CA2351642A1 - Immune reactivity to expressed activated oncogenes for diagnosis and treatment of malignancy - Google Patents
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Abstract
Methods for the detection, monitoring and treatment of malignancies are disclosed. Detection of the proliferation of T
cells in response to in vitro exposure to a protein expression product of an acivated oncogene or cancer-related gene associated with a malignancy, or detection or immunocomplexes formed between the protein expression product and antibodies in body fluid, allows the diagnosis of the presence of a malignancy. The present invention also discloses methods and compositions for treating a malignancy.
cells in response to in vitro exposure to a protein expression product of an acivated oncogene or cancer-related gene associated with a malignancy, or detection or immunocomplexes formed between the protein expression product and antibodies in body fluid, allows the diagnosis of the presence of a malignancy. The present invention also discloses methods and compositions for treating a malignancy.
Description
IMMUNE REACTIVITY TO EXPRESSED ACTIVATED ONCOGEN~S
FOR DIAGNOSIS AND TREATMENT OF MALIGNANCY
Technical Field The present invention is generally directed toward the detecaion, monitoring, and treatment of malignancies through the use of a cancer patient's own l0 immune reactivity 1to the protein expression product of an activated oncogene or cancer-related gene associated with malignancy.
Bac around of the Invs:ntion Despite ~snormous investments of financial and human resources, cancer remains one of the major causes of death. A common characteristic of malignancies is uncontrolled cell growth. Cancer cells appear to have undergone a process of transformation from the normal phenotype to a malignant phenotype capable of autonomous growth. Mutation of somatic cell genes is considered to be a common primary event that results in the transformation of ;normal cells to malignant cells. The malignant phenotypic characteristics encoded by the mutated genes are passed on during cell division to the progeny of the transformed cells. Various genes involved with transformation have been designated as oncogenes.
Oncogenes were originally identified as components of the genetic material a~f oncogenic viruses. The homologous genes on human chromosomes are commonly termed oncogenes or proto-oncogenes.
Ongoing research involving oncogenes has identified at least forty oncogenes operative in malignant cells and responsible for, or associated with, transformation. Oncogenes have been classified into different groups based on the putative function or location of their gene products (such as the protein expressed by the oncogene).
Proto-oncogenes are believed to be essential for certain aspects o;f normal cellular physiology. Certain proto-oncogenes appear to be activated to a cellular oncogene through quantitative mechanisms that result from increased or deregulated expression of an essentially normal gene product. For example, the myc gene family has been associated with initiation and/or progression of certain human lymplZOmas and carcinomas, whose transforming activation is the result of quantitative mechanisms.
Alternatively, other proto-oncogenes appear to be activated to transforming cellular oncogenes through qualitative mechanisms, including mutation in the coding sequence of the gene. This creates a gene product with an altered primary structure and biochemical properties as a result of one or more differences in the amino acid sequence of the protein. For example, the ras gene family, causally associated with the most common forms of human malignancy (e.g., colon cancer) is activated as a result of single codon changes.
Studies to develop cancer therapies have, in general, focused on the use of characteristic differences between normal and malignant cells. Mutated, translocated or otherwise ovE:rexpressed proto-oncogenes and the products of such .genes represent potential identifiable characteristic differences between normal and malignant cells. The identified differences have been utilized in attempts to develop diagnostic assays or therapeutic regimens.
An approach to developing a diagnostic assay has been to attempt to quantify the expressed product of an oncogene in tissue or body fluids, utilizing antibodies directed toward the unique or abnormal oncogene product.
In general, xenogeneic antibodies have been raised against the abnormally expressed proto-oncogene product. Problems in the development of diagnostic assays based on detecting abnormal oncogene products include the following factors:
(1) a lack of ant~:bodies with high specificity, affinity and selectivity for the abnormal product; (2) only small amounts of abnormal oncogene product may be released by tumor cells; (3) oncogenic products may be released only intermittently by tumor cells; (4) the oncogene product may be absorbed out of the body fluid by antibody or may be formed into immune complexes; and (5) the free antigen may be rapidly cleared or degraded.
Due to the difficulties in the current approaches to cancer diagnosis and therapy, there is a need in the art for improved methods and compositions.
The present invention fills this need, and further provides other related advantages.
Summary of the Invention Briefly stated, the present invention provides a variety of methods for the detection of a malignancy in a warm-blooded animal, wherein an activated oncogene or cancer-related gene: is associated with the malignancy.
The methods may be used on a one time basis when a malignancy is suspected or on a periodic basis to monitor an individual with an elevated risk of acquiring a malignancy. In on~a embodiment, the method comprises the steps of (a) inc»lbating T cells, isolated from a warm-blooded animal, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy; and (b) detecting the presence or absence of proliferation of the T cells, thereby determining the presence or absence of the malignancy. In another embodiment, the method comprises the steps of: (a) contacting a body fluid, suspected of containing antibodies specific for a protein expression product of an activated oncogene or cancer-related gene associated with the malignancy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
FOR DIAGNOSIS AND TREATMENT OF MALIGNANCY
Technical Field The present invention is generally directed toward the detecaion, monitoring, and treatment of malignancies through the use of a cancer patient's own l0 immune reactivity 1to the protein expression product of an activated oncogene or cancer-related gene associated with malignancy.
Bac around of the Invs:ntion Despite ~snormous investments of financial and human resources, cancer remains one of the major causes of death. A common characteristic of malignancies is uncontrolled cell growth. Cancer cells appear to have undergone a process of transformation from the normal phenotype to a malignant phenotype capable of autonomous growth. Mutation of somatic cell genes is considered to be a common primary event that results in the transformation of ;normal cells to malignant cells. The malignant phenotypic characteristics encoded by the mutated genes are passed on during cell division to the progeny of the transformed cells. Various genes involved with transformation have been designated as oncogenes.
Oncogenes were originally identified as components of the genetic material a~f oncogenic viruses. The homologous genes on human chromosomes are commonly termed oncogenes or proto-oncogenes.
Ongoing research involving oncogenes has identified at least forty oncogenes operative in malignant cells and responsible for, or associated with, transformation. Oncogenes have been classified into different groups based on the putative function or location of their gene products (such as the protein expressed by the oncogene).
Proto-oncogenes are believed to be essential for certain aspects o;f normal cellular physiology. Certain proto-oncogenes appear to be activated to a cellular oncogene through quantitative mechanisms that result from increased or deregulated expression of an essentially normal gene product. For example, the myc gene family has been associated with initiation and/or progression of certain human lymplZOmas and carcinomas, whose transforming activation is the result of quantitative mechanisms.
Alternatively, other proto-oncogenes appear to be activated to transforming cellular oncogenes through qualitative mechanisms, including mutation in the coding sequence of the gene. This creates a gene product with an altered primary structure and biochemical properties as a result of one or more differences in the amino acid sequence of the protein. For example, the ras gene family, causally associated with the most common forms of human malignancy (e.g., colon cancer) is activated as a result of single codon changes.
Studies to develop cancer therapies have, in general, focused on the use of characteristic differences between normal and malignant cells. Mutated, translocated or otherwise ovE:rexpressed proto-oncogenes and the products of such .genes represent potential identifiable characteristic differences between normal and malignant cells. The identified differences have been utilized in attempts to develop diagnostic assays or therapeutic regimens.
An approach to developing a diagnostic assay has been to attempt to quantify the expressed product of an oncogene in tissue or body fluids, utilizing antibodies directed toward the unique or abnormal oncogene product.
In general, xenogeneic antibodies have been raised against the abnormally expressed proto-oncogene product. Problems in the development of diagnostic assays based on detecting abnormal oncogene products include the following factors:
(1) a lack of ant~:bodies with high specificity, affinity and selectivity for the abnormal product; (2) only small amounts of abnormal oncogene product may be released by tumor cells; (3) oncogenic products may be released only intermittently by tumor cells; (4) the oncogene product may be absorbed out of the body fluid by antibody or may be formed into immune complexes; and (5) the free antigen may be rapidly cleared or degraded.
Due to the difficulties in the current approaches to cancer diagnosis and therapy, there is a need in the art for improved methods and compositions.
The present invention fills this need, and further provides other related advantages.
Summary of the Invention Briefly stated, the present invention provides a variety of methods for the detection of a malignancy in a warm-blooded animal, wherein an activated oncogene or cancer-related gene: is associated with the malignancy.
The methods may be used on a one time basis when a malignancy is suspected or on a periodic basis to monitor an individual with an elevated risk of acquiring a malignancy. In on~a embodiment, the method comprises the steps of (a) inc»lbating T cells, isolated from a warm-blooded animal, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy; and (b) detecting the presence or absence of proliferation of the T cells, thereby determining the presence or absence of the malignancy. In another embodiment, the method comprises the steps of: (a) contacting a body fluid, suspected of containing antibodies specific for a protein expression product of an activated oncogene or cancer-related gene associated with the malignancy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
(b) incubating the body fluid under conditions and for a time sufficient t:a allow immunocomplexes to form; and (c) detecting the presence or absence of one or more immunocomplexes formed between the protein expression product and antibodies in the body fluid specific for the protein expression product, thereby determining the presence or absence of the malignancy.
In another aspect, the present invention provides methods for monitoring the effectiveness of l0 cancer therapy in a warm-blooded animal with a malignancy, wherein an acti~at.ed oncogene or cancer-related gene is associated with the malignancy. In one embodiment, the method comprises the steps of: (a) contacting a first body fluid sample,, taken from the warm-blooded animal prior to initiation of therapy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
(b) incubating the body fluid under conditions and for a time sufficient t,o allow immunocomplexes to form;
(c) detecting immuriocomplexes formed between the protein expression product and antibodies in the body fluid specific for the protein expression product; (d) repeating steps (a), (b), and (c) on a second body fluid sample taken from the animal subsequent to the initiation of therapy; and (e) comparing the number of immunocomplexes detected in the first and second body fluid samples, thereby monitoring the effectiveness of the therapy in the animal.
The present invention is also directed toward methods for treating a malignancy in a warm-blooded animal, wherein an activated oncogene or cancer-related gene is associated with the malignancy. In one embodiment, the method comprises the steps of:
(a) isolating T cells from a warm-blooded animal;
(b) incubating the 'T cells in the presence of at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy, such WO 9l/11719 PCT/US91/00497 that the T cells proliferate; and (c) administering to the warm-blooded animal an effective amount of the proliferated T cells. In another embodiment, the method comprises the step; of: (a) isolating T cells from a 5 warm-blooded animal; (b) incubating the T cells in the presence of at Ieasi: one protein expression product of an activated oncagene or cancer-related gene associated with the malignancy, svuch that the T cells proliferate;
(c) cloning one or more cells that proliferated in the presence of the protein expression product; and (d) administering to the warm-blooded animal an effective amount of the cloned. T cells. In a third embodiment, the method comprises immunizing the animal with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy.
Within a related aspect, the present invention provides anti-cancer therapeutic compositions comprising T cells proliferated: in the presence of at least one protein expression product of an activated oncogene or cancer-related gene associated with a malignancy, in combination with a physiologically acceptable carrier or diluent. Such T cells are useful in the manufacture of a medicament for treai_.ing a malignancy in a warm-blooded animal.
brief Description of the Drawing's Figure 1 graphically illustrates that specif is T cell responses to point-mutated ras peptide can be generated by in vivo priming on day 0 with 50 ~tg of a peptide of 12 amino acid length constructed identically to murine ras-p21 (amino acids 5-16) but with the substitution of arginine for the glycine normally found at position 12 (i.e., position 12 of ras-p21 but position 8 of the peptide). This peptide is denoted as ras p5-16(R12] or as ras-R12. Mice were immunized with ras-R12 peptide plus adjuvant or with adjuvant alone. After 10 days, the draining lymph nodes were harvested and lymphocytes stimulated in vitro with ras-R12 peptide or as specificity controls with similar peptides substituted with serine, cysteine or glycine. at position 12 as opposed to arginine, denoted as ras-S12, ras-C12 and ras-G12, respectively.
Four days later, cultures were pulsed with [3H]-thymidine (3HTdR) for 8 hours, as described in Example 1 below.
Results are represented as ~CPM.
Figure 2 shows that ras-peptide specific T cells grown long-term invitm in response to intermittent stimulation by ras-peptide retain specific function.
T cells derived from mesenteric lymph nodes of C57BL/6 mice primed to ras-R12 (as defined for Figure 1 above) were cultured long-term in vitro in response to intermittent stimulation With ras-R12 peptide (5 ~cg/ml) on irradiated 86 spleen cells (3000 rad) as antigen presenting cells.
The specificity of the T cell line was tested on day 85 by stimulating with graded concentrations of various ras peptides (as defined for Figure 1 above) or with trypsinized OVA (TOVA) containing several potential immunogenic peptides. Data is represented as 3HTdR uptake in counts per minutE: (c.p.m.).
Figure :3 graphically illustrates that T cells specific for a ras peptide respond specifically to intact ras p21 protein containing the same residue substitution.
This peptide, denoted as p5-1'7 [R12 ] , consists of 13 amino acid residues and was constructed identically to marine ras-p21 (amino acids 5-17) but with the substitution of arginine (R) for the glycine (G) normally found at position 12 of ras~-p21. Cloned B6 T cells specific for the Arg-12 ras peptide were cultured with irradiated B6 spleen cells and either p5-17[R12] or intact ras p21 proteins bearing the designated amino acid at position-12 (1 ~tg/ml). Proliferative responses were measured after four days (as described in Figure 1). The data represent the mean of triplicate determinations of the c.p.m. of incorporated 3HTdR. The standard deviations were <10% of the c.p.m.
WO 91/11719 PCT/LS9l/0(1497 Figure 4 shows that specific T call responses can ba generated by in vivo priming With a peptide of 13 amino acid length constructed identically to marina ras-p21 (amino acids 54-66) but with the substitution of laucina (L) for the glutamina (Q) normally found at position 61 of ras-p21. This peptide is denoted as p54-66[L61] or [L61]. C3H/HeN mica ware immunized twice subcutanaously at two weak intervals with Ribi adjuvant alone or adjuvant emulsified with p54-66[L61] ras peptide ( 100 ~tg) . Two weeks altar the f final immunization, spleen calls from mica in;Iactad with adjuvant alone (open bar) or adjuvant plus p54~~66[L61] ras peptide (black bar) ware harvested and tested in vitnv for prolifarativa response to the indicated p51-c~6 ras peptide ( 100 ~tg/ml) . Tha peptide containing the substitution of lysine (R) for glutamina at position 61 is denoted as [R51]. The data represent the mean of triplica~ta determinations of the c.p.m. of incorporated 38TdR ~ S.D.
Pigura 5 graphically illustrates that intact ras pZi[L61] protein is specifically recognized by a ras peptide-induced T cell lira. Specific T cell lines and clones ware ganas~atad and maintained as described in Example 2 below. A T call line which exhibits specific reactivity to the ras peptide p54-66[L61] (penal 1~) and a T call clone which exhibits specific reactivity to the ras peptide ps-17[R1~] (panel B) ware stimulated with (a) no antiq~n, (b~ sensitising ras peptide, or (c) intact ras pZi[h~1J protein. The ra ps4-66[L61] specific T call lira wss of C38 origin and the ras p5-17[R12] specific T
call clone was of B6 origin. Prol~ifarativa assays ware psrtormad with acynganaic irrsdiatad spleen calls as antigen presenting cells (as described in Figure 1).
Stimulating peptides and proteins ware used at a concentration of 5 Wg/ml. The ras pZi[L61] protein was produced in E coli H8101 using the prokaryotic expression plasmid plat-L9 and purif iad by sequential DFaE-Saphacal WO 91/11'19 PCT/US91/00497 and Ssphadex~ colLnmn chromatography. The data represent the mean c.p.m. of incorporated 3HTdR ~ S.D.
Detailed Descri~ticn of the Invention Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter.
Activated oncoa~ene - as used herein, racers to proto-oncogenes that have bscome activated, leading to the expression of protein produets with amino acid sequences other than those of the protein products expressed by the normal proto-oncoganas.
Cancer-ry,lated Qene - as used herein, reters to any altered gsne (other than an oncogena) associated with the devalopnent o=~ maintenance o! the malignant phenotype.
Examples of such altered genes include mutants of the p53 tusor supprassor gene.
Protein exflression product - as used herein, includes proteins, polypeptidsa, and peptides; and may be an intact molecule, a fragaent thereof, or a functional equivalent thereat; and may ba genetically engineered.
Proliferation o! T cells - as used herein, includes the multiplication of T cells a: wall as the stisulation o! T calls leading to multiplication, i.a., the im3.tiation of events leading to mitosis and aitosis itself. Methods for detecting proliferation of T calls are discussed below.
As noted above, the present invention is directed toward aethods and compositions for the diagnosis, monitoring and treataant of malignancies in a wars-bloodsd animal, wherein activated oncogenss or cancer-related genes are associated with the malignancies.
The disclosure o1.' the present invention shows that the protein expression products of activated oncoganes and cancer-related genes can be recognized by thymus-dependant lymphocytes (hereinafter "T cells") and, therefore, the autochthonous immune response can be utilized to diagnose and treat malignancies expressing such protein expression products.
Activation of proto-oncogenes is associated with or leads to transformation and expression of the malignant phenotype. Activation by mechanises such as mutation or chromosomal translocation (gene rearrangement) creates a gene product with altered primary structure, i.e., a protein expzession product with one or more amino acid differences relative to the normal protein expressed by the proto-oncogene. Mutation mechanisms include point mutation of nucleotides, recombination, deletion and insertion. Examples of activation of proto-oncogenes through mutation include activation of ras and neu proto-oncogenes. Chromosomal translocation results in a fused protein, such as that associated with chronic myeologenous leukemia. Similarly, cancer-related genes express abnormal protein products with altered amino acid sequences. For example, mutation of the p53 tumor suppressor gene results in amino acid substitutions.
As discla~sed within the present invention, protein products with altered primary structure expressed by activated oncoc~enes and cancer-related genes are recognized by T cel:Ls. Such abnormal protein expression products "turn over" within cells, i.e., undergo a cycle wherein a protein is synthesized, functions and then is degraded and replaced by a newly synthesized molecule.
The ensuing peptide fragments from the degraded protein bind to major histo~compatibility complex (M>IC) antigens.
By display of an abnormal peptide bound to MHC antigen on the cell surface and recognition by host T cells of the combination of abno~.-mal peptide plus self MHC antigen, a malignant cell will be immunogenic to T cells. The exquisite specificity of the T cell receptor enables individual T cells to discriminate between fragments of proteins which differ by a single amino acid residue.
During t:he immune response to an abnormal peptide, T cells expressing a T cell receptor with high affinity binding o:E the peptide-MHC complex will bind to the peptide-MIiC complex and thereby become activated and 5 induced to prolife:rate. In the first encounter with an abnormal peptide, small numbers of immune T cells will proliferate and differentiate into effector and memory T cells . The primary immune response will occur in vivo but is not detected in vitro . Subsequent encounter with the same 10 antigen by the memory T cell will lead to a faster and more intense immune response. The secondary response will occur either in vivo or in vitro. The in vitro response is easily gauged by measuring the degree of proliferation of the T cell population re-exposed in the antigen.
Proliferation of ttie T cell population in response to a particular antigen :is considered to be indicative of prior exposure or priming to the antigen.
Within one aspect of the present invention, a malignancy in which an activated oncogene or cancer related gene is associated with the malignancy may be detected. An immune response to an abnormal protein expressed by an act:W ated oncogene or cancer-related gene, once generated, can be long-lived and can be detected long after immunization, regardless of whether the protein is present or absent in the body at the time of testing. In one embodiment, prior exposure of a warm-blooded animal, such as humans, to the protein expression product of an activated oncogene ~~r cancer-related gene can be detected by examining for the presence or absence of T cell proliferative responses. More specifically, T cells isolated from an :individual by routine techniques are incubated with a protein expression product of an activated oncogene or cancer-related gene. Examples of oncogenes include ras, src, abl, fgr, rel, yes, fes, net, mos, raf, erb B, erb A, fms, neu, roe, kit, sea, sir, myc, myb, for, ski, jun and ets. Alternatively, more than one protein expression product can be examined with the T cell sample. It may be desirable to incubate individual aliquots of a T' cell sample with only one protein expression product: if such a protein interferes with another protein expression product.
Detection of the prolif eration of T cells may be accomplished by a, variety of known techniques. For example, T cell proliferation can be detected by measuring the rate of DNA synthesis. T cells which have been stimulated to proliferate exhibit an increased rate of DNA
l0 synthesis. A typical way to measure the rate of DNA
synthesis is, for example, by pulse-labeling cultures of T cells with tritiated thymidine, a nucleoside precursor which is incorporated into newly synthesized DNA. The amount of tritia.ted thymidine incorporated can be determined using a liquid scintillation spectrophotometer.
Other ways to detect T cell proliferation include measuring increaseaa in interleukin-2 (IZi-2) production, Ca2+ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
A reprea~entative example of an activated oncogene that exp:cesses a protein product capable of immunizing T cells is the ras oncogene. Ras proto-oncogenes encode a highly conserved family of 2lKd proteins (189 amino acids in length) collectively designated as "p21". p21 proteins bind to the inner aspect of the cell membrane, associate with guanosine nucleotides and have intrinsic GTPase activity. p21 proteins have been implicated as important intermediary signalling proteins which regulate cell growth and differentiation. Ras oncogenes were first detected as genetic material i:n the transforming Harvey and Kirsten marine sarcoma retroviruses. In animals, carcinogens induce very specific and predictable mutations, usually involving colons 1:2, 13, 59 and/or 61, which impair the intrinsic GTPase a<aivity of the p21 molecule and confer transforming activity. The human genome contains at least three ras proto-oncogene homologs of the viral ras oncogene denoted ;as K-ras, H-ras and N-ras which are located on three separate human chromosomes. Each of the three ras genes c:an become activated through specific mutation. Mutations of one of the three ras proto-oncogenes occurs commonly in a variety of human malignancies including pancreas adenocarcinoma, thyroid follicular carcinoma, colon adenocarcinoma, seminoma, lung adenocarcinoma, liver adeno carcinoma, melanoma, myeloid leukemia, and myel.oma. The disclosure of the present invention shows that once mutated, the expressed protein product of any of the three ras genes by virtue of a single amino acid substitution may be recognized by autochthonous T cel:Ls.
For exaimple, within the present invention, peptides consisting of 12 or 13 amino acid residues which corresponded to the amino acid sequence from residues 5 16, 5-17, or 4-16 of p21 were constructed to contain either the normal glycine (termed Gly-12 or G-12), or arginine (termed Arg-12 or R-12 ) , or serine (termed Ser-12 or S-12), or cysteine (termed Cys-12, or C-12). C57BL/6 mice were immunized with a single dose of the Arg-12 peptide. Lymphocytes from immunized mice were subsequently tested for proliferative response to the above peptides in wu~rc~ and, as shown in Figure 1, were found to proliferate specifically in response to the Arg-12 peptide, but not the Gly-12, Cys-12, or Ser-12 peptides.
In addition, the lymphocytes from mice immunized with the Arg-12 peptide were shown (Figure 3) to proliferate specifically in response to stimulation by the intact p21 protein containing this same amino acid substitution (Oncogene Science, 7:nc., Manhasset, New York).
Another example of a peptide suitable within the present invention is ras p54-66[L61]. This peptide consists of 13 amine acid residues which correspond to the amino acid sequence from residues 54-66 of p21, except leucine (L) has been substituted for the glutamine (Q) normally found at position 61. Lymphocytes from mice immunized with p54-66[L61] were found to proliferate in vitro, as shown in Figure 4, specifically in response to the Leu-61 peptide, but: not to Gln-61 or Lys-61 peptides. In addition, the lymphocytes from mice immunized with the Leu-61 peptide were shown (Figure 5) to proliferate specifically in response to stimulation by the intact p21 protein containing the same amino acid substitution.
Similarly,, proteins (or peptides based upon or derived therefrom) other than p21 may be isolated or constructed by a variety of known techniques. It will be appreciated by those skilled in the art that it may be desirable to increase the length of an overall peptide or of the native flanking regions to facilitate the induction of T cell responses. As discussed above, protein expression products of activated oncogenes other than ras, or cancer-related genes (i.e., with an amino acid sequence different from that of the proteins expressed by normal proto-oncogenes ar normal genes) are suitable for use within the methods described herein.
For therapeutic purposes, T cells that proliferate in the: presence of one or more protein expression products of activated oncogenes or cancer-related genes can be expanded in number either in vitro or in vivo. Proliferation of such T cells in viho may be accomplished in a ~rariety of ways. For example, the T cells can be re-exposed to one or more protein expression products,. It may be desirable to repeat the exposure of T cells to the protein to induce proliferation. As shown in Figure 2, protein expression product-specific T cells can be grown to large numbers in vitro with retentia~n of specificity in response to intermittent restimulation with the immunizing peptide.
Alternatively, one or more T cells that proliferate in the presence of a protein expression can be expanded in number by cloning. Methods for cloning cells are well known in tl~e art. For example, T cell lines may be established in v~~tro and cloned by limiting dilution.
Responder T cells are purified from the peripheral blood of sensitized patients by density gradient centrifugation and sheep red cell. rosetting and established in culture by stimulating with the nominal antigen in the presence of irradiated autologous filler cells. In order to generate CD4+ T cell lines,. intact ras p21 protein bearing the relevant amino acid substitution is used as the antigenic stimulus and autologous peripheral blood lymphocytes (PBL) or lymphoblastoid cell lines (LCL) immortalized by infection with Epstein Barr virus are used as antigen presenting cells. 7n order to generate CD8+ T cell lines, autologous antigen-presenting cells transfected with an expression vector wJzich produces relevant mutated ras p21 protein are used as stimulator cells. Established T cell lines are cloned 2-4. days following antigen stimulation by plating stimulated T' cells at a frequency of 0.5 cells per well in 96-well flat:-bottom plates with 1 x 106 irradiated PBL or LCL cells and recombinant interleukin 2 (rIL2) (50 U/ml). Wells with established clonal growth are identified at approximately 2-3 weeks after initial plating and restimu:Lated with appropriate antigen in the presence of autologous antigen-presenting cells, then subsequently expanded by the addition of low doses of rIL2(10 U/ml) 2-3 days following antigen stimulation. T
cell clones are maintained in 24-well plates by periodic restimulation with antigen and rIL2 approximately every tWO weeks.
Regardless of how an individual s T cells are proliferated in vitro, 'the T cells may be administered to the individual for therapeutic attack against a tumor. Thus, a patient s own T cells (autochthonous T cells) can be used as reagents to mediate specific tumor therapy.
Typically, about 1 '..K 109 to 1 X 1011 T cells/M2 will be administered intravenously or intracavitary, e.g., in pleural or peritoneal cavities, or in the bed of a resected tumor. Tt will be evident to those skilled in the art that the rnimber and frequency of administration WO 91/11719 PCT/US9l/O(k197 will be dependent upon the response of the patient.
Suitable carriers ar diluents for T cells include physiological saline or sera. It will be recognized by one skilled in the art that the composition should be 5 prepared in sterile form.
T cells ma!,r also be proliferated in vivo. For example, immunization of an individual with one or more protein expression products of activated oncogenes or cancer-related genes can induce continued expansion in the 10 number of T cells necessary for therapeutic attack against a tumor. It may be desirable to administer the protein expression product repetitively.
The present= invention also discloses that the protein expression products of activated oncogenes or 15 cancer-related genes, in addition to being immunogenic to T cells, appear to stimulate B-cells to produce antibodies capable of recognizing these proteins. Detection of such antibodies provides .another way to diagnose a malignancy in which an activated oncogene or cancer-related gene is associated with the. malignancy. Antibodies specific (i.e., which exhibit;. a binding affinity of about 107 liters/mole or better) for one or more protein expression products may be found in a variety of body fluids including sera and ascites. Detection of one or more immunocomplexes fonaed between a protein expression product and antibodies specific for the protein may be accomplished by a variety of known techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISA).
Suitable immunoassays include the double monoclonal antibody sandwich immunoassay technique of David et al. (U. S:. Patent 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., ~adloimmunoassay Methods, E. and S. Livingstone, Edinburgh, 1970); the "western blot"
method of Cordon et al. (U. S. Patent 4,452,901);
immunoprecipitation of labeled ligand (Brown et al., ,~
WO 91/11719 PCT/US91/00.~97 ~~iol. Chem. x:4980-4983, 1980); enzyme-linked immunosorbent assays. as described by, for example, Rainas and Ross (.T. Eliol. Chem. x:5154-5160, 1982);
immunocytochamical techniques, including the use of fluorochromas (Brooks at al., ~li~F.,~~ ~mmunol. ~: 477, 1980); and nautralixation of activity (Mown-Pope et al., pt'OC. Nat Acad. ~ci. L~sA x:2396~2400 (19s4~~, all of which era hereby incorporated by reference. In addition to the immunoassays describsd abovs, a number o! othsr immunoassays are available, including those described in U.S. Patent NOS.: 3,817,827; 3,850,75?; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876.
For dstaction purposes, the protein expression products ("antigens") may sitbar be labeled or unlabeled.
Whsn unlabelsd, the antigens find use in agglutination assays. In addition, unlabeled antigsns can be ussd in combination with labelsd molscules that era reactive with immunocomplexas, or in combination with labeled antibodies (sscond antibodies) that era rsactivs with the antibody directed against the protein axprasaion product, such as antibodiss specific !or immunoglobulin. 711ternatiwly, the antigens can be directly labeled. Wharf they are labeled, the rsportar group can include radioisotopes, fluorophores, ensymes, lu~inescers, or dye particles.
These and other labels are well known in the art and era describmd, for example. in the tolloving Q.S. patents:
3,766,161; 3,791,937;; 3,817,837; 3,996,345; and 4,233,402.
Typically in an ELISa assay, antigen is adsorbed to the surface of a microtitar well.. Rssidual protein binding sites on the surface era then blocked with an appropriate agent, such as bovine swum albumin (HSA), heat-inactivated normal goat serum (NGS), or HLOTTO
(buffered solution o! nonfat dry milk which also contains a prassrvativs, salts, and an antifoaming agent). The well is than inc:ubatad with a sample suspected of containing specific antibody. The sample can be applied WO 91/11', 19 PCTII.'S91/0049"
neat, or, more often, it can be diluted, usually in a buttered solution which contains a small amount (0.1-5.0~
by weight) of protsin, such as BSA, NGS, or BLOTTO. Attar incubating for a sufficient length o! tine to allow specific binding to occur, the well is washed to remove unbound protein and then incubated with an' anti-species specific imaunoglobulin antibody labeled with a reportsr group. Ths reporter group can bs chosen from a variety of enzymes, including horseradish peroxidase, bata-galactosidase, al~calins phosphatase, and glucoss oxidase.
Sufficient time i;: allowed for specific binding to occur, then the well is .again washsd to remove unbound conjugate, and the substrate for the anzyms is added. Color is allowed to develop and the optical density of the contents of the wall is determined visually or instrumentally.
In one prsfarrsd embodiment of this aspect o!
the present invention, a reporter group is board to the protein expression product. The step o! detecting ~~~pl~,~ involves removing substantially any unbound protein expression product and then detecting the presence or absence of the reporter group.
In another preferred sabodisent, a reporter group is bound to a second antibody capable of binding to the antibodies specific for a protein expression product.
The step of detecting ia~unoco~plexes involves (a) loving substantially any unbound antibody, ~e second antibody, (c) removing substantially any unbound sscond antibody and than (d) detecting the presents or absence o! the reporter group. where the antibody specific for the protein expression product is darivsd from a human, the second antibody is an anti-human antibody.
In a 'third preferred aabodiunt for detecting immunocomplaxss, a reporter group is bound to a molecule capable of binding to the imoaunoco~plexes. The step o!
detecting involves (a) adding the molecule, (b) rssoving substantially any unbound molecule, and den (c) detecting the presence or absence of the reporter group. An example of a molecule capable of binding to the immunocomplexes is protein A.
It will be evident to one skilled in the art that a variety of methods for detecting the immunocomplexes may be employed within the present invention. Reporter groups suitable for use in any of the methods include radioisotopes, fluorophores, enzymes, luminescers, and dye particles.
l0 In a related aspect of the present invention, detection of immunocomplexes formed between a protein expression product of an activated oncogene, or cancer-related gene, and antibodies in body fluid which are specific for the protein may be used to monitor the effectiveness of cancer therapy. Samples of body fluid taken from an individual prior to and subsequent to initiation of therapy may be analyzed for the immunocomplexes by the methodologies described above.
Briefly, the number of immunocomplexes detected in both samples are compared. A substantial change in the number of immunocomplexes in the second sample (post-therapy initiation) relative to the first sample (pre-therapy) reflects successful therapy.
The following examples are offered by way of illustration and na~t by way of limitation.
Example 1 ~~ i r~_; tat,'_nn, of SDeci:f is Class-IT Restricted ~-Cell Reseo~se to Mutated Ras Oncoaene Products A. Immunization C57BL/6 and H6.C-H-2bm12 mice (Jackson Laboratories, Bar Harbor, Me.) were inoculated with a synthetic peptide (Amino acid sequence: KLVWGARGVGK;
Microbiological Associates, Bethesda, Md.) which corresponds to residue 5-16 of the p21 protein product of mutated ras proto-oncogene encoding a residue 12 substitution of Arginine (R) for Glycine (G). The peptide was solubilized in distilled water at 1 mg/ml, emulsified in complete Freunds adjuvant (Sigma Co., St. Louis, Mo.) at a ration of 1:1, then injected subcutaneously into the base of the tail using a 25 gauge needle. Alternatively peptide was emulsified wits Ribi MPL+TDM+CWS adjuvant (Ribi Immunochem. Res., Hamilton, Mt.) and injected - subcutaneously into both hindquarters. Total amount of peptide injected was 50 erg per mouse. Seven days later animals were sacrificed and draining periaortic and inguinal lymph nodes: were removed. Lymph nodes, suspended in buffered saline in petri dishes, were teased apart with 18 gauge needles. Dislodged lymphocytes were collected and washed with buffered saline then suspended at 1 X 106 cells per ml in culture medium for use in proliferation assays.
B. P_r01_?feratlon~ssav Lymphocytes obtained from immunized mice were suspended in cultures medium (consisting of RPMI 1640 Gibco supplemented with l0% fetal calf serum, 2 mM L-glutamine, 100 U/ml streptomycin, 100 U/ml penicillin and 2.5 X 10-5 M 2-mercaptoethanol.) and were plated in 96-well flat bottom microtiter plates (Costar Co., Cambridge, Mass.) at 1 X 105 cells per well. Syngeneic spleen cells which had been irradiated to 3000 tads were added at 2 X 105 cells per well to serve as antigen presenting cells. The immunizing peptide and indicated control peptides (50 ~Cg/ml) were added to triplicate wells (final volume of 200 ~1/well). ThE: plates were incubated at 37°C in humidified air containing 5% C02 for 72 hours then pulsed for 6 hr with 1 uC:i pet well of tritiated thymidine (3H-TdR; 20 Ci/mmol from NEN Products, Boston, Ma.).
Lymphocytes from individual wells were collected onto filter paper disks using a multi-channel harvester then transferred to scintillation fluid in individual counting tubes. ~ emission was measured using a Beckmann liquid scintillation spectrophotometer. The data presented in 5 Figure 1 represents the mean of triplicate determinations.
Example 2 Generation of T CP » r.;rac and Clones With S~ecificitv 10 fir the Protein l.~roduct o,~ I~~'ated Ras Proto-Oncog~ene Lymphocytes obtained from draining lymph nodes of immunized mice a.s described above were plated (4 X 106 cells per well) in 24-well culture plates (Costar Co.) 15 with irradiated syngeneic spleen cells (10,000 rads; 2 X
106 cells per well) and immunizing peptide (5 ~tg/well) to give a final volume of 2 ml (culture medium). Plates were cultured for 5 day's (37°C, 5~ Co2), then split 1:2 onto replicate plates. Aftsr ten days lymphocytes were 20 restimulated by plating 5 X 105 cultured lymphocytes with 4 X 106 irradiated syngeneic spleen cells and peptide at 5 ~tg/ml. After three days, individual wells were redistributed into 2-4 wells of culture medium containing 10 units of Interleukin-2 (human recombinant from Hoffmann-LaRoche) to facilitate cell expansion. Lines were restimulated with antigen and subsequently IL-2 as described above, every 3-4 weeks.
Immune lymphocytes which had been twice stimulated with antigen invibo were cloned at day 13 following initiation of culture (3 days following Ag restimulation) by plating immune T cells at a frequency of 0.5 cells per well. in 96-well flat-bottom plates with irradiated syngeneic spleen cells (1 x 106 cells per well) and Interleukin-2 at 50 U/ml. Wells with clonal growth were expanded and tested for immune specificity as shown in Figure 2. Specific T cell clones were maintained as described for T cell lines.
21.
From the foregoing, it will be evident that, although specific embodiments of the invention have been described herein far purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.
In another aspect, the present invention provides methods for monitoring the effectiveness of l0 cancer therapy in a warm-blooded animal with a malignancy, wherein an acti~at.ed oncogene or cancer-related gene is associated with the malignancy. In one embodiment, the method comprises the steps of: (a) contacting a first body fluid sample,, taken from the warm-blooded animal prior to initiation of therapy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
(b) incubating the body fluid under conditions and for a time sufficient t,o allow immunocomplexes to form;
(c) detecting immuriocomplexes formed between the protein expression product and antibodies in the body fluid specific for the protein expression product; (d) repeating steps (a), (b), and (c) on a second body fluid sample taken from the animal subsequent to the initiation of therapy; and (e) comparing the number of immunocomplexes detected in the first and second body fluid samples, thereby monitoring the effectiveness of the therapy in the animal.
The present invention is also directed toward methods for treating a malignancy in a warm-blooded animal, wherein an activated oncogene or cancer-related gene is associated with the malignancy. In one embodiment, the method comprises the steps of:
(a) isolating T cells from a warm-blooded animal;
(b) incubating the 'T cells in the presence of at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy, such WO 9l/11719 PCT/US91/00497 that the T cells proliferate; and (c) administering to the warm-blooded animal an effective amount of the proliferated T cells. In another embodiment, the method comprises the step; of: (a) isolating T cells from a 5 warm-blooded animal; (b) incubating the T cells in the presence of at Ieasi: one protein expression product of an activated oncagene or cancer-related gene associated with the malignancy, svuch that the T cells proliferate;
(c) cloning one or more cells that proliferated in the presence of the protein expression product; and (d) administering to the warm-blooded animal an effective amount of the cloned. T cells. In a third embodiment, the method comprises immunizing the animal with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy.
Within a related aspect, the present invention provides anti-cancer therapeutic compositions comprising T cells proliferated: in the presence of at least one protein expression product of an activated oncogene or cancer-related gene associated with a malignancy, in combination with a physiologically acceptable carrier or diluent. Such T cells are useful in the manufacture of a medicament for treai_.ing a malignancy in a warm-blooded animal.
brief Description of the Drawing's Figure 1 graphically illustrates that specif is T cell responses to point-mutated ras peptide can be generated by in vivo priming on day 0 with 50 ~tg of a peptide of 12 amino acid length constructed identically to murine ras-p21 (amino acids 5-16) but with the substitution of arginine for the glycine normally found at position 12 (i.e., position 12 of ras-p21 but position 8 of the peptide). This peptide is denoted as ras p5-16(R12] or as ras-R12. Mice were immunized with ras-R12 peptide plus adjuvant or with adjuvant alone. After 10 days, the draining lymph nodes were harvested and lymphocytes stimulated in vitro with ras-R12 peptide or as specificity controls with similar peptides substituted with serine, cysteine or glycine. at position 12 as opposed to arginine, denoted as ras-S12, ras-C12 and ras-G12, respectively.
Four days later, cultures were pulsed with [3H]-thymidine (3HTdR) for 8 hours, as described in Example 1 below.
Results are represented as ~CPM.
Figure 2 shows that ras-peptide specific T cells grown long-term invitm in response to intermittent stimulation by ras-peptide retain specific function.
T cells derived from mesenteric lymph nodes of C57BL/6 mice primed to ras-R12 (as defined for Figure 1 above) were cultured long-term in vitro in response to intermittent stimulation With ras-R12 peptide (5 ~cg/ml) on irradiated 86 spleen cells (3000 rad) as antigen presenting cells.
The specificity of the T cell line was tested on day 85 by stimulating with graded concentrations of various ras peptides (as defined for Figure 1 above) or with trypsinized OVA (TOVA) containing several potential immunogenic peptides. Data is represented as 3HTdR uptake in counts per minutE: (c.p.m.).
Figure :3 graphically illustrates that T cells specific for a ras peptide respond specifically to intact ras p21 protein containing the same residue substitution.
This peptide, denoted as p5-1'7 [R12 ] , consists of 13 amino acid residues and was constructed identically to marine ras-p21 (amino acids 5-17) but with the substitution of arginine (R) for the glycine (G) normally found at position 12 of ras~-p21. Cloned B6 T cells specific for the Arg-12 ras peptide were cultured with irradiated B6 spleen cells and either p5-17[R12] or intact ras p21 proteins bearing the designated amino acid at position-12 (1 ~tg/ml). Proliferative responses were measured after four days (as described in Figure 1). The data represent the mean of triplicate determinations of the c.p.m. of incorporated 3HTdR. The standard deviations were <10% of the c.p.m.
WO 91/11719 PCT/LS9l/0(1497 Figure 4 shows that specific T call responses can ba generated by in vivo priming With a peptide of 13 amino acid length constructed identically to marina ras-p21 (amino acids 54-66) but with the substitution of laucina (L) for the glutamina (Q) normally found at position 61 of ras-p21. This peptide is denoted as p54-66[L61] or [L61]. C3H/HeN mica ware immunized twice subcutanaously at two weak intervals with Ribi adjuvant alone or adjuvant emulsified with p54-66[L61] ras peptide ( 100 ~tg) . Two weeks altar the f final immunization, spleen calls from mica in;Iactad with adjuvant alone (open bar) or adjuvant plus p54~~66[L61] ras peptide (black bar) ware harvested and tested in vitnv for prolifarativa response to the indicated p51-c~6 ras peptide ( 100 ~tg/ml) . Tha peptide containing the substitution of lysine (R) for glutamina at position 61 is denoted as [R51]. The data represent the mean of triplica~ta determinations of the c.p.m. of incorporated 38TdR ~ S.D.
Pigura 5 graphically illustrates that intact ras pZi[L61] protein is specifically recognized by a ras peptide-induced T cell lira. Specific T cell lines and clones ware ganas~atad and maintained as described in Example 2 below. A T call line which exhibits specific reactivity to the ras peptide p54-66[L61] (penal 1~) and a T call clone which exhibits specific reactivity to the ras peptide ps-17[R1~] (panel B) ware stimulated with (a) no antiq~n, (b~ sensitising ras peptide, or (c) intact ras pZi[h~1J protein. The ra ps4-66[L61] specific T call lira wss of C38 origin and the ras p5-17[R12] specific T
call clone was of B6 origin. Prol~ifarativa assays ware psrtormad with acynganaic irrsdiatad spleen calls as antigen presenting cells (as described in Figure 1).
Stimulating peptides and proteins ware used at a concentration of 5 Wg/ml. The ras pZi[L61] protein was produced in E coli H8101 using the prokaryotic expression plasmid plat-L9 and purif iad by sequential DFaE-Saphacal WO 91/11'19 PCT/US91/00497 and Ssphadex~ colLnmn chromatography. The data represent the mean c.p.m. of incorporated 3HTdR ~ S.D.
Detailed Descri~ticn of the Invention Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter.
Activated oncoa~ene - as used herein, racers to proto-oncogenes that have bscome activated, leading to the expression of protein produets with amino acid sequences other than those of the protein products expressed by the normal proto-oncoganas.
Cancer-ry,lated Qene - as used herein, reters to any altered gsne (other than an oncogena) associated with the devalopnent o=~ maintenance o! the malignant phenotype.
Examples of such altered genes include mutants of the p53 tusor supprassor gene.
Protein exflression product - as used herein, includes proteins, polypeptidsa, and peptides; and may be an intact molecule, a fragaent thereof, or a functional equivalent thereat; and may ba genetically engineered.
Proliferation o! T cells - as used herein, includes the multiplication of T cells a: wall as the stisulation o! T calls leading to multiplication, i.a., the im3.tiation of events leading to mitosis and aitosis itself. Methods for detecting proliferation of T calls are discussed below.
As noted above, the present invention is directed toward aethods and compositions for the diagnosis, monitoring and treataant of malignancies in a wars-bloodsd animal, wherein activated oncogenss or cancer-related genes are associated with the malignancies.
The disclosure o1.' the present invention shows that the protein expression products of activated oncoganes and cancer-related genes can be recognized by thymus-dependant lymphocytes (hereinafter "T cells") and, therefore, the autochthonous immune response can be utilized to diagnose and treat malignancies expressing such protein expression products.
Activation of proto-oncogenes is associated with or leads to transformation and expression of the malignant phenotype. Activation by mechanises such as mutation or chromosomal translocation (gene rearrangement) creates a gene product with altered primary structure, i.e., a protein expzession product with one or more amino acid differences relative to the normal protein expressed by the proto-oncogene. Mutation mechanisms include point mutation of nucleotides, recombination, deletion and insertion. Examples of activation of proto-oncogenes through mutation include activation of ras and neu proto-oncogenes. Chromosomal translocation results in a fused protein, such as that associated with chronic myeologenous leukemia. Similarly, cancer-related genes express abnormal protein products with altered amino acid sequences. For example, mutation of the p53 tumor suppressor gene results in amino acid substitutions.
As discla~sed within the present invention, protein products with altered primary structure expressed by activated oncoc~enes and cancer-related genes are recognized by T cel:Ls. Such abnormal protein expression products "turn over" within cells, i.e., undergo a cycle wherein a protein is synthesized, functions and then is degraded and replaced by a newly synthesized molecule.
The ensuing peptide fragments from the degraded protein bind to major histo~compatibility complex (M>IC) antigens.
By display of an abnormal peptide bound to MHC antigen on the cell surface and recognition by host T cells of the combination of abno~.-mal peptide plus self MHC antigen, a malignant cell will be immunogenic to T cells. The exquisite specificity of the T cell receptor enables individual T cells to discriminate between fragments of proteins which differ by a single amino acid residue.
During t:he immune response to an abnormal peptide, T cells expressing a T cell receptor with high affinity binding o:E the peptide-MHC complex will bind to the peptide-MIiC complex and thereby become activated and 5 induced to prolife:rate. In the first encounter with an abnormal peptide, small numbers of immune T cells will proliferate and differentiate into effector and memory T cells . The primary immune response will occur in vivo but is not detected in vitro . Subsequent encounter with the same 10 antigen by the memory T cell will lead to a faster and more intense immune response. The secondary response will occur either in vivo or in vitro. The in vitro response is easily gauged by measuring the degree of proliferation of the T cell population re-exposed in the antigen.
Proliferation of ttie T cell population in response to a particular antigen :is considered to be indicative of prior exposure or priming to the antigen.
Within one aspect of the present invention, a malignancy in which an activated oncogene or cancer related gene is associated with the malignancy may be detected. An immune response to an abnormal protein expressed by an act:W ated oncogene or cancer-related gene, once generated, can be long-lived and can be detected long after immunization, regardless of whether the protein is present or absent in the body at the time of testing. In one embodiment, prior exposure of a warm-blooded animal, such as humans, to the protein expression product of an activated oncogene ~~r cancer-related gene can be detected by examining for the presence or absence of T cell proliferative responses. More specifically, T cells isolated from an :individual by routine techniques are incubated with a protein expression product of an activated oncogene or cancer-related gene. Examples of oncogenes include ras, src, abl, fgr, rel, yes, fes, net, mos, raf, erb B, erb A, fms, neu, roe, kit, sea, sir, myc, myb, for, ski, jun and ets. Alternatively, more than one protein expression product can be examined with the T cell sample. It may be desirable to incubate individual aliquots of a T' cell sample with only one protein expression product: if such a protein interferes with another protein expression product.
Detection of the prolif eration of T cells may be accomplished by a, variety of known techniques. For example, T cell proliferation can be detected by measuring the rate of DNA synthesis. T cells which have been stimulated to proliferate exhibit an increased rate of DNA
l0 synthesis. A typical way to measure the rate of DNA
synthesis is, for example, by pulse-labeling cultures of T cells with tritiated thymidine, a nucleoside precursor which is incorporated into newly synthesized DNA. The amount of tritia.ted thymidine incorporated can be determined using a liquid scintillation spectrophotometer.
Other ways to detect T cell proliferation include measuring increaseaa in interleukin-2 (IZi-2) production, Ca2+ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
A reprea~entative example of an activated oncogene that exp:cesses a protein product capable of immunizing T cells is the ras oncogene. Ras proto-oncogenes encode a highly conserved family of 2lKd proteins (189 amino acids in length) collectively designated as "p21". p21 proteins bind to the inner aspect of the cell membrane, associate with guanosine nucleotides and have intrinsic GTPase activity. p21 proteins have been implicated as important intermediary signalling proteins which regulate cell growth and differentiation. Ras oncogenes were first detected as genetic material i:n the transforming Harvey and Kirsten marine sarcoma retroviruses. In animals, carcinogens induce very specific and predictable mutations, usually involving colons 1:2, 13, 59 and/or 61, which impair the intrinsic GTPase a<aivity of the p21 molecule and confer transforming activity. The human genome contains at least three ras proto-oncogene homologs of the viral ras oncogene denoted ;as K-ras, H-ras and N-ras which are located on three separate human chromosomes. Each of the three ras genes c:an become activated through specific mutation. Mutations of one of the three ras proto-oncogenes occurs commonly in a variety of human malignancies including pancreas adenocarcinoma, thyroid follicular carcinoma, colon adenocarcinoma, seminoma, lung adenocarcinoma, liver adeno carcinoma, melanoma, myeloid leukemia, and myel.oma. The disclosure of the present invention shows that once mutated, the expressed protein product of any of the three ras genes by virtue of a single amino acid substitution may be recognized by autochthonous T cel:Ls.
For exaimple, within the present invention, peptides consisting of 12 or 13 amino acid residues which corresponded to the amino acid sequence from residues 5 16, 5-17, or 4-16 of p21 were constructed to contain either the normal glycine (termed Gly-12 or G-12), or arginine (termed Arg-12 or R-12 ) , or serine (termed Ser-12 or S-12), or cysteine (termed Cys-12, or C-12). C57BL/6 mice were immunized with a single dose of the Arg-12 peptide. Lymphocytes from immunized mice were subsequently tested for proliferative response to the above peptides in wu~rc~ and, as shown in Figure 1, were found to proliferate specifically in response to the Arg-12 peptide, but not the Gly-12, Cys-12, or Ser-12 peptides.
In addition, the lymphocytes from mice immunized with the Arg-12 peptide were shown (Figure 3) to proliferate specifically in response to stimulation by the intact p21 protein containing this same amino acid substitution (Oncogene Science, 7:nc., Manhasset, New York).
Another example of a peptide suitable within the present invention is ras p54-66[L61]. This peptide consists of 13 amine acid residues which correspond to the amino acid sequence from residues 54-66 of p21, except leucine (L) has been substituted for the glutamine (Q) normally found at position 61. Lymphocytes from mice immunized with p54-66[L61] were found to proliferate in vitro, as shown in Figure 4, specifically in response to the Leu-61 peptide, but: not to Gln-61 or Lys-61 peptides. In addition, the lymphocytes from mice immunized with the Leu-61 peptide were shown (Figure 5) to proliferate specifically in response to stimulation by the intact p21 protein containing the same amino acid substitution.
Similarly,, proteins (or peptides based upon or derived therefrom) other than p21 may be isolated or constructed by a variety of known techniques. It will be appreciated by those skilled in the art that it may be desirable to increase the length of an overall peptide or of the native flanking regions to facilitate the induction of T cell responses. As discussed above, protein expression products of activated oncogenes other than ras, or cancer-related genes (i.e., with an amino acid sequence different from that of the proteins expressed by normal proto-oncogenes ar normal genes) are suitable for use within the methods described herein.
For therapeutic purposes, T cells that proliferate in the: presence of one or more protein expression products of activated oncogenes or cancer-related genes can be expanded in number either in vitro or in vivo. Proliferation of such T cells in viho may be accomplished in a ~rariety of ways. For example, the T cells can be re-exposed to one or more protein expression products,. It may be desirable to repeat the exposure of T cells to the protein to induce proliferation. As shown in Figure 2, protein expression product-specific T cells can be grown to large numbers in vitro with retentia~n of specificity in response to intermittent restimulation with the immunizing peptide.
Alternatively, one or more T cells that proliferate in the presence of a protein expression can be expanded in number by cloning. Methods for cloning cells are well known in tl~e art. For example, T cell lines may be established in v~~tro and cloned by limiting dilution.
