in an identical
manner as the standard antigen battery.
[0174] Tuberculin syringes pre-loaded with 10 .mu.g of peptide in
100 .mu.L of IFA are injected intradermally in the
extremities/areas of the body away from sites of prior
immunizations with the peptide vaccines.
[0175] Lymph node aspiration: If there is clinical evidence of
lymph node enlargement in the region of immunization, a fine needle
aspiration biopsy of the lymph nodes is performed. The collected
specimens are studied for frequency of CTLs directed against the
peptide vaccine.
9 Sample Schedule (in weeks): Cycle length is 21 days Cycle Cycle
of Rx Pre-Rx 1 2 3 4 5 6 7 8 Week of study -- 0 3 6 9 12 15 18 24
Treatment X X X X X X X X CBC, CHEM X X X X X X X X Immunologic
tests X X X X X X X X Injection site reaction X X X X X X X DTH X X
Node aspirate.sup.1 X .sup.1Only if node is clinically
enlarged.
[0176] Sample Preparation: Peripheral blood is collected in purple
top Vacutainer tubes. A special study refer card is generated to
identify these specimens. The samples are kept at room temperature
until use, which will be within 2 hours of collection.
[0177] Vaccine Information
[0178] Melanoma Peptides
[0179] Storage and Stability:
[0180] Store intact vials frozen at -10.degree. to -20.degree. C.
The Tyrosinase peptide is labeled for refrigerated storage, but can
be frozen to allow storage with the other peptides; freezing does
not negatively effect the stability of the solution. Thawed vials
may be stored for up to 6 hours at refrigerated temperatures. Once
thawed, the peptide is used within 3 hours when kept at room
temperature. Long-term stability of the peptides in intact vials is
under evaluation through an on-going stability testing program.
[0181] Montanide ISA 51 Adjuvant (Incomplete Freund's Adjuvant,
IFA)
[0182] Montanide ISA 51, NSC 675756, is an oil-based adjuvant
product similar to Incomplete Freund's Adjuvant which, when mixed
with a water-based solution on 1:1 w/w ratio, forms a water-in-oil
emulsion. It consists of a highly purified oil, Dralcol VR, and a
surfactant, mannide oleate. Montanide ISA 51 is manufactured by
Seppic, Inc., and is provided in amber glass ampoules containing 3
mL of the solution. Montanide ISA 51 is provided through the Cancer
Therapy Evaluation Program, NCI. Montanide ISA 51 is an
Investigational New Drug.
[0183] Mode of Action:
[0184] Acts to enhance immune response to vaccination.
[0185] Storage and Stability:
[0186] The solution is stored at controlled room temperature.
Exposure to cold temperatures may result in a clouded solution,
which should be discarded. An expiration date is printed on the
ampoule label.
[0187] Compatibility Notes:
[0188] This solution can be filtered as it's being withdrawn from
the glass ampoule to remove any glass particles. The oil will break
down the rubber tip of the plunger on syringes; it is advisable to
use a different syringe for each ampoule. Do not allow the
Montanide ISA 51 to be in direct contact with the rubber tip of the
plunger for more time than is necessary to withdraw the solution
and inject it into the peptide vial. Fresh syringes can be used to
withdraw the emulsified vaccine from the vaccine vial. Once the
emulsion is made, there is less interaction of the oil directly
with the rubber tip of the plunger.
[0189] GM-CSF (Leukine.RTM., Sargramostatin)
[0190] For this protocol, the lyophyllized version of Leukine is
used. Each vial contains 250 mcg of sargramostim, 40 mg mannitol,
10 mg sucrose, NF and 1.2 mg tromethamine.
[0191] Preparation and Storage:
[0192] Aseptically reconstitute a 250 mcg vial with 1.0 mL
Bacteriostatic Water for Injection, USP. During reconstitution, the
diluent is directed at the side of the vial and the contents gently
swirled to avoid foaming. Do not agitate or shake. The
reconstituted Leukine solution is clear, colorless, isotonic with a
pH of 7.4.+-.0.3, and contains 250 mcg/ml of sargramostim. When
reconstituted in this manner, the solution is stable for up to 20
days when stored at 4-8.degree. C. Do not freeze. Unused vials
should be stored as per the manufacturer's direction.
[0193] Vaccine Preparation Instructions
[0194] General Vaccine Preparation Information
[0195] Emulsify each peptide or peptide/GM-CSF mixture individually
with Montanide ISA-51. Prepare the vials as directed for each group
below. Place the vials upside down in a tube platform holder of a
vortex machine and vortex at highest speed for 12 minutes. This
will minimize the amount of emulsion adhering to the inside surface
of the vial. Because neither the peptide solution nor the Montanide
ISA-51 contain preservatives or bacteriostatics, the prepared
peptide vaccines should be administered as soon as possible.
[0196] Group A
[0197] Remove the peptide vial from the freezer and allow thawing
at room temperature. Once thawed, add 1.2 mL of Montanide ISA to
the vial and vortex.
[0198] Group B
[0199] Remove one vial of the peptide from the freezer and allow
thawing at room temperature. Reconstitute a vial of GM-CSF
(sargramostatin), 250 mcg, with 1 mL Bacteriostatic Water for
Injection, USP, yielding a concentration of 250 mcg/mL. Add 0.25 mL
of this solution (62.5 mcg) to the peptide vial. Add 1.25 mL of
Montanide ISA 51 to the peptide vial. Load two tuberculin syringes
with equal volumes of this solution prior to use. Discard unused
GM-CSF.
