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Definitions

• the invention relates to methods for diagnosing the predisposition onset and course of disease.
• the invention also relates to methods for treating disease. Further, the invention relates to reagents which are useful for the diagnosis and treatment of such diseases.
• TCR ⁇ T-cell receptor ⁇ -chain
• This locus is a gene complex containing variable (V), diversity (D), and joining (J) gene segments which participate in somatic cell rearrangement with a constant (C) region gene segment to encode the ⁇ -chain of the T-cell receptor (Chien et al. (1984) Nature 309. 322-326).
• V variable
• D diversity
• J joining
• C constant region gene segment to encode the ⁇ -chain of the T-cell receptor
• the TCR ⁇ locus resides at 7q35 (Isobe et al. (1985) Science 228, 580).
• this complex spans more than 600 kb and contains 70 to 80 variable region segments (Concannon et al. (1986) Proc. Natl. Acad. Sci.
• V region genes are adjacent to two tandemly organized regions each of which include a D and a C gene segment separated by a cluster of six or seven J region gene segments (Tunnacliffe et al. (1985) Nucleic Acids Res. 13, 6651-6661; Toyonaga et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8624-8628).
• the translated ⁇ -chain polypeptide pairs with a T-cell receptor ⁇ -chain can be expressed on the surface of the T-cell (reviewed by Kronenberg et al. (1986) Annu. Rev. Immunol. 4., 529-591).
• the T-cell receptor then functions in recognizing antigen in the context of a self major histocompatibility molecule (Dembic et al. (1986) Immunol. Today 7, 308).
• the co-recognition of antigen and major histocompatibility molecules by a T-cell must be specific and carefully regulated, since improper immune regulation fosters autoimmunity.
• T-cells play a pivotal role in the differentiation and regulation of effector mechanisms within the immune system (Paul et al. (1977) Science 195, 1293-1300).
• TCR genes may be candidates for genetic elements contributing to the susceptibility to MS and other immunologically mediated disorders. Support for this possibility has been obtained from animal studies which have identified associations between experimental demyelinating disorders and TCR genes. For example, chronic-relapsing experimental encephalomyelitis (EAE) (Mokhtarian et al. (1984) Nature 309, 356-358), and Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (Lipton (1975) Infect. Immun. 11, 1147-1155; Melvold et al. (1987) J. Immunol.
• EAE experimental encephalomyelitis
• TMEV Theiler's murine encephalomyelitis virus
• the invention includes methods for diagnosing predisposition to a disease.
• Such methods include obtaining a DNA test sample from an animal, such as a human, and detecting in that sample a target DNA sequence correlated with a susceptibility gene for the disease.
• the target DNA sequence comprises a DNA sequence contained within or in close proximity to a genomic DNA sequence encoding a variable region of a T-cell antigen receptor ⁇ -chain.
• the target DNA sequence comprises a restriction fragment length polymorphism (RFLP) linked to one or more V ⁇ segments of a T-cell antigen receptor ⁇ -chain. The detection of such an RFLP provides an indication that a first susceptibility gene for the disease is present in the genome of the individual.
• RFLP restriction fragment length polymorphism
• the detection of a first target DNA sequence associated with a T-cell antigen receptor chain may be combined with the detection of a second target DNA sequence associated with the variable region of a different T-cell antigen receptor chain. Further, such detection of target DNA sequences, which may, for example, define ⁇ and ⁇ -chain haplotypes, may be combined with determining the Major Histocompatibility Complex (MHC) haplotype as a further indication of the predisposition of that individual to a particular disease.
• MHC Major Histocompatibility Complex
• the invention also includes methods for diagnosing the onset or monitoring the course of a disease.
• Such methods include obtaining a T-cell nucleic acid sample from an animal such as a human and detecting in such a sample a target nucleic acid sequence correlated with the disease.
• the first target nucleic acid sequence is formed of DNA or RNA which corresponds to a DNA sequence contained within or in close proximity to a rearranged genomic DNA sequence encoding a variable region of a T-cell antigen receptor.
• Such variable regions include ⁇ and ⁇ variable regions of the T-cell antigen receptor.
• the detection of a first target nucleic acid sequence for a specific variable region or gene segment of a particular T-cell antigen receptor chain may be combined with the detection of a second target nucleic acid sequence correlated with the same disease.
• This second target nucleic acid sequence is also formed of an RNA or DNA and corresponds to a DNA sequence contained within or in close proximity to a rearranged genomic DNA sequence encoding a specific variable region or gene segment of a different T-cell antigen receptor chain.
• Methods for diagnosing the onset or monitoring of the course of a disease also include obtaining a suitable sample containing T-cell antigen receptor from an animal, such as a human, and detecting in such a T-cell receptor sample a first target polypeptide sequence correlated with an disease.
• This polypeptide sequence is contained within a variable region of a T-cell antigen receptor.
• variable regions include ⁇ and ⁇ variable regions of the T-cell antigen receptor.
• this detection of a first target polypeptide sequence may be combined with the detection of a second target polypeptide sequence, also correlated with the disease, wherein the second polypeptide sequence comprises a specific variable region of a different T-cell antigen receptor chain correlated with the disease.
• target nucleic acid and polypeptide sequences correlated with disease which include for example specific ⁇ and ⁇ variable regions or segments, may be combined with determining the MHC expression of the individual as a further indication of the onset or cause of the disease.
• the invention also includes methods and reagents for treating disease.
• the disease is correlated with at least one T-cell clone containing a specific variable region in the T-cell receptor of the T-cell clone.
• the method comprises treating an afflicted animal, such as a human, with at least one antibody which is reactive with the specific variable region of that T-cell receptor.
• the invention includes antibodies for detecting and treating disease correlated with at least one T-cell clone containing a specific variable region or specific variable segment in the T-cell receptor of the T-cell clone.
• FIG 1 is a schematic representation of the four types of myelin basic protein (MBP) T-cell receptors in B10.PL mice.
• the types are defined on the basis of the combination of ⁇ and ⁇ T-cell receptor chains.
• the bars represent T-cell receptor heterodimers with ⁇ chains on the left and ⁇ chains on the right. Numbers beside each bar indicate variable (V) and joining (J) gene segments (V on top, J on the bottom) constituting complete genes encoding each change (diversity gene segments for the b chain are not shown).
• the circles with different shading represent T-cells with different profiles of antigen fine specificity.
• the numbers below each type indicate the total number and percentage of T-cells determined to belong to each category.
• Figure 2 identifies the nucleotide sequences of human ⁇ chain cDNAs. The clones from which nucleotide sequences were determined are indicated at the left. Clones designated PL were derived from a peripheral lymphocyte cDNA library. Other clones derived from established T-cell lines are labelled with the name of the cell line except for V ⁇ YT35, which is derived from a MOLT-3 cell line (Eyanagi (1984) Nature 308, 145- 149). Clone ATL21 (Ikuta, et al. (1985) Proc. Nat. Acad. Sci. U.S.A. 82 7701-7705) was derived from a genomic sequence and introns have been edited out in this alignment. Additional germ-line V ⁇ gene segment sequences homologous to PL2.13, PL3.9 and PL5.10 have been published, but are not shown here (Ikuta, supra). The sequences are listed in the order V ⁇ 1-V ⁇ 15, as in Figure 3.
• Figure 3 is the amino acid sequence of murine and human V ⁇ genes.
• Human (h) and murine (m) V ⁇ gene-segment nucleotide sequences were translated and aligned to maximize homology at the amino acid level.
• Amino acids conserved at the 75% level are indicated by asterisks.
• V ⁇ 2.1 and V ⁇ 2.2 differ only in the leader sequence and hence appear identical in this comparison. Amino acids are identified by the single-letter code.
• Figure 4 is a Southern blot analysis displaying the most common restriction patters for T-cell receptor V/3 gene segment subfamilies 1 through 14. Two different digests are shown for each subfamily. Vertical lines indicate the estimated numbers of gene segments within individual subfamilies. In some cases, estimates of subfamily sizes were made based on additional hybridization data not shown. Subfamilies V ⁇ 12 and V ⁇ 13 appear to share common members as indicated by nucleotide sequence data.
• FIG. 5 depicts the segregation of polymorphic V ⁇ and C ⁇ alleles.
• Each chromosome indicates the alleles detected at the V ⁇ 8, V ⁇ 11 and C ⁇ loci ordered top to bottom, respectively. Numbers below schematic chromosomes are for subject identification.
• Figure 6 demonstrates linkage disequilibrium between the alleles at the V ⁇ 8 and V ⁇ 11 loci in humans.
• Southern blots display the hybridization patters for both homozygotes and heterozygotes at each locus. Allelic frequencies are indicated to the left. Fragment size is indicated to the right.
• the distribution of haplotypes in a population of 52 unrelated individuals generated by genotyping at the V ⁇ 8, V ⁇ 11 and C ⁇ loci are indicated in the lower part of the figure. The distribution of haplotypes expected was calculated from the allelic frequencies.
• Figure 7 depicts a restriction enzyme map for the murine V ⁇ locus.
• Figure 8A depicts the nucleotide sequence of TCR ⁇ cDNA clones derived from 70 hybridomas specific for the MVP peptide 1-9NAc.
• Figure 8B depicts the translated protein sequence of two distinct V ⁇ sequences from Figure 8A.
• Figure 9 depicts the cell surface expression of a V ⁇ 8 chain by T-cells specific for the MBP peptide 1-9NAc and the in vivo elimination of V ⁇ 8 plus lymphocytes following i.p. injection of F23.1 antibody.
• T H PL172.10 and PL212.6 and T H clone 2C6 were analyzed for T-cell cell surface expression using quantitative fluorescence of flow cytometry.
• the T-cells were stained with the monoclonal antibody of 23.1 (anti-V/38) or 500.A.A2 all by fluorescein isothyoceinate (FITC) -conjugated goat anti-mouse IgG.
• FITC fluorescein isothyoceinate
• Figure 10 depicts the inhibition of MBP response with anti-TCR monoclonal antibodies.
• Figure 11 is a histogram demonstrating the down modulation of V ⁇ 8.2 T-cells following in vivo antibody injections of an EAE mouse.
• Figure 12 depicts the effect of in vivo monoclonal antibody treatment on the response of EAE mice to MBP.
• Figure 13 depicts the comparison of the response to MBP and monoclonal antibody treated mice with and without EAE.
• Figure 14 demonstrates treatment with anti-V ⁇ monoclonal antibody results in reversal of MBP-induced paralysis in EAE mice.
• Figure 15 depicts the allelic frequencies of 38 Caucasian, unrelated MS patients compared to 100 normal individuals.
• the hybridization pattern at each locus is displayed by Southern blot for both homozygotes and heterozygotes. Allelic frequencies are indicated to the left of each pattern and fragment size to the right. Allelic frequencies of MS patients are indicated first followed by those determined for normal individuals in parentheses. Asterisks (*) indicate invariant bands.
• T-cells play an important role in human diseases involving the immune system, such as, for example, autoimmune diseases.
• T-cells are believed to act as the causative agents that incorrectly recognize the body itself as foreign.
• the action of T-cells is mediated through antigen recognition by the cell antigen receptors present on the T-cell surface.
• T-cell antigen receptors TCARs
• TCRs T-cell receptors
• the present invention is based, in part, on the discovery that certain restriction fragment length polymorphisms (RFLPs) linked to specific V ⁇ gene segments are in linkage disequilibrium with at least one putative susceptibility gene for multiple sclerosis.
• the invention is also based, in part, on the discovery that clonal populations of T-cells containing specific variable regions in both the ⁇ and ⁇ -chain of the TCAR are present in a mouse model system for multiple sclerosis. See Figure 1. In this mouse model system, treatment with a monoclonal antibody specific for a particular V ⁇ segment in the TCAR of a clonal population of T-cells associated with murine experimental encephalomyelitis (EAE) reduced incidence of the disease.
• EAE murine experimental encephalomyelitis
• the invention is based upon the discovery that a multiple sclerosis susceptibility gene is located in the human genome centromeric to VjSll and likely between V ⁇ 8 and V ⁇ 11. Since there are at least six TCAR V/3 genes between V ⁇ 8 and V ⁇ 11 (Lai et al. (1988) Nature331, 543-546, it is believed that a gene encoding a V ⁇ segment in this chromosomal region will be found to be a multiple sclerosis susceptibility gene.
• T cells can be studied to identify TCAR disease correlations. These correlations can then be used to produce diagnostic, therapeutic or disease monitoring procedures.
• Relevant diseases include but are not limited to autoimmune diseases, neoplastic diseases, infectious diseases, hypersensitivity, transplantation and graft-versus-host disease, and degenerative nervous system diseases.
• Autoimmune diseases include but are not limited to arthritis such as rheumatoid arthritis, type I diabetes, juvenile diabetes, multiple sclerosis, thyroiditis, myasthenia gravis, systemic lupus erythematosis, Sjogren's syndrome, Grave's disease, Addison's disease, Goodpasture's syndrome, scleroderma, dermatomyositis, myxoedema, pernicious anemia, autoimmune atrophic gastritis, and autoimmune hemolytic anemai.
• arthritis such as rheumatoid arthritis, type I diabetes, juvenile diabetes, multiple sclerosis, thyroiditis, myasthenia gravis, systemic lupus erythematosis, Sjogren's syndrome, Grave's disease, Addison's disease, Goodpasture's syndrome, scleroderma, dermatomyositis, myxoedema, pernicious anemia, autoimmune atrophic gas
• Neoplastic diseases include but are not limited to lymphoproliferative diseases such as leukemias, lymphomas, Non-Hodgkin's lymphoma, and Hodgkin's lymphoma, and cancers such as cancer of the breast, colon, lung, liver, pancreas, etc.
• Infectious diseases include but are not limited to viral infections caused by viruses such as HIV, HSV, EBV, CMV, Influenza, Hepatitis A, B, or C; fungal infections such as those caused by the yeast genus Candida; parasitic infections such as those caused by schistosomes, filaria, nematodes, trichinosis or protozoa such as trypanosomes causing sleeping sickness, plasmodium causing malaria or leishmania causing leischmaniasis; and bacterial infections such as those caused by mycobacterium, corynebacterium, or staphylococcus.
• Hypersensitivity diseases include but are not limited to Type I hypersensitivities such as contact with allergens that lead to allergies.
• Type II hypersensitivities such as those present in Goodpasture's syndrome, myasthenia gravis, and autoimmune hemolytic anemia
• Type IV hypersensitivities such as those manifested in leprosy, tuberclosis, sarcoidosis and schistosomiasis.
• Degenerative nervous system diseases include but are not limited to multiple sclerosis and Alzheimer's disease.
• a "disease” is any immunological disease which is mediated, at least in part, by a T-cell clonal population. Such diseases are capable of afflicting any animal containing an immune system.
• the preferred animal of interest is the human being.
• Other preferred animals include domesticated animals such as equine, bovine, ovine, porcine, canine, feline and murine species. Diseases in such other species may be analogous to those identified in humans or be uniquely characterized for a particular species or group of species.
• the methods and reagents of the present invention to diagnose and treat disease are useful to the medical and veterinary disciplines.
• T-cell antigen receptor specific reagents such as antibodies or nucleic acids, are used herein for the diagnosis, treatment, and monitoring of disease.
• the first step in this process is to correlate the presence of a specific TCAR with a disease state. This correlation may either involve the expression of a particular TCAR on a subset of disease related cells or may involve an analysis of the TCAR genes that are inherited by the individual and their relationship to predisposing that individual to developing a particular disease. Once such a correlation is made, it is used to detect or diagnose that disease state in patients.
• TCAR reagents may further be used to treat the disease state, in their capacity to specifically modulate the action of the T-cells they identify. They may also be conjugated with various toxins to further modulate the targeted T-cells.
• TCAR reagents are used to monitor the disease treatment to determine whether the treatment is effective and the disease is in remission, or to determine whether the patient has relapsed with a return of the disease state, or to determine whether the patient remains in a stable, disease free condition. During the monitoring period, the TCAR reagents are useful both diagnostically to indicate the presence or absence of disease, as well as therapeutically to treat the disease.
• the diagnosis" of disease in humans or other animals as described herein requires a correlation between specific TCARs with each disease. This correlation is determined by the methods and reagents described herein.
• the diagnosis, treatment and monitoring of disease is based upon disease correlations determined by comparing the TCARs in disease related samples with suitable baseline samples.
• the disease correlations and resulting clinical procedures can be based upon quantitative as well as qualitative differences in the TCARs expressed in the different samples.
• a relative change in the value of a correlating TCAR in serially obtained samples during the course of a treatment is of value; as is the absolute value at each point in the series.
• TCAR reagents In a clinical setting, disease diagnosis is made using a variety of procedures and TCAR reagents. Such diagnosis involves the use of TCAR specific reagents that are labeled with detectable moieties such as biotin, radiolabels, fluorescent labels, or metal ions to name a few. These reagents include either nucleic acid probes or antibodies and can be used in a variety of clinical procedures including but not limited to imaging analysis, RFLP analysis, PCR analysis, fluorescent cytometry, fluorescent microscopy, in situ hybridization, nuclear magnetic resonance analysis, ELISA analysis, etc. Diagnostic procedures can be either performed in vitro or in vivo.
• kits are useful in performing the diagnosis. Such kits include the necessary TCAR reagents coupled with the appropriate detectable marker for the analysis, suitable standards, solid phase components such as microscope slides, microtiter dishes, or beads as necessary, other active components such as enzymes and substrates useful for detection, etc. If multiple TCAR reagents are required to perform the diagnosis, kits include each such reagent; as for example, MHC, V ⁇ or V ⁇ specific reagent (s).
• Treatment of Disease Based Upon TCARs The treatment of disease based upon TCARs in humans or animals is most effective when only those T-cell subsets involved in the disease are specifically modulated. Treatment of larger T-cell subsets can also be effective, but has the potential disadvantage of modulating non-disease specific T-cells as well. In practice, it may be necessary to modulate a subset of normal T-cells having variable region sequences common to those used by the T-cell clone (s) which mediate the disease. Effective immunotherapies based upon TCAR reagents involve either the ablation or deletion of disease specific T-cells, or alternatively, the induced proliferation of specific T-cells.
• the dose of the TCAR reagent for example, an antibody specific for a particular TCAR, should be predetermined by in vitro techniques known in the art, such that the cells are either correctly stimulated or else correctly targeted for removal.
• the mode of treatment can involve either acute or chronic treatment conditions. This in turn will lead to treatment regimens involving either one time bolus administrations, continuous administrations or repeated administrations.
• the dose forms can include, but are not limited to injectable forms applied by either intraperitoneal, intramuscular, or intravenous injection, slow release forms, such as by those delivered in transplantable forms, on patches, or in other colloidal forms.
• the reagent itself needs to be properly formulated, as for example, a humanized antibody combined with various sugars, buffers or stability causing compounds that extend the stability or half-life of the reagent.
• the reagent can first be modified to increase or decrease the amount of carbohydrates complexed to it, or alternatively to complex it with reagents such as polyethylene glycol (PEG).
• reagents such as polyethylene glycol (PEG).
• pharmaceutical compositions comprising the therapeutic reagent in the appropriate buffers, salts and pH are required.
• the same reagents that are used to diagnose or treat disease can also be used to monitor the effectiveness of a disease therapy or to monitor the progression of the disease throughout phases of remission, relapse or stable periods.
• the initial disease correlation is made on those TCARs that are expressed by the arthritic T-cell clone (s).
• TCAR reagent (s) based on this correlation can then be used to diagnose this arthritis and to treat it.
• the TCAR reagent(s) can be used to track the extent of the elimination of the arthritic cell subset to the point where all the cells are gone and the disease is in remission.
• the TCAR reagent(s) can be periodically used to test for the reappearance of the arthritic cells.
• the patient is periodically rediagnosed to determine whether the individual is in a stable remission, has relapsed and needs to be retreated, or is undergoing a flareup of acute disease.
• the TCAR reagents are also useful in combined diagnostic and therapeutic procedures.
• T-cell Antigen Receptors Correlation of T-cell Antigen Receptors with Disease
• diagnosis, treatment and monitoring of T-cell related diseases depends upon the correlation of specific TCARs with disease states.
• An infinite spectrum of T-cells exists in the body that are involved in recognizing an equally infinite spectrum of disease related antigens.
• the specificity of this recognition is accomplished by the interaction of specific TCARs with disease antigens.
• the diversity of the TCARs themselves is also very high. This diversity is created by the multiplicity of TCAR germline segments, the combinatorial joining of these different segments, the junctional flexibility and
• these disease specific samples include cells from the peripheral blood, synovial fluid, pleural fluid or spinal fluid as well as those that have infiltrated tissues, synovium, skin lesions or components of the nervous system. Such infiltrated cells can be studied either directly in tissue sections or else allowed to diffuse from the tissues and studied directly or expanded in tissue culture.
• the cells that have actually infiltrated a synovium are preferable to the whole population of cells present in the peripheral blood when trying to analysis rheumatoid arthritis specific TCARs.
• the peripheral blood or spinal fluid is the sample of choice.
• Correlations and diagnosis of a predisposition to disease can be made using restriction fragment length polymorphisms (RFLPs) of inherited TCAR genes.
• RFLPs restriction fragment length polymorphisms
• Any DNA test sample containing genomic DNA can be used for this analysis.
• the analysis is much less complicated if the DNA test sample does not contain a substantial amount of T-cells. This is due to the fact that the rearranged TCAR genes observed in T-cells, complicate the analysis of the presence or absence of different TCAR alleles.
• the samples of choice are peripheral blood B cells or fibroblasts.
• additional samples are also useful. These include samples that can be used to obtain comparison values.
• One such comparison sample is a similar sample taken from a normal individual who does not have the disease of interest. The normal individual's sample can be used as a measure of the relative increase or decrease of the TCAR in the diseased patient's sample.
• Other useful samples include samples taken from the same patient before the onset of disease or before the initiation of therapy. For rheumatoid arthritis patients, for example, a sample taken from the same patient during a stable period is extremely useful to obtain a value to compare with a sample taken during an acute flareup period.
• samples taken from the same patient before the therapy begins, during the therapy, and during periods of remission and relapse are all valuable. These longitudinal or serial samples can be used to show the amount of change observed in the relevant TCAR marker.
• any of the TCAR reagents described herein can be used to identify disease correlations useful for disease diagnosis and prognosis.
• the disease correlations fall into two categories. The first category involves correlations between the expression of certain TCARs and the presence of certain diseases.
• the second category involves prognosis based upon the analysis of the inherited TCARs in an individual's genomic DNA to determine whether the actual TCAR genes that were inherited can predispose that individual to develop a disease. In the later case, the disease itself might not yet be manifested and the TCARs that correlate with the disease might not yet be expressed.
• the methodologies described infra. illustrate some of the ways in which disease correlations with TCAR reagents can be made; others will be known by those skilled in the art.
• T cell becomes activated and clonally expands resulting in a subset of T-cells each of which expresses the same
• TCAR This TCAR is an identifying characteristic of the disease specific T-cell subset. If more than one, but a limited number of T-cells, become activated by the disease specific antigen (s), then multiple clones expand resulting in an oligoclonal subset of T-cells. The oligoclonal subset is still only expressing a limited number of TCARs which in turn can be used to identify the oligoclonal subset.
• Techniques that are useful for identifying disease correlations must be able to detect the disease specific subset or oligoclonal subset of T-cells in the presence of all of the other T-cells that are present in the sample. Some of the techniques that can be used are described infra.
• RNA is isolated from the T-cells in a sample and used to synthesize complementary DNA (cDNA) which is in turn cloned into bacterial cells to produce the library.
• cDNA complementary DNA
• the library represents the complete set of TCARs that were expressed in the sample at approximately the same proportions as expressed.
• An analysis of the clones in the library by sequencing the TCAR cDNAs or hybridizing them to known TCAR probes, for example, indicates which TCARs are expressed most often. This is a characteristic indicative of a disease expanded subset of cells each expressing the same TCAR.
• One advantage of a cDNA library is that it is possible to analyze each V, D, J, and C region expressed by the dominantly expressed TCARs to determine their significance in the correlation to disease.
• TCAR specific primers in a polymerase chain reaction (PCR) (U.S. Patent 4,683,195 by Mullis et al.).
• PCR polymerase chain reaction
• the RNAs obtained from a disease specific sample are first used to synthesize cDNA.
• the cDNA is in turn amplified with TCAR specific primers by PCR to produce highly TCAR enriched DNAs.
• DNAs can then be sequenced or labelled and then used to probe a nitrocellulose filter that has been spotted with a panel of known TCAR genes.
• TCAR sequences or genes are deemed to be indicative of the oligoclonal or disease specific subset of T-cells.
• a one-sided PCR procedure can be used which is analogous to that described by Loh E.Y. et al. (1989) Science 243, 217-220.
• the disease sample itself can be analyzed directly by in situ hybridization.
• the TCAR RNA being expressed inside each individual T-cell can be detected by hybridizing those cells to TCAR specific gene probes.
• the sample is divided into multiple samples that are in turn probed with a panel of TCAR genes. This panel can include V, J DJ, or C region specific probes. If a cell is expressing a particular TCAR RNA, it will be detected as a positive when hybridized to the corresponding TCAR gene probe.
• Genomic DNA can be prepared from the disease sample and analyzed by Southern analysis using a panel of TCAR gene probes. Each probe will detect the unrearranged and rearranged TCAR genes in each T-cell of the sample. Since most of the non-disease T-cells will be present at very low percentages, the rearranged bands corresponding to these T-cells will be too weak to see above background by Southern blot analysis. For an expanded disease T-cell subset or oligoclonal subset, however, the rearranged TCARs will be present at higher levels, and are detectable as band(s) distinct from the unrearranged bands.
• Anti-TCAR antibodies can be used to screen disease specific T-cell samples for the expression of specific TCARs.
• the antibodies can either react directly with the intact TCAR on live cells, or the cells can be pretreated with ethanol or other suitable reagents, so that antibodies reactive with denatured TCAR can be used.
• the sample is divided into multiple portions and each portion in analyzed by FLOW with one antibody or an antibody pool. The antibodies that react with the greatest proportion of cells in the sample are deemed to represent the disease specific cells.
• samples can also be analyzed by fluorescent microscopy instead of fluorescent cytometry.
• TCARs are inherited as a multigene family. For each
• TCAR gene one allele will be inherited maternally, and one allele will be inherited paternally. Differences in the alleles inherited for each TCAR gene can be defined by restriction fragment length polymorphisms
• TCAR haplotypes can be defined for each individual by identifying which alleles (or RFLPs) are observed in the genomic DNA of that individual. This is preferably done by Southern analysis, where the individual's genomic DNA is digested with various restriction enzymes known to define a RFLP of interest, and then hybridized to a panel of TCAR gene probes. Once the DNA sequence surrounding the restriction enzyme site responsible for the RFLP is known, then oligonucleotides can be synthesized corresponding to flanking sequences around the site and the technique of PCR can also be used.
• a disease correlation is then made by determining whether the inheritance of particular RFLPs for TCARs (or TCAR gene haplotypes) occur more often in patients manifesting the disease than in those where the disease is not present.
• Family studies are especially useful in this type of analysis, as the inheritance of a TCAR gene haplotype can be traced throughout the generations of the family and correlations made between affected and unaffected individuals. From this analysis it becomes possible to predict that an individual, although not currently manifesting the disease, is predisposed to developing the disease, based upon that person's inheritance of the disease indicating TCAR haplotype.
• variable regions of TCARs For disease correlations, a RFLP involving the variable regions of TCARs will be preferred over those involving the constant regions. It is the variable regions of TCARs that interact with antigens and are, thus, disease specific. There are only 2 ⁇ constant regions and 1 ⁇ constant regions of TCARs that are used by the majority of T-cells.
• At least one putative "susceptibility gene" for the disease may be present in the animal at risk for the disease.
• susceptibility genes include, but are not limited to, T-cell receptor genes encoding the variable region of ⁇ and/or ⁇ -chains of the TCAR.
• Such variable regions in the human ⁇ -chain of the T-cell receptor include V ⁇ gene segments", J ⁇ gene segments and D ⁇ gene segments.
• V ⁇ gene segments include V ⁇ gene segments
• J ⁇ gene segments and D ⁇ gene segments In the case of human TCAR ⁇ -chains
• such variable genes include those encoding V ⁇ gene segments and J ⁇ gene segments.
• Such V, J or D gene segment may comprise normal or abnormal genes.
• a V ⁇ gene segment may be normal in that when it is found in a population, there is little or no variation in its nucleotide and amino acid sequence.
• the inheritance of such a "normal” gene may result in a haplotype indicative of a predisposition to a disease.
• an abnormal gene segment i.e. one in which there is substantial allelic variation between individuals, may define a haplotype wherein the inheritance of the "abnormal" gene segment causes a predisposition to a disease.
• target DNA sequences are typically contained within or in close physical proximity to a genomic DNA sequence encoding a variable region of a TCAR ⁇ or ⁇ -chain.
• Target DNA sequences are "correlated" with a susceptibility gene if it is demonstrated that each is in linkage disequilibrium with such a susceptibility gene.
• a target DNA sequence is in close proximity to a variable region if it is in linkage disequilibrium with a DNA sequence encoding a variable region or segment of a TCAR chain. Linkage disequilibrium is determined by methods well known to those skilled in the art and as described herein.
• more than one susceptibility gene may be responsible for a particular disease.
• one or more second target DNA sequences correlated with a second susceptibility gene are also detected as an indication of the predisposition of an individual to that disease.
• the Major Histocompatibility Complex (MHC) haplotype may provide a further indication of predisposition to a particular disease. For example, in some populations, approximately 80% of the individuals afflicted with multiple sclerosis have an MHC haplotype of DR2 + .
• a first target DNA sequence for example, within the variable region of a T-cell receptor ⁇ -chain correlated to a first susceptibility gene defines a ⁇ -chain haplotype indicative of the predisposition of the animal to that disease.
• a second susceptibility gene for a particular disease is also identified as a part of the etiology of a particular disease, e.g. a susceptibility gene located within the T-cell receptor ⁇ -chain variable region locus
• an ⁇ -chain haplotype is defined by a second target DNA sequence as a further indication of predisposition to such a disease.
• predisposition is not to be construed as an absolute indication that a particular disease will develop. Rather, such a determination identifies the risk of an individual for developing such a disease.
• an individual may have haplotypes indicative of a predisposition to MS but may not develop symptoms of such a disease unless exposed to other environmental factors required to precipitate the onset of the disease, e.g., a viral infection, etc.
• a preferred DNA test sample is genomic DNA from an individual animal.
• the target DNA sequence correlated with a susceptibility gene lies outside the structural region encoding a segment of a variable region of a T-cell antigen receptor chain such as the ⁇ or ⁇ -chain.
• the target DNA sequence may comprise a unique sequence which encodes or destroys a DNA sequence recognized by a particular restriction endonuclease.
• the target DNA sequence is detected by digesting genomic DNA with the appropriate restriction endonuclease.
• the digested genomic material may be separated on the basis of size by electrophoresis and probed with a labelled oligonucleotide, e.g., a labelled V ⁇ or V ⁇ cDNA to determine if the cleavage site of the restriction endonuclease correlated with the susceptibility gene is present in the genomic DNA.
• a labelled oligonucleotide e.g., a labelled V ⁇ or V ⁇ cDNA
• the presence or absence of a target DNA sequence encoding an RFLP may be detected by using an oligonuleotide ligase assay procedure. Landegren U. et al. (1988) Science 241, 1077-1080; Landegren, U. et al. (1988) Science 242, 229-347.
• Target DNA sequences other than those encoding RFLPs may also be used.
• a unique sequence in close proximity with a variable region gene may be in linkage disequilibrium with a susceptibility gene. To the extent that such a sequence does not encode an
• RFLP such a unique sequence may be detected in genomic DNA by the polymerase chain reaction (PCR) (Sakai et al. (1986) Nature 329, 166).
• PCR polymerase chain reaction
• two oligonucleotide primers complementary to opposite strands each located at or near each of the ends of the unique DNA sequence defining the target DNA are used in conjunction with a DNA polymerase to exponentially synthesize the target DNA sequence.
• amplified target DNA sequences may thereafter be detected by an appropriately labelled probe.
• the target DNA sequence may be contained within a susceptibility gene for a particular disease.
• the sequence of all or part of the susceptibility gene will be known.
• it may be detected by standard hybridization techniques.
• the above described PCR techniques may be used to detect the target sequence contained within the susceptibility gene.
• the susceptibility gene itself encodes an RFLP the presence of the susceptibility gene may be detected or confirmed by RFLP analysis with an appropriate probe for the susceptibility gene or other gene sequences linked to the RFLP.
• the target sequence correlated to a susceptibility gene is linked to one or more gene segments encoding the variable region of a ⁇ or ⁇ TCAR polypeptide chain.
• appropriate probes for such variable region gene segments may be used in conjunction with an appropriate restriction endonuclease.
• a panel of cDNA probes for the V ⁇ subfamilies are used. See Figure 2 and Concannon et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6589-6602.
• V ⁇ gene segments cDNA probes for the V ⁇ subfamilies are used. See Klein M. et al. (1987) Proc. Natl. Acad Sci. USA 84, 6884-6888.
• specific RFLPs associated with 15 subfamilies of V ⁇ gene segments were used to determine if a correlation existed between such RFLPs and a predisposition to multiple sclerosis in humans.
• various haplotypes defined by RFLPs associated with V ⁇ 8 and V ⁇ 11 were used to detect a correlation between such RFLPs and a putative multiple sclerosis susceptibility gene. Based on the extent of linkage disequilibrium measured between these RFLPs and MS, it was determined that this putative MS susceptibility gene is located between gene segments V ⁇ 8 and V ⁇ 11 in the human genome.
• the susceptibility gene has yet to be completely characterized, it is believed to be a V ⁇ gene segment which is used in a T-cell antigen receptor of a T-cell clone which mediates the onset and course of MS.
• the identification of individuals having a haplotype indicative of the presence of one or more of these RFLPs provides an indication of the presence of the multiple sclerosis susceptibility gene in that individual.
• the T-cell receptor comprises an ⁇ and ⁇ -chain which recognizes antigen in the context of an MHC molecule
• such other genetic components may reside in the MHC locus and/or a variable region locus encoding the T-cell receptor ⁇ -chain.
• an RFLP within or in close proximity to the V ⁇ l6 gene segment has been correlated with susceptibility to MS based upon the linkage disequilibrium between this RFLP and MS.
• a putative second MS susceptibility gene is therefore located within the a chain variable region gene locus.
• an ⁇ -chain haplotype may also be defined for an individual which provides by itself or in combination with an indication of an increased risk of that individual for developing MS.
• a test sample comprises either a T-cell nucleic acid sample or a T-cell polypeptide test sample.
• a "T-cell nucleic acid test sample” comprises DNA or RNA corresponding to rearranged genomic DNA from T-cells. Such rearranged genomic DNA encodes the ⁇ -chains and ⁇ - chains used by the population of differentiated T-cell to form TCAR RNAs and polypeptides.
• a T-cell nucleic acid test sample typically is an appropriate tissue or fluid sample containing T-cells.
• T-cells in such a sample may if necessary, be further purified by standard methods known to those skilled in the art to facilitate analysis of the nucleic acids present in the T-cell population.
• Such T-cell nucleic acid sample contain rearranged genome DNA encoding ⁇ and ⁇ TCAR chains used by the numerous T-cell clones present in the individual. It is the detection of a particular nucleic acid sequence correlated with the disease encoding either a specific variable region of a T-cell receptor ⁇ and/or ⁇ -chain of the TCAR within the T-cell population which allows for the diagnosis of the onset or course of the disease.
• FIG. 1 is a schematic representation of four types of T-cell receptors and B10.PL mice afflicted with EAE. As can be seen, in the ⁇ -chain, V ⁇ 2.3 and V ⁇ 4.2 are each associated with a J ⁇ 39 gene segment.
• Northern analysis of mRNA extracted from T-cells with probes corresponding to the VJ region encoded by V ⁇ 2.3 /J ⁇ 39 and V ⁇ 4.2 /J ⁇ 39 can be used to detect the presence of two of the four T-cell clones responsible for EAE in mice.
• a similar analysis can be performed for the various segments defining the ⁇ -chain.
• the EAE specific T-cell clones can be detected with even greater accuracy by detecting expression of the specific V ⁇ and V ⁇ variable regions of the TCARs of these clones.
• other diseases may be diagnosed in a similar manner once the particular variable region segments are identified for each T-cell population responsible for the particular disease.
• the rearranged genomic DNA contained within one or more T-cell clones which mediate a particular di'sease may contain an RFLP generated upon rearrangement or which is a remnant of an RFLP from the undifferentiated DNA. If this is the case, detection in a T-cell nucleic acid sample of such an RFLP as a target sequence correlated with the T-cell variable region may be used to diagnose the onset or course of the T-cell mediated disease.
• T-cell antigen receptor test sample is any sample from an animal which contains T-cells.
• the sample containing T-cells may be treated according to standard procedures known to those skilled in the art to enrich for T-cells.
• the test sample or T-cell enriched sample may be treated with appropriate detergents and purified by methods well known to those in the art to obtain purified or partially purified T-cell antigen receptor.
• the T-cell antigen receptor test sample is analyzed to determine if a first target polypeptide sequence comprising the variable region of a TCAR chain correlated with a disease is present by a specific variable region within ⁇ and/or ⁇ -chains.
• a first target polypeptide sequence is "correlated" with disease if a specific variable region or segment within the variable region of a T-cell antigen receptor polypeptide chain is found to be common amongst a population or subpopulation afflicted with the same malady.
• an analogous second target polypeptide sequence also correlated with the particular disease and comprising a specific variable region or segment in a different TCAR chain may be used to detect the onset and course of the disease.
• T-cells associated with EAE in murine species express a limited repertoire of TCAR a and ⁇ -chains.
• the onset and course of EAE in such an animal is monitored by assaying, for example, for V ⁇ 2.3, V ⁇ 4.2, V ⁇ 8.2 and/or V ⁇ 13.
• Such detection is typically with monoclonal antibodies for these particular segments of the variable region and may also include antibodies directed to particular J ⁇ , J ⁇ and D ⁇ gene segments associated with the variable region of T-cell receptors that are responsible for this disease.
• T-cell nucleic acids or polypeptide samples may be detected by the methods described herein for analyzing T-cell nucleic acids or polypeptide samples.
• some diseases are acquired based on exposure to environmental factors such as various pathogens including viruses, bacteria and parasites. In such cases, there may be no genetic predisposition to such diseases.
• Such a situation does not preclude a predictable T-cell response to a particular antigen to produce one or more clonal populations of T-cells which in addition to mediating an immunological response to the foreign antigen also mediate an response.
• reagents are provided for diagnosing or treating a disease characterized-by a clonal population of T-cells containing one or more specific variable regions or segments in the TCAR.
• Such reagents include nucleic acid probes for diagnosis and antibodies for diagnosis and treatment.
• Such antibodies may be polyclonal antibodies but are preferably monoclonal antibodies which are reactive with the specific variable regions or segments correlated with the disease.
• the antibody is typically labelled so that its binding with a particular TCAR may be detected.
• Such antibodies can also be used to correlate a particular TCAR with a disease.
• Polyclonal or monoclonal antibodies can be produced by procedures known in the art, but the characteristics of the antibody will depend upon the immunogen and screening procedures used to produce it.
• antibodies specific to particular T-cell antigen receptor segments can be used in unmodified form or conjugated to radionuclides or toxins by means well known in the art, and used to deliver the conjugated substances to targeted T-cells.
• F23.1 antibodies can be conjugated to a toxin, such as Ricin, by the method of Bumol (1983) Proc. Natl. Acad. Sci. 80, 529. Briefly, monoclonal antibodies reactive with the desired T-cell receptor segment, such as V ⁇ 8, are prepared by conventional and well-known means.
• the antibodies are purified and combined with excess (6 mol/mol) N-succinimydyl 3-(2-pyridyldithio) propionate (Pharmacia, Uppsala, Sweden) in PBS. After 30 minutes incubation at room temperature, the solution is dialyzed against PBS.
• the modified antibodies are conjugated with an appropriate toxin, such as diphtheria toxin A chain.
• Other toxins such as ricin A can also be employed.
• the diphtheria toxin A chain is isolated as detailed in Bumol, supra.
• the modified antibodies are mixed with excess (3 mol/mol) reduced diphtheria toxin A chain (10% of the total volume), allowed to react for 36 hours at 4°C, and concentrated by chromatography on Sephadex G-200.
• the product is applied to a Sephadex G200 column (1.0 x 100 cm), allowed to equilibrate and eluted with PBS.
• the toxin-conjugated antibodies are administered to the animal, preferably by inter-peritoneal injections of approximately 40 ⁇ g of purified antibody conjugate or as determined to be appropriate on the basis of patient weight, severity of disease and other such factors. Injections are repeated at intervals, preferably approximately every three days.
• RNA sequences encoding TCARs can be used to diagnose predisposition or onset of a disease. Since the discovery of TCARs, many of the genes of this multigene family have been identified, cloned and" sequenced. It is currently estimated that there are 50-100 V/3s, 2 D/3s, 13 J ⁇ s, 2 C ⁇ s, over 100 V ⁇ s, over 50 J ⁇ s, and 1 C ⁇ gene segments that are combined to make TCAR heterodimers. In addition, gene segments encoding the variable an constant regions of ⁇ and ⁇ chains of TCARs used by a small class of T-cells are known.
• any of these sequences can be used to determine disease correlations and diagnose predisposition, onset or course of a disease.
• synthetic DNAs or RNAs can be produced by procedures well known in the art to produce nucleic acid sequences that correspond to portions of the complete gene or RNA sequences. These synthetic sequences may be either short oligonucleotide sequences, complete gene sequences or sequences corresponding to all or part of any combination of the V, D, J, or C gene segments.
• Either polyclonal or monoclonal antibodies can be produced by procedures known in the art. However, the characteristics of the antibody will depend on the immunogen and screening procedure used to produce it.
• TCAR containing samples Animals can be immunized to produce antibodies with a variety of TCAR containing samples. These include (1) peptide sequences that correspond to portions of a TCAR (U.S. pending patent applications. Serial Number 726,502 filed April 24, 1985 and Serial Number 176,706 filed April 1, 1988), (2) whole T-cells that express a unique TCAR heterodimer on their surface e.g.
• TCAR polypeptide that has been purified from T-cells by well known procedures such as immunoprecipitation, (4) recombinantly expressed and purified TCAR protein produced from expression systems, including but not limited to yeast, eukaryotic, or prokaryotic cells, and (5) either whole cells or TCAR produced by them, where the cells have been transfected with specific TCAR genes of interest and are expressing the transfected genes in a soluble or membrane bound form.
• the characteristics of the final antibodies are dependent upon the screening procedures used to obtain them. If the screening procedure is designed to identify only those antibodies that recognize denatured protein, then the resulting antibodies that are selected by the screening procedure may not generally react with the intact TCAR polypeptide present on live cells. If the antibodies are to be used for diagnostic procedures, this may or may not make any difference, as a diagnostic procedure that relies on detecting denatured TCAR protein can be chosen. If the antibodies are to be effective therapeutically, however, it is important that they recognize the intact TCAR present on the patient's immune system T-cells.
• Screening procedures that can be used to screen hybridoma cells expressing different anti-TCAR antibodies include among others (1) enzyme linked immunosorbent essays (ELISA), (2) flow cytometry (FLOW) analysis, (3) immunoprecipitation, and (4) the ability to comodulate the CD3 antigen (part of the TCAR-CD3 complex present on the surface of T-cells) off of the surface of cells.
• ELISA enzyme linked immunosorbent essays
• FLOW flow cytometry
• the comodulation and FLOW screening procedures are preferred for the selection of antibodies that are able to recognize intact TCAR on live cells due to the inherent properties of these techniques.
• the immunoprecipitation and ELISA procedures are preferred for the identification of antibodies to inactive or denatured TCAR.
• formats of an ELISA that can be used to screen for anti-TCAR antibodies can be envisioned by one skilled in the art. These include, but are not limited to, formats comprising purified, synthesized or recombinantly expressed TCAR polypeptide attached to the solid phase or bound by antibodies attached to the solid phase or formats comprising the use of whole T-cells or T-cell lysate membrane preparations either attached to the solid phase or bound by antibodies attached to the solid phase. Samples of hybridoma supernatants would be reacted with either of these two formats, followed by incubation with, for instance, a goat anti-mouse immunoglobulin complexed to an enzyme- substrate that can be visually identified.
• FLOW Screening Assay Supernatants of antibody producing hybridomas can be screened by FLOW in a number of different ways as is known by one skilled in the art.
• One screening procedure involves the binding of potential antibodies to a panel of T-cells that express well-known TCARs on their surface. Generally antibodies that react with intact TCAR are detected by this analysis, but the
• T-cells can be fixed slightly with ethanol, in which case antibodies reacting with denatured TCAR polypeptide can be identified.
• FLOW assays can also be formatted where potential antibodies are screened by their ability to compete with the binding of a known antibody for the TCAR present on a T-cell.
• the antibodies to be screened do not react with intact TCAR, they can be screened by their ability to immunoprecipitate a known TCAR as analyzed by
• Hybridoma cell supernatants can be pooled and then screened by this technique. Using this assay, it is possible to identify the chain of the TCAR heterodimer that the anti-TCAR antibodies are recognizing.
• the TCAR normally exists on the surface of T-cells as a complex with the chains of the CD3 complex.
• an antibody binds to this TCAR:CD3 complex, the complex in turn becomes internalized in the T-cell and disappears from the cell surface.
• T-cells are reacted with an antibody specific to the TCAR and the complex becomes internalized, a further reaction with an anti-
• CD3 specific antibody will result in substantially no detection of CD3 bearing cells by FLOW analysis.
• This comodulation screening procedure detects antibodies that are able to interact with intact TCAR on the surface of T-cells, and thus, is preferred for the selection of anti-TCAR antibodies that have the most useful characteristics for TCAR therapeutic reagents for treating disorders.
• the TCAR heterodimer comprises a b chain subunit made up of V ⁇ , D ⁇ , J ⁇ , and C ⁇ gene segments and an ⁇ -chain subunit made up of V ⁇ , J ⁇ , and C ⁇ gene segments.
• Antibodies to the TCAR can recognize epitopes in either the b chain or the a chain regions, as well as combined epitopes made up of some ⁇ -chain and some ⁇ -chain sequences.
• the epitopes can consist of linear stretches, amino acids from either chain or alternatively can arise from conformational sequences where the amino acids in the epitope come together from different parts of the receptor sequence.
• TCAR specific antibodies can be subclassified as follows. Some antibodies will be anti-C ⁇ , anti-V ⁇ , anti-J ⁇ , anti-DJ ⁇ , anti-V ⁇ , anti-J ⁇ , or anti-C ⁇ specific to name a few of the possible subclassifications.
• V region antibodies for example, can be further divided into those anti-V region antibodies that are specific for each of the subfamilies of V ⁇ or V ⁇ , such as anti-V ⁇ 1 specific or anti-V ⁇ 8 specific antibodies. These subfamily specific antibodies can be subdivided even further into antibodies that will recognize only individual members of each subfamily, such as for example anti-V ⁇ 8.1 specific antibodies.
• Antibodies can also be produced that react with shared regions of the TCAR, such as anti-V ⁇ 2J ⁇ 2.3 specific antibodies. A similar approach can be used to characterize antibodies to TCARs comprising ⁇ and ⁇ subunits.
• Anti-TCAR Antibody TCAR specific antibodies can also be classified as anti-idiotypic, anti-clonotypic, anti-major framework. or anti-minor framework antibodies. This classification is based more on the functional characteristics of the antibody than on the molecular site that the antibody reacts with.
• anti- idiotypic antibodies are characterized by the antibody's ability to react with the "idiotypic" region, i.e., the region of the receptor that makes it unique from other receptors. This is analogous to the idiotypic regions of immunoglobulins, which are regions of the immunoglobulin heterodimer that interact with antigen.
• Anti-clonotypic antibodies are so defined, because they reacted only with the TCAR present on an individual T-cell clone.
• Anti-major a framework antibodies for example, are distinguished by their ability to recognize all a bearing TCARs, such as would also be true of anti-Ca region antibodies.
• Anti-minor framework antibodies react with a subset of T-cells, such that they can be used to subdivide the total population of T-cells detected by anti-major framework antibodies into subsets. These subsets can then be even further subdivided by anti-idiotypic antibodies.
• Antibodies that are useful as diagnostic or therapeutic reagents can consist of all of these types of TCAR specific antibodies, but some types are expected to be more effective than others depending upon the circumstances under which they are used.
• anti-clonotypic antibodies are used therapeutically in disease states which are characterized by the over proliferation of a single T-cell clone.
• anticlonotypic reagents are not the preferred reagents for treating diseases that arise from not just one T-cell clone, but from a handful of different clones.
• anti-clonotypic reagents are generally useful for treatment protocols that are effective only in a single patient.
• anti-V region specific or anti- minor framework specific reagents that react with a handful of disease specific clones that exist in multiple patients with the same disease state (but perhaps heterogeneous MHC antigens) are preferred therapeutic reagents.
• Such an antibody is used to treat the whole subgroup of patients, not just individual patients.
• Any TCAR specific antibody can also be characterized by its ability to recognize either denatured TCAR polypeptide or nondenatured polypeptide as would exist on live cells. Depending upon the diagnostic procedure to be used, either type of antibody is useful, but for antibodies to be therapeutically effective, they preferably react with the intact, nondenatured receptor that exists on the cells circulating throughout the patients immune system.
• the isotype of the anti-TCAR specific antibody is also important.
• an antibody of a specific isotype may be preferable to one of a different isotype.
• the IgG2a isotype reacts with Fc receptors on cells of the reticuloendothelial system and is more readily removed from the circulation and sequestered in the spleen than other isotypes. Such an antibody that has reacted with a target cell may result in the more efficient removal of the target cell from the site of active disease.
• some isotypes (such as IgG2a) are more effective in antibody dependent cell cytotoxicity reactions than others.
• antibodies of the IgG isotype are preferable to those of the IgM isotype because they have higher binding affinities.
• the desired isotype of an antibody may be selected by screening potential antibodies by an ELISA assay designed to select the isotype of interest.
• the solid phase can be coated with goat anti- mouse IgG Fc specific antibodies, if it is desired to select for antibodies having the IgG isotype.
• the isotype switch can be done by repeatedly selecting for the isotype of interest using magnetic beads (super paramagnetic iron oxide particles, Biomag beads purchased from Advanced Magnetics, Inc.) coated with a goat anti-mouse antibody preparation including all isotype classes.
• the IgG2a binding sites on the coated magnetic beads are first blocked with an irrelevant antibody of the IgG2a isotype. All cells producing antibodies of differing isotypes will then be bound by the beads and removed magnetically, resulting in an enrichment of cells producing the IgG2a isotype. These cells can then be cloned by limiting dilution, and using commercially available anti-isotypic reagents in an ELISA assay, the IgG2a producing clones can be identified.
• Antibodies can be produced in many different animal hosts, including but not limited to, rabbits, mice, and rats. When these antibodies are used therapeutically in humans, they are recognized to varying degrees as foreign and an immune response is generated in the patient.
• One approach to overcome this problem is to immunosuppress the patient in addition to the antibody treatment.
• Another approach is to produce chimeric antibody molecules that combine a mouse variable region domain and a human constant region domain. Such chimeras produce a less marked immune response than non-chimeric antibodies.
• a further approach is to use the techniques of recombinant DNA technology to "humanize" the antibodies. Such humanized antibodies retain the hypervariable regions required for the antigen recognition sites, but all other regions including the framework regions of the variable domain are of human origin. These humanized antibodies are preferable for immunotherapy in that they minimize the effects of an immune response. This in turn leads to a lowering of any concomitant immunosuppression and to increased long term effectiveness in, for instance, chronic disease situations or situations requiring repeated antibody treatments.
• fragments of the antibodies may also be used. Fragments such as F(ab') 2 .
• Fab' or Fab fragments can be generated by enzymes such as pepsin or papain and reducing agents, as is routinely known in the art. These fragments retain the antigen binding domains present in the parent molecules.
• These types of molecules can also be produced by recombinant DNA technology where the variable region domains are ligated and expressed as single chain polypeptides.
• Whole antibody molecules or their fragments can also be produced as immunoconjugates.
• conjugates contain another active group that may be a toxin, cytotoxic compound or radionuclide to name but a few (Copending U.S. Patent Application, Serial Number 07/229,288, filed August 5, 1988).
• the antibody can thus be used to target the active toxin or cytotoxic compound to the specific disease site involved.
• T-cells have been implicated in many different T-cell mediated diseases, another level of treatment involves modulating the entire T-cell population. Although this is more specific than broad immunosuppression therapies, it is still a non-specific therapy that results in the loss of the beneficial functions of some T-cell subsets along with the loss of the deleterious effects of the disease subset of T-cells.
• the preferred immunotherapy herein is one that affects substantially only disease related T-cells and not any other T-cells.
• T-cells are identified by the expression of specific TCARs.
• various levels of immunotherapies can be developed by subdividing the whole T-cell population into those subsets of T-cells that are disease related by virtue of the expression of disease related TCARs.
• Immunotherapies based upon anti-constant region TCARs are no more specific than the anti-CD3 immunotherapies, as both therapies affect the whole population of T-cells.
• immunotherapies based upon anti-variable TCAR subfamily reagents (anti-V ⁇ 6, for instance) target only that subset of T-cells expressing that TCAR variable region subfamily and as a consequence are more specific.
• the preferred immunotherapies are those that substantially affect only the subset of T-cells that are disease related and which do not impair non-disease related T-cells and their functions. Although less preferable, immunotherapies are acceptable which do adversely affect some non-disease related T-cells provided the patient's immune system is not compromised.
• the discussion presented herein for immunotherapy is also applicable for the preferred diagnostic procedures using antibodies and procedures designed to monitor disease therapy and progression using antibodies.
• T-cells To develop the most specific disease diagnostic, therapeutic and monitoring methods, it is necessary to subdivide the population of T-cells into those that are disease specific. This is done by analyzing the TCAR expression and detecting which ones are disease related. A preferred way to accomplish this is by developing a TCAR expression matrix.
• the first element in the matrix involves the major histocompatibility antigens (MHC).
• MHC major histocompatibility antigens
• Many T-cell mediated diseases such as diseases, are correlated with the expression of certain MHC molecules.
• disease can be subdivided into subsets expressing different MHC molecules.
• the next level in the matrix is to determine which ⁇ chain variable region TCARs are being expressed in the different MHC subgroups. This will result in the further subdivision of each MHC subgroup into further divisions.
• the next level in the matrix is to determine which ⁇ -chain variable region TCARs are being expressed.
• the combination of V ⁇ , V ⁇ and MHC results in further subdivisions of the (MHC + V ⁇ ) groups. Even further subdivisions are obtained by analyzing the expression of specific D and/or J TCAR gene segments.
• Disease correlations can be made at any of the levels of the matrix, but the more levels that are analyzed, the more specific the correlation will be. Diagnostic, therapeutic and monitoring strategies can also be developed at any level in the matrix, but again, using more levels results in more specific methods.
• High-molecular-weight DNA was isolated from a panel of 30 lymphoblastic cell lines (LG series) derived from the offspring of consanguineous marriages and homozygous for class I and class II MHC antigens (Gatti, et al. (1979) Tissue Antigens 13, 35). DNA samples from lymphoblastoid cell lines representing 69 additional unrelated individuals were supplied through the courtesy of CEPH (Centre pour L'Etude du Polymorphisme Humain), a Paris-based gene-mapping consortium. DNAs derived from some additional cell lines and from some tissue samples were also examined. Rearrangement of the C3 genes was not observed in any of these DNA samples.
• Nucleic acid probes corresponding to members of human V/3 gene segment subfamilies V ⁇ 1 through V ⁇ 14 were isolated from subclones described by Concannon et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6598 and correspond to that set forth in Figure 2 (nucleotide sequence) and Figure 3 (amino acid sequence). For subfamilies 5 and 7, probes corresponding to two different subfamily members were used. Gel-purified inserts containing V ⁇ -specific sequences were labeled by random priming with ⁇ -[ 32 P]triphosphates (Feinberg, et al. (1984) Anal. Biochem. 137. 266) to specific activities ranging from 5 x 10 8 to 1 x 10 9 cpm/ ⁇ g and were used without further purification.
• Hybridizations were carried out for 12-15 hours at 37°C in 50% formamide, 5 x SSC (1 X SSC is 0.15 M NaCl/0.015 M sodium citrate), 0.02 M sodium phosphate, pH 6.7, 100 ⁇ g/ml sheared denatured salmon sperm DNA, 0.5% nonfat powdered dry milk, 10% dextran sulfate, 1% SDS, and 1-2 ng/ml of probe. Filters were washed in 2 X SSC and 0.1% SDS at 60°C and exposed to Kodak XAR-5 X-ray film for 12 to 120 h. Probe was removed for subsequent rounds of hybridization by washing twice in boiling .01 X SSC for 15 minutes on a room temperature rocker platform.
• V ⁇ Gene Segment Subfamilies Probes representing each of the human T-cell antigen receptor V ⁇ gene subfamilies V ⁇ 1 through V ⁇ 14 were hybridized, to germline DNA from -100 unrelated individuals. For 30 of these individuals (the LG series cell lines), this hybridization analysis was carried out with four different restriction enzymes. This allowed for estimation of the size of the various V ⁇ gene segment subfamilies with greater accuracy because it avoided problems of interpretation caused by polymorphism in outbred human populations. As shown in Figure 4, the 14 V ⁇ subfamilies contain at least 48 V ⁇ gene segments. As in the mouse (Patten, et al. (1984) Nature (Lond.) 312, 40; Barth et al.
• V ⁇ gene representation By statistical analysis of V ⁇ gene representation in cDNA libraries, the expressed human V ⁇ gene repertoire has been estimated to be ⁇ 59 genes with 95% confidence (Concannon et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6598).
• the 48 genes identified herein by hybridization approach this number and yet do not include three additional members of the V/36 subfamily described by Ikuta et al. (1985) Proc. Natl. Acad. Sci. USA 82, 7701, and those members contained in subfamilies V ⁇ 15, V ⁇ 16, V ⁇ 17 and V ⁇ 18 (a total of at least eight members) (Tillinghast," et al. (1986) Science (Wash. DC) 223. 879 and Kimura et al.
• V ⁇ genes in the human genome exceeds that estimated by statistical analysis of expressed genes.
• Some of the V ⁇ gene segments detected by hybridization will correspond to pseudogenes that cannot be productively rearranged, or are not transcribed in populations of mature T-cells (Siu, et al. (1986) Proc. Natl. Acad. Sci. USA 164,1600). Therefore, these gene segments would not be represented in cDNA libraries from peripheral lymphocytes.
• V8/Bam HI and V ⁇ 12/Bam HI Two of the probe/enzyme combinations (V8/Bam HI and V ⁇ 12/Bam HI) appeared to detect the same polymorphic Bam HI site. Subsequent restriction mapping of a cosmid clone which hybridized to both the V ⁇ 8 and V ⁇ 12 probes indicated that these probes were hybridizing to V ⁇ gene segments which flanked a single polymorphic Bam HI restriction site.
• V ⁇ 8/Bam HI and V ⁇ ll/Bam HI Two of the polymorphisms which were identified and displayed the minimal three hybridization patterns consistent with the segregation of two alleles at each locus (V ⁇ 8/Bam HI and V ⁇ ll/Bam HI) were tested for segregation.
• the V ⁇ 8 and V ⁇ 11 probes were hybridized sequentially to Bam Hi-digested genomic DNA from members of several three-generation families, one of which is shown in Figure 5. To increase the informativeness of markers in this genomic area, the segregation of a previously reported polymorphism detected with a C ⁇ probe (Robinson et al. (1985) Proc. Natl. Acad. Sci.
• the V ⁇ 8 probe hybridized to two or three bands when washed under high stringency (1 x SSC/0.1% SDS at 65°C), an invariant band of 3.3 kb containing the V ⁇ 8.2 gene segment, and a polymorphic band of either 23 or 2.0 kb containing the V ⁇ 8.1 gene segment. In the analysis of 100 unrelated individuals, the 23-kb allele occurred at a frequency of 46.4% and the 2.0-kb allele at a frequency of 53.5%.
• the V ⁇ 11 probe hybridized to an invariant band of 12 kb and polymorphic bands of either 25 and/or 20 kb in 100 unrelated individuals.
• the 25-kb allele occurred at a frequency of 47.4% and the 20-kb allele at a frequency of 52.6%.
• the C ⁇ probe hybridized to bands of either 10 or 9.0 kb in the same panel of individuals.
• the 10- kb allele occurred at a frequency of 55.9% and the 9-kb allele at a frequency of 44.1%.
• a diagram of this family and the results obtained by hybridization with V ⁇ 8, V ⁇ 11, and C ⁇ probes are shown in Figure 5.
• haplotypes created by the three probes described above, V ⁇ 8, V ⁇ 11, and C ⁇ are potentially informative markers for following genetic segregation because the alleles at each locus are approximately equal in frequency, and hence one might expect to see each of the theoretically possible nine haplotypes in outbred human populations with high frequencies.
• the loci could be very close from a genetic standpoint, either because of close physical linkage, or recent generation of alleles.
• the finding herein of allelic frequencies approaching 50% in a variety of populations in a panel of unrelated individuals containing representatives of a number of different populations, and the finding of the same alleles among these representatives, provides support for the argument that this effect is probably not due to recent mutation.
• T-Cell Receptor V Genes in Murine Autoimmune Encephalomyelitis and Antibody Therapy
• T-cells responding to MBP were isolated from the draining popliteal, inguinal, and paraaortic lymph nodes of eight-week-old B10.
• PL mice ten days after subcutaneous injection of 200 ⁇ g MBP emulsified in complete Freund's adjuvant according to the method of Kimoto et al. (1980) J. Exp. Med. 152, 759-770.
• MBP was prepared from rat brains using the method of Smith (1969) J. Neurochem. 16, 83-92. Lymph node cells were initially cultured at 3 x 10 6 cells/ml in serum-free HL- 1 Ventrex medium containing 20 ⁇ g/ml rat MBP.
• T-cell blasts Five days later responding T-cell blasts were cultured at 5 x 10 5 cells/ml in complete culture medium containing 20 ⁇ g/ml rat MBP, 2 x 10 6 cells/ml syngeneic irradiated (3000 rad) spleen cells as a source of antigen presenting cells (APC) and 10% supernatant from concanavalin A-stimulated rat spleen cells according to the method of Gillis et al. (1978) J. Immunol. 120, 2027-2032.
• T-cells were routinely restimulated with MBP, APC and CAS every 12-14 days. Clones were derived by limiting dilution in 96-well flat bottom microtiter rates at 0.3 cells per well.
• Complete culture medium consisted of Dulbecco's Modified Eagle's Media supplemented with 4.5 g/l glucose, 2mM glutamine, 100 ⁇ g/ml streptomycin, 100 ⁇ g/ml penicillin, 5 10 -5 M 2- mercaptoethanol and 10% fetal bovine serum (cDMEM).
• Three T-cell lines specific for MBP (BML-1 , 1, and 3 ) were established from three separate groups of B10.PL mice (3-4 individual mice per group) immunized with rat MBP emulsified in complete Freund's adjuvant (200 ⁇ g/mouse).
• the lines were found to proliferate specifically to an acetylated N-terminal peptide, 1-9NAc, indicating the immunodominance of this epitope in the B10.PL response to MBP.
• These T H cells were also uniformly restricted to the IAu class II molecule as their proliferation was specifically blocked with an anti-I-A u b monoclonal antibody (10-2.16, Oi et al. (1978) Clin. Top.
• Hybridomas were generated by fusing MBP-reactive B10.PL helper T-cell blasts with the AKR thymoma BW5147 as previously described in Kappler et al. (1981) J. Exp. Med. 153, 1198-1214. Three separate fusions were performed involving independent lines of T-cells (BML 1, 2 and 3), each derived from 3-4 individual mice and passaged for 2-8 weeks prior to fusion. Hybrids were screened for their ability to secrete IL-2 in response to both native MBP and a synthetic acetylated N- terminal MBP derivative (1-9NAc) previously shown to induce EAE in Pl/J mice according to the method of
• RNA for Northern blot analysis was prepared by resuspension of the cells in guanidinium thiocyanate followed by centrifugation through a CsCl cushion according to the method of Chirgwin et al. (1979) Biochemistry 18. 5294-5299.
• Poly (A) + RNA was selected on oligo(dT)-cellulose columns and 10 ⁇ g was electrophoresed on a 1% agarose formaldehyde gel. Conditions for blotting hybridization and washing were the same as those for Southern blots.
• 36 additional T H cells were analyzed by Northern and Southern blots.
• V ⁇ 4.2-V ⁇ 39 genes were identified as a unique -14 kb bands upon digestion with Hind III and -0.7 kb bands with Eco RI when genomic blots were probed with a V ⁇ 4 cDNA probe and a V ⁇ 39 oligonucleotide probe (data not shown).
• genomic arrangement of the V ⁇ and J ⁇ gene segments are not yet known, the fact that all the V ⁇ 2 expressing hybridomas (including those whose V ⁇ 2.3-J ⁇ 30 genes were sequenced) exhibit identical sized V-J DNA fragments upon digestion with the Hind III restriction enzyme (likewise for Eco RI and Bam HI) suggests that all these T H cells utilize the same V ⁇ 2.3 and J ⁇ 39 gene segments.
• V ⁇ Genes The V ⁇ genes of 37 additional T H cells were analyzed by Southern and Northern blots using the methods described for the V ⁇ genes (results not shown).
• Northern blot analyses demonstrate a total of 26 T H cells positive for the V ⁇ 8 and J ⁇ 26 gene segments.
• Southern blot analyses of these cells with the restriction enzymes Eco RI and Hind III identify common 1.6 and 9.3 kb DNA bands, respectively, cohybridizing to probes for the V/38 gene segment and J ⁇ 2 gene segment cluster (results not shown). These DNA bands also cohybridize to an oligonucleotide probe for the J ⁇ 2.6 gene segment (data not shown).
• the restriction enzyme map in Figure 7 shows the V ⁇ 8.1 gene to be located on a 1.3 kb Hind III fragment and the V ⁇ 8.2 and V ⁇ 8.3 genes on the same 9.5 kb Hind III fragment.
• V ⁇ Genes Double-stranded cDNA was synthesized from 5 ⁇ g polyA+ RNA using the Amersham cDNA Synthesis System (RPN.1256). cDNA libraries were constructed by adding synthetic Eco RI linkers and cloning into the Eco RI site of lambda gt10 with the Amersham cDNA Cloning System (RPN.1257). Libraries contained 1-3 x 10 7 total recombinants and were screened with C ⁇ , C ⁇ and specific V ⁇ -region probes at a concentration of 10 5 cpm/ml.
• cDNA libraries were constructed from nine T H hybridomas from one cell line specific for the 1-9NAC encephalitogenic peptide and screened these with probes for the constant region of the a (C ⁇ ) or b (C ⁇ ) genes of the T-cell receptor. Fifteen C ⁇ and 15 C ⁇ cDNA clones were isolated from each library. Clones were sequenced which (1) possessed inserts greater in length than 1.0 kb and (2) failed to hybridize to probes for the rearranged V ⁇ and V ⁇ genes of the BW517 tumor parent, V ⁇ l (Chien et al. (1984) Nature 312, 31-35, v ⁇ l6, V ⁇ 1 (Barth et al.
• V ⁇ 2.3 The V ⁇ 2 gene segment is newly described and is designated as V ⁇ 2.3 because it is highly similar to two other previously sequenced members of the V ⁇ 2 subfamily, TA39 (V ⁇ 2.1) and TA19 (V ⁇ 2.2) (93%) and 96% similar, respectively (Arden et al. (1985) Nature 316. 783-787).
• V ⁇ 4 gene sequenced is identical to a previously sequenced member of this subfamily, TA28 (V ⁇ 4.2) (Arden et al. (1985) supra).
• a subfamily is a set of V gene segments that cross hybridize and generally are 75% or more similar to one another (Crews et al. (1981) Cell 25, 59-66).
• the J ⁇ 39 element utilized by both V ⁇ 2.3 and V ⁇ 4.2 genes as depicted in Figure 1 has been reported previously (TA39; Arden et al. (1985) supra).
• V ⁇ Genes The V ⁇ genes from six T H hybridomas specific for peptide 1-9NAc using the methods described for the V ⁇ genes. The results of the DNA sequence analysis demonstrated that five of the six Vgenes use the same V ⁇ 8.2 and J ⁇ 2.6 gene segments, whereas the sixth employs different gene segments (V ⁇ 13 and J ⁇ 2.2) (results not shown). At least four of the V ⁇ 8 genes have arisen from unique rearrangements, since they possess unique junctional nucleotide sequences. The fifth was derived from a distinct clone exhibiting a unique additional rearrangement.
• Antibody Treatment A. Antibodies Specific for V ⁇ 8 Chains Block the Recognition of the MBP Peptide 1-9NAc bv V/38T H Cells In Vitro
• F23.1 is extremely effective in blocking the antigen recognition of V ⁇ 8.2T H hybridomas specific for the N- terminal MBP peptide 1-9NAc (results not shown).
• the addition of F23.1 antibody produced virtually complete suppression of interleukin-2 (IL-2) release by V ⁇ 8.2-expressing hybridomas PL127.6 and PL414 in response to the MBP peptides 1-9NAc and pM1-20 (>98% inhibition,
• V ⁇ 13 rather than V ⁇ 8 chains (Table III).
• no significant IL-2 suppression was exhibited by a V ⁇ 8- negative MBP-specific SJL/J T-cell hybridoma, 5317.1.
• T H hybridomas were isolated from B10. PL or SJL/J mice injected with rat BMP. Lymph node cells were Isolated from the popliteal and inguinal lymph nodes of individual B10. PL or (B10.PLxSJL/J)
• mice 10 days after subcutaneous injection of 50 ⁇ g MBP peptide dissolved in phosphate-buffered saline and emulsified in an equal volume of complete Freund's adjuvant supplemented with 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). B10. PL mice were injected with the N- terminal peptide 1-9NAc and F1 mice with the peptide 1-9NAc plus the C-terminal peptide recognized by SJL/J (H-2 S ) MBP-specific T H cells, pM87-98 (Kono et al. (1988) J. Exp. Med. (in press). b T-cell receptor genes are from Table 1.
• V ⁇ 2 refers to rearranged gene segments V ⁇ 2.3-J ⁇ 39, V ⁇ 4 to gene segments V ⁇ 2.3-J ⁇ 39, V ⁇ 8 to V ⁇ 8 .2/3 ⁇ 2.
• b and V ⁇ 13 to V ⁇ 13/J ⁇ 2.2.
• the precise T-cell receptor genes used by hybridoma SJ17.1 are unknown, but V ⁇ 8 ⁇ -chains are not expressed on the cell surface as determined by fluorescence flow cytometry with the F23.1 monoclonal antibody. This is consistent with the fact that the SJL/J mouse genome has a deletion of approximately half of the known V ⁇ genes, including all of the V ⁇ 8 genes (Behlke et al. (1986) Nature 322, 379-382).
• N-terminal MBP peptides 1-9NAc and pM1-20 were used at final concentrations of 10 ⁇ M and 0.2 ⁇ M, respectively, and the C-terminal MBP peptide at a concentration of 10 ⁇ M.
• Purified protein derivative (PPD) was used at a concentration of 100 ⁇ g/ml.
• d T-cells were treated with 35 ⁇ g/ml F23.1 antibody (anti-V ⁇ 8) or 34-5-8S antibody (anti-D d ,
• HTdR uptake for hybridomas represented the proliferation of the IL-2-dependent cell line HT-2 after transfer of supernatant from hybridoma cultures (see legend to Figure 6.
• 3 HTdR uptake for lymph node cells represented an 18 hr. pulse of 4 day primary cultures containing 4x10 5 lymph node cells and antigen.
• F23.1 antibodies were used to inhibit the proliferation of lymph node cells from MBP peptide-injected mice.
• lymph node cells are capable of adoptively transferring EAE, when injected back into irradiated (350 R) B10.PL mice after a five day period of restimulation with the peptide in vitro (data not shown).
• B10.PL mice were injected with 200 ⁇ g of MBP peptide 1-9NAc in complete Freund's adjuvant (CFA) containing purified protein derivative (PPD) and 10 days later, the draining lymph nodes were removed and cultured in vitro in the presence or absence of F23.1 antibody.
• CFA complete Freund's adjuvant
• PPD purified protein derivative
• F1 mice B10.PL X SJL/J
• MBP peptides 1-9NAc and pM87-98 The latter peptide is specifically recognized by F1 and SJL/J (H- 2') T H cells capable of causing EAE. Since the SJL/J genome has deleted approximately 50% of its V ⁇ gene segments, including all of the V ⁇ 8 gene segments (Behlke et al. (1986) Proc. Natl. Acad. Sci. USA 83, 767-771), all SJL/J T-cells are V ⁇ 8-negative.
• FIG. 9 presents representative data from a typical experiment. Spleen cells were removed at various times following intraperitoneal injection of F23.1 antibody and subjected to quantitative fluorescence flow cytometry using the F23.1 antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Within 24 hours the level of V ⁇ 8 expression on individual cells had decreased by about 5-fold (see Figure 9D). By 72 hours, the number of detectable V ⁇ 8-positive spleen cells was essentially zero (not significantly different from background staining due to the FITC antibody alone, Figure 9D), and this depletion remained complete for up to 7 days post-injection (Figure 9E).
• FITC fluorescein isothiocyanate
• mice which developed the disease in both treatment and control groups did not differ. Thus, once escape from antibody protection occurred, it was as severe in the treated as in the untreated animals. Analysis of spleen cells from mice treated with the F23.1 antibody who developed EAE demonstrated that treatment had removed most V ⁇ 8-positive spleen cells. Therefore, it appears that the mice which develop EAE despite treatment with anti-V ⁇ 8 antibody may expand V ⁇ 13 or other non-V ⁇ 8-positive T H cells specific for the MBP 1-9NAc epitope. Antibody treatment for T-cells expressing V ⁇ 13 TCAR is described in Example 3. EXAMPLE 3
• mice Female mice (6-10 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME) or bred at the California Institute of Technology animal facility.
• Myelin Basic Protein MBP was prepared from frozen rat or mouse brains purchased from Pel-Freeze Biologicals (Rogers, AR) according to a previously published protocol (Smith (1987) J. Neurochem 16, 83-92).
• MBP was emulsified with an equal volume of complete Freund's adjuvant supplemented with 4 mg/ml H37Ra (Difco Laboratories, Inc., Detroit, MI). Mice were immunized with 150 ⁇ g of MBP in each of the hind footpads. For induction of EAE mice were also given an intravenous dose of 75 ng of purified pertussis toxin (List Biological Laboratories, Inc., Campbell, CA) at 24 and 72 hours after MBP immunization.
• Anti-V ⁇ 8.2 (F23.2) was a gift from Michael Bevan.
• Anti-V ⁇ 13 (MR12-4) was donated by Osami Kanagawa.
• the antibodies were purified by Protein A chromatography and then biotinylated with NHS-LC-Biotin (Pierce, Rockford, IL) according to the manufacturers recommendations. Biotinylated antibodies were diluted in phosphate-buffered saline (PBS) containing 5% fetal calf serum and 1 mM Hepes. 1 X 10 6 cells were stained with 20 ⁇ g/ml of antibody at 37°C for 20 minutes.
• PBS phosphate-buffered saline
• Example 2 The analysis of 33 independent MBP-specific B10PL- derived T hybridomas in Example 2 detected the use of only V ⁇ 8.2 and V ⁇ 13 gene segments (see Figure 1). To measure the contribution of T-cells expressing these two V-region genes in the response to MBP (Smith (1987)
• Anti-TCAR V-region antibodies can be used to deplete specific T-cells.
• various concentrations of anti-V ⁇ 8.2 and anti-V ⁇ 13 were injected into the peritoneal cavity of B10.PL mice.
• Antibody concentrations in unpurified ascites were determined by comparison to a IgGl standard in an ELISA.
• the antibodies were then diluted and injected without further purification in a total volume of 0.1 ml per mouse. Seventy-two hours later, peripheral T-cells were purified over nylon wool and analyzed by fluorescence flow cytometry.
• V ⁇ 8.2 and V ⁇ 13 T-cells Following in vivo antibody treatment.
• F23.2 V ⁇ 8.2-specific
• MR12-4 V ⁇ 13-specific
• Various concentrations of either F23.2 (V ⁇ 8.2-specific) or MR12-4 (V ⁇ 13-specific) were injected into groups of 3 B10.PL mice. Three days later, lymph node cells were purified over nylon wool and analyzed by quantitative fluorescence flow cytometry. Cells were stained with either biotinylated F23.2 or biotinylated MR12-4 followed by FITC-conjugated streptavidin.
• Anti-V ⁇ 8.2 injection reduced, but did not eliminate, reactivity to MBP. The remaining response was inhibited by anti-V ⁇ 13 but not significantly hindered by addition of anti-V ⁇ 8.2.
• Anti-V ⁇ 13 injection did not lead to a significant reduction in MBP reactivity. However, the response was no longer significantly inhibited by anti