Responder T cells are purified from the peripheral blood of sensitized patients by density gradient centrifugation and sheep red cell. rosetting and established in culture by stimulating with the nominal antigen in the presence of irradiated autologous filler cells. In order to generate CD4+ T cell lines,. intact ras p21 protein bearing the relevant amino acid substitution is used as the antigenic stimulus and autologous peripheral blood lymphocytes (PBL) or lymphoblastoid cell lines (LCL) immortalized by infection with Epstein Barr virus are used as antigen presenting cells. 7n order to generate CD8+ T cell lines, autologous antigen-presenting cells transfected with an expression vector wJzich produces relevant mutated ras p21 protein are used as stimulator cells. Established T cell lines are cloned 2-4. days following antigen stimulation by plating stimulated T' cells at a frequency of 0.5 cells per well in 96-well flat:-bottom plates with 1 x 106 irradiated PBL or LCL cells and recombinant interleukin 2 (rIL2) (50 U/ml). Wells with established clonal growth are identified at approximately 2-3 weeks after initial plating and restimu:Lated with appropriate antigen in the presence of autologous antigen-presenting cells, then subsequently expanded by the addition of low doses of rIL2(10 U/ml) 2-3 days following antigen stimulation. T
cell clones are maintained in 24-well plates by periodic restimulation with antigen and rIL2 approximately every tWO weeks.
Regardless of how an individual s T cells are proliferated in vitro, 'the T cells may be administered to the individual for therapeutic attack against a tumor. Thus, a patient s own T cells (autochthonous T cells) can be used as reagents to mediate specific tumor therapy.
Typically, about 1 '..K 109 to 1 X 1011 T cells/M2 will be administered intravenously or intracavitary, e.g., in pleural or peritoneal cavities, or in the bed of a resected tumor. Tt will be evident to those skilled in the art that the rnimber and frequency of administration WO 91/11719 PCT/US9l/O(k197 will be dependent upon the response of the patient.
Suitable carriers ar diluents for T cells include physiological saline or sera. It will be recognized by one skilled in the art that the composition should be 5 prepared in sterile form.
T cells ma!,r also be proliferated in vivo. For example, immunization of an individual with one or more protein expression products of activated oncogenes or cancer-related genes can induce continued expansion in the 10 number of T cells necessary for therapeutic attack against a tumor. It may be desirable to administer the protein expression product repetitively.
The present= invention also discloses that the protein expression products of activated oncogenes or 15 cancer-related genes, in addition to being immunogenic to T cells, appear to stimulate B-cells to produce antibodies capable of recognizing these proteins. Detection of such antibodies provides .another way to diagnose a malignancy in which an activated oncogene or cancer-related gene is associated with the. malignancy. Antibodies specific (i.e., which exhibit;. a binding affinity of about 107 liters/mole or better) for one or more protein expression products may be found in a variety of body fluids including sera and ascites. Detection of one or more immunocomplexes fonaed between a protein expression product and antibodies specific for the protein may be accomplished by a variety of known techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISA).
Suitable immunoassays include the double monoclonal antibody sandwich immunoassay technique of David et al. (U. S:. Patent 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., ~adloimmunoassay Methods, E. and S. Livingstone, Edinburgh, 1970); the "western blot"
method of Cordon et al. (U. S. Patent 4,452,901);
immunoprecipitation of labeled ligand (Brown et al., ,~
WO 91/11719 PCT/US91/00.~97 ~~iol. Chem. x:4980-4983, 1980); enzyme-linked immunosorbent assays. as described by, for example, Rainas and Ross (.T. Eliol. Chem. x:5154-5160, 1982);
immunocytochamical techniques, including the use of fluorochromas (Brooks at al., ~li~F.,~~ ~mmunol. ~: 477, 1980); and nautralixation of activity (Mown-Pope et al., pt'OC. Nat Acad. ~ci. L~sA x:2396~2400 (19s4~~, all of which era hereby incorporated by reference. In addition to the immunoassays describsd abovs, a number o! othsr immunoassays are available, including those described in U.S. Patent NOS.: 3,817,827; 3,850,75?; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876.
For dstaction purposes, the protein expression products ("antigens") may sitbar be labeled or unlabeled.
Whsn unlabelsd, the antigens find use in agglutination assays. In addition, unlabeled antigsns can be ussd in combination with labelsd molscules that era reactive with immunocomplexas, or in combination with labeled antibodies (sscond antibodies) that era rsactivs with the antibody directed against the protein axprasaion product, such as antibodiss specific !or immunoglobulin. 711ternatiwly, the antigens can be directly labeled. Wharf they are labeled, the rsportar group can include radioisotopes, fluorophores, ensymes, lu~inescers, or dye particles.
These and other labels are well known in the art and era describmd, for example. in the tolloving Q.S. patents:
3,766,161; 3,791,937;; 3,817,837; 3,996,345; and 4,233,402.
Typically in an ELISa assay, antigen is adsorbed to the surface of a microtitar well.. Rssidual protein binding sites on the surface era then blocked with an appropriate agent, such as bovine swum albumin (HSA), heat-inactivated normal goat serum (NGS), or HLOTTO
(buffered solution o! nonfat dry milk which also contains a prassrvativs, salts, and an antifoaming agent). The well is than inc:ubatad with a sample suspected of containing specific antibody. The sample can be applied WO 91/11', 19 PCTII.'S91/0049"
neat, or, more often, it can be diluted, usually in a buttered solution which contains a small amount (0.1-5.0~
by weight) of protsin, such as BSA, NGS, or BLOTTO. Attar incubating for a sufficient length o! tine to allow specific binding to occur, the well is washed to remove unbound protein and then incubated with an' anti-species specific imaunoglobulin antibody labeled with a reportsr group. Ths reporter group can bs chosen from a variety of enzymes, including horseradish peroxidase, bata-galactosidase, al~calins phosphatase, and glucoss oxidase.
Sufficient time i;: allowed for specific binding to occur, then the well is .again washsd to remove unbound conjugate, and the substrate for the anzyms is added. Color is allowed to develop and the optical density of the contents of the wall is determined visually or instrumentally.
In one prsfarrsd embodiment of this aspect o!
the present invention, a reporter group is board to the protein expression product. The step o! detecting ~~~pl~,~ involves removing substantially any unbound protein expression product and then detecting the presence or absence of the reporter group.
In another preferred sabodisent, a reporter group is bound to a second antibody capable of binding to the antibodies specific for a protein expression product.
The step of detecting ia~unoco~plexes involves (a) loving substantially any unbound antibody, ~e second antibody, (c) removing substantially any unbound sscond antibody and than (d) detecting the presents or absence o! the reporter group. where the antibody specific for the protein expression product is darivsd from a human, the second antibody is an anti-human antibody.
In a 'third preferred aabodiunt for detecting immunocomplaxss, a reporter group is bound to a molecule capable of binding to the imoaunoco~plexes. The step o!
detecting involves (a) adding the molecule, (b) rssoving substantially any unbound molecule, and den (c) detecting the presence or absence of the reporter group. An example of a molecule capable of binding to the immunocomplexes is protein A.
It will be evident to one skilled in the art that a variety of methods for detecting the immunocomplexes may be employed within the present invention. Reporter groups suitable for use in any of the methods include radioisotopes, fluorophores, enzymes, luminescers, and dye particles.
l0 In a related aspect of the present invention, detection of immunocomplexes formed between a protein expression product of an activated oncogene, or cancer-related gene, and antibodies in body fluid which are specific for the protein may be used to monitor the effectiveness of cancer therapy. Samples of body fluid taken from an individual prior to and subsequent to initiation of therapy may be analyzed for the immunocomplexes by the methodologies described above.
Briefly, the number of immunocomplexes detected in both samples are compared. A substantial change in the number of immunocomplexes in the second sample (post-therapy initiation) relative to the first sample (pre-therapy) reflects successful therapy.
The following examples are offered by way of illustration and na~t by way of limitation.
Example 1 ~~ i r~_; tat,'_nn, of SDeci:f is Class-IT Restricted ~-Cell Reseo~se to Mutated Ras Oncoaene Products A. Immunization C57BL/6 and H6.C-H-2bm12 mice (Jackson Laboratories, Bar Harbor, Me.) were inoculated with a synthetic peptide (Amino acid sequence: KLVWGARGVGK;
Microbiological Associates, Bethesda, Md.) which corresponds to residue 5-16 of the p21 protein product of mutated ras proto-oncogene encoding a residue 12 substitution of Arginine (R) for Glycine (G). The peptide was solubilized in distilled water at 1 mg/ml, emulsified in complete Freunds adjuvant (Sigma Co., St. Louis, Mo.) at a ration of 1:1, then injected subcutaneously into the base of the tail using a 25 gauge needle. Alternatively peptide was emulsified wits Ribi MPL+TDM+CWS adjuvant (Ribi Immunochem. Res., Hamilton, Mt.) and injected - subcutaneously into both hindquarters. Total amount of peptide injected was 50 erg per mouse. Seven days later animals were sacrificed and draining periaortic and inguinal lymph nodes: were removed. Lymph nodes, suspended in buffered saline in petri dishes, were teased apart with 18 gauge needles. Dislodged lymphocytes were collected and washed with buffered saline then suspended at 1 X 106 cells per ml in culture medium for use in proliferation assays.
B. P_r01_?feratlon~ssav Lymphocytes obtained from immunized mice were suspended in cultures medium (consisting of RPMI 1640 Gibco supplemented with l0% fetal calf serum, 2 mM L-glutamine, 100 U/ml streptomycin, 100 U/ml penicillin and 2.5 X 10-5 M 2-mercaptoethanol.) and were plated in 96-well flat bottom microtiter plates (Costar Co., Cambridge, Mass.) at 1 X 105 cells per well. Syngeneic spleen cells which had been irradiated to 3000 tads were added at 2 X 105 cells per well to serve as antigen presenting cells. The immunizing peptide and indicated control peptides (50 ~Cg/ml) were added to triplicate wells (final volume of 200 ~1/well). ThE: plates were incubated at 37°C in humidified air containing 5% C02 for 72 hours then pulsed for 6 hr with 1 uC:i pet well of tritiated thymidine (3H-TdR; 20 Ci/mmol from NEN Products, Boston, Ma.).
Lymphocytes from individual wells were collected onto filter paper disks using a multi-channel harvester then transferred to scintillation fluid in individual counting tubes. ~ emission was measured using a Beckmann liquid scintillation spectrophotometer. The data presented in 5 Figure 1 represents the mean of triplicate determinations.
Example 2 Generation of T CP » r.;rac and Clones With S~ecificitv 10 fir the Protein l.~roduct o,~ I~~'ated Ras Proto-Oncog~ene Lymphocytes obtained from draining lymph nodes of immunized mice a.s described above were plated (4 X 106 cells per well) in 24-well culture plates (Costar Co.) 15 with irradiated syngeneic spleen cells (10,000 rads; 2 X
106 cells per well) and immunizing peptide (5 ~tg/well) to give a final volume of 2 ml (culture medium). Plates were cultured for 5 day's (37°C, 5~ Co2), then split 1:2 onto replicate plates. Aftsr ten days lymphocytes were 20 restimulated by plating 5 X 105 cultured lymphocytes with 4 X 106 irradiated syngeneic spleen cells and peptide at 5 ~tg/ml. After three days, individual wells were redistributed into 2-4 wells of culture medium containing 10 units of Interleukin-2 (human recombinant from Hoffmann-LaRoche) to facilitate cell expansion. Lines were restimulated with antigen and subsequently IL-2 as described above, every 3-4 weeks.
Immune lymphocytes which had been twice stimulated with antigen invibo were cloned at day 13 following initiation of culture (3 days following Ag restimulation) by plating immune T cells at a frequency of 0.5 cells per well. in 96-well flat-bottom plates with irradiated syngeneic spleen cells (1 x 106 cells per well) and Interleukin-2 at 50 U/ml. Wells with clonal growth were expanded and tested for immune specificity as shown in Figure 2. Specific T cell clones were maintained as described for T cell lines.
21.
From the foregoing, it will be evident that, although specific embodiments of the invention have been described herein far purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.
Claims (13)
1. A method for the detection of a malignancy in a warm-blooded animal, wherein an activated oncogene or cancer-related gene is associated with the malignancy, comprising the steps of:
(a) incubating T cells, isolated from a warm-blooded animal, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy; and (b) detecting the presence or absence of proliferation of the T cells, thereby determining the presence or absence of the malignancy.
(a) incubating T cells, isolated from a warm-blooded animal, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy; and (b) detecting the presence or absence of proliferation of the T cells, thereby determining the presence or absence of the malignancy.
2. The method of claim 1 wherein the step of detecting comprises measuring the rate of DNA synthesis of the T cells.
3. A method for the detection of a malignancy in a warm-blooded animal, wherein an activated oncogene or cancer-related gene is associated with the malignancy, comprising the steps of:
(a) contacting a body fluid, suspected of containing antibodies specific for a protein expression product of an activated oncogene or cancer-related gene associated with the malignancy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
(b) incubating the body fluid under conditions and for a time sufficient to allow immunocomplexes to form; and (c) detecting the presence or absence of one or more immunocomplexes formed between the protein expression product and antibodies in the body fluid specific for the protein expression product, thereby determining the presence or absence of the malignancy.
(a) contacting a body fluid, suspected of containing antibodies specific for a protein expression product of an activated oncogene or cancer-related gene associated with the malignancy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
(b) incubating the body fluid under conditions and for a time sufficient to allow immunocomplexes to form; and (c) detecting the presence or absence of one or more immunocomplexes formed between the protein expression product and antibodies in the body fluid specific for the protein expression product, thereby determining the presence or absence of the malignancy.
4. A method for monitoring the effectiveness of cancer therapy in a warm-blooded animal with a malignancy, wherein an activated oncogene or cancer-related gene is associated with the malignancy, comprising the steps of:
(a) contacting a first body fluid sample, taken from the warm-blooded animal prior to initiation of therapy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
(b) incubating the body fluid under conditions and for a time sufficient to allow immunocomplexes to form;
(c) detecting immunocomplexes formed between the protein expression product and antibodies in the body fluid specific for the protein expression product;
(d) repeating steps (a), (b), and (c) on a second body fluid sample taken from the animal subsequent to the initiation of therapy; and (e) comparing the number of immunocomplexes detected in the first and second body fluid samples, thereby monitoring the effectiveness of the therapy in the animal.
(a) contacting a first body fluid sample, taken from the warm-blooded animal prior to initiation of therapy, with at least one protein expression product of an activated oncogene or cancer-related gene associated with the malignancy;
(b) incubating the body fluid under conditions and for a time sufficient to allow immunocomplexes to form;
(c) detecting immunocomplexes formed between the protein expression product and antibodies in the body fluid specific for the protein expression product;
(d) repeating steps (a), (b), and (c) on a second body fluid sample taken from the animal subsequent to the initiation of therapy; and (e) comparing the number of immunocomplexes detected in the first and second body fluid samples, thereby monitoring the effectiveness of the therapy in the animal.
5. The method of any one of claims 3 or 4 wherein a reporter group is bound to a second antibody capable of binding to the antibodies, and wherein the step of detecting comprises (a) removing substantially any unbound antibody, (b) adding the second antibody, (c) removing substantially any unbound second antibody, and (d) detecting the presence or absence of the reporter group.
6. The method of claim 5 wherein the second antibody is an anti-human antibody.
7. The method of any one of claims 3 or 4 wherein a reporter group is bound to a molecule capable of binding to the immunocomplexes, and wherein the step of detecting comprises (a) adding the molecule, (b) removing substantially any unbound molecule, and (c) detecting the presence or absence of the reporter group.
8. The method of claim 7 wherein the molecule capable of binding to the immunocomplexes is protein A.
9. The method of any one of claims 3 or 4 wherein a reporter group is bound to the protein expression product, and wherein the step of detecting comprises removing substantially any unbound protein expression product and thereafter detecting the presence or absence of the reporter group.
10. The method of any one of claims 5, 7, or 9 wherein the reporter group is selected from the group consisting of radioisotopes, fluorophores, enzymes, luminescers, and dye particles.
11. The method of any one of claims 1-10 wherein the protein expression product of an activated oncogene is a protein encoded by an oncogene selected from the group consisting of ras, src, abl, fgr, rel, yes, fes, net, mos, raf, erb B, erb A, fms, neu, ros, kit, sea, sis, myc, myb, fos, ski, jun and ets.
12. T cells isolated from a warm-blooded animal, said T cells proliferated in the presence of at least one protein expression product of an activated oncogene or cancer-related gene associated with a malignancy, for use in the manufacture of a medicament for treating a malignancy in the warm-blooded animal.
13. The T cells of claim 12 wherein the protein expression product of the activated oncogene is a protein encoded by an oncogene selected from the group consisting of ras, src, abl, fgr, rel, yes, fes, net, mos, raf, erb B, erb
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US47064590A | 1990-01-26 | 1990-01-26 | |
| US470,645 | 1990-01-26 | ||
| CA002074720A CA2074720C (en) | 1990-01-26 | 1991-01-24 | Immune reactivity to expressed activated oncogenes for diagnosis and treatment of malignancy |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002074720A Division CA2074720C (en) | 1990-01-26 | 1991-01-24 | Immune reactivity to expressed activated oncogenes for diagnosis and treatment of malignancy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2351642A1 true CA2351642A1 (en) | 1991-08-08 |
Family
ID=25675372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002351642A Abandoned CA2351642A1 (en) | 1990-01-26 | 1991-01-24 | Immune reactivity to expressed activated oncogenes for diagnosis and treatment of malignancy |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2351642A1 (en) |
-
1991
- 1991-01-24 CA CA002351642A patent/CA2351642A1/en not_active Abandoned
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