[0200] Group C
[0201] Remove one vial of the peptide from the freezer and allow
thawing at room temperature. Reconstitute a vial of GM-CSF
(sargramostatin), 250 mcg, with 1 mL Bacteriostatic Water for
Injection, USP, yielding a concentration of 250 mcg/nL. Add 0.4 mL
of this solution (100 mcg) to the peptide vial. Add 1.25 ML of
Montanide ISA 51 to the peptide vial. Load two tuberculin syringes
with equal volumes of this solution prior to use. Discard unused
GM-CSF.
[0202] Vaccine Administration Information
[0203] Dose Specifics:
[0204] Each peptide vaccine will consist of a total volume of 1-1.5
nL containing 1 mg of the peptide, 1 mL of Montanide ISA 51 (1.0
mL) and, for Group B, .about.50 .mu.g GM-CSF (1.25 mL), or, for
Group C, 100 .mu.g GM-CSF (1.5 mL).
[0205] Administration:
[0206] Vaccinations are given on day 0 of each treatment cycle. Due
to the large volume, each peptide vaccine is administered in 2
shots in a contiguous location (a total of 2 shots per vaccination
session). The peptide vaccines should be injected in the vicinity
of one of the major nodal basins. This basin must not have been
dissected. Subsequent immunizations should be rotated among
available nodal basins (both arms or thighs).
[0207] Site and date of administration are recorded in the
treatment record.
[0208] Nursing Implication:
[0209] Inform patient of possible side effects. Answer any
questions.
[0210] Do not premedicate unless absolutely necessary.
Acetaminophen is the preferred analgesic if needed, but
nonsteroidal anti-inflammatory agents or aspirin may be used.
Diphenhydramine may be used to reduce erythema if needed.
[0211] Monitor for acute reactions. Outpatients may need to be
observed for 1-2 hours after receiving first dose.
[0212] Vaccine Side Effects
[0213] Because of the low dose of GM-CSF used and the slow-release
nature of the vaccine emulsion, side effects normally seen with
systemic treatment doses of GM-CSF should not play a factor in this
vaccination treatment. Expected side effects are related to the
peptides (vitiligo) and Montanide ISA 51. It is possible that the
GM-CSF may potentiate the reaction seen at the injection site.
[0214] Dermatology/Skin: Injection site reaction, rare granuloma
formation, possible development or worsening of pre-existing
vitiligo, rash.
[0215] Hepatic: Transient rises in liver transaminases.
[0216] Constitutional: Low-grade fever.
[0217] OcularNisual: Possible damage to the eye (no reported
incidences to date).
[0218] Statistical Considerations and Methodology:
[0219] Study design: This is a pilot study designed to determine
the immunological effects of tumor vaccine (differentiation antigen
peptide) challenge in the presence of various concentrations of
immune adjuvants GM-CSF and incomplete Freund's adjuvant. The
secondary goals of the study examine the toxicity profile of each
treatment combination and report the number of objective responses
observed.
[0220] The study design chosen for this proposal is a stratified
randomized design with type of dominant disease (see section 5.0)
as the sole stratification factor. This design was chosen rather
than that of a traditional phase I design as the dose levels of
each reagent are quite low (GM-CSF doses are below 10% of commonly
used doses). At these levels, the anticipated number of toxicities
and their severity are thought to be minimal. Toxicities will be
carefully monitored and accrual will be suspended if 2 or more of
the first six patients experience a grade 4 hematologic toxicity
lasting for 5 or more days or a rise in serum creatinine to levels
2 or more times that of their baseline value.
[0221] Accrual: Ten patients with stage 1V malignant melanoma are
randomized to each one of the 3 treatment combinations. The total
number of eligible patients to be accrued is 30. Patients are
allocated to each dose level using a dynamic allocation procedure
that balances the marginal distribution of type of dominant disease
between treatment combinations.
[0222] Endpoints: The primary end points for this pilot study are
the changes in tumor antigen peptide specific immune responses (in
vitro and DTH) from pretreatment levels. PBLs are tested with a
panel of monoclonal antibodies to estimate the percentage of
CD4.sup.+ T cells, CD8+T cells, B cells, monocytes, and dendritic
cells. Double staining with PE-labeled tetramers and FITC-labeled
anti-CD8 monoclonal antibody is performed in the presence of Fc
receptor specific antibody and the percentage of double stained
CTLs will be obtained. Forty-eight hours after injection, each
injection sight is inspected for the extent of erythema and
induration.
[0223] Secondary end points include the number and severity of
hematologic and non-hematologic toxicities observed and the
proportion of objective responses observed. All patients meeting
the eligibility criteria who have signed a consent form and who
have begun treatment are considered evaluable for the estimation of
the objective response proportion. The objective response
proportion is estimated by the number of patients with an objective
status of complete response or partial response on two consecutive
tumor evaluations at least 6 weeks apart divided by the total
number of patients evaluable for response.
[0224] Analysis plan: No formal hypothesis testing is performed in
this study. Agreement between the two methods used to estimate CTL
frequency is assessed using the graphical methods proposed by Bland
and Altman. The analysis plan for the gp-100 peptide antigen and
GM-CSF is as follows: Plots are constructed of the of the percent
of CD4+ T cells, CD8+T cells, B cells, monocytes, dendritic cells
and double stained CTLs present against time. Also, plots of the
percent change in these factors from their pretreatment levels
against time will be constructed. These plots enable visual
assessment of patterns of change and variability within a treatment
regimen and between treatment regimens. The extent of erythema and
induration 48 hours after injection is plotted against time for the
peptide antigen and GM-CSF to assess whether extent of erythema and
induration is a function of the number of injections or peptide
antigen/GM-CSF pair.
[0225] Optimal immunization strategies are those which produce a 5
fold increase from pretreatment level 6 in most of these variables,
which is maintained at least 6 weeks in 7 out of 10 patients
treated at that level, with little toxicity and a small degree of
erythema/induration.
Other Embodiments
[0226] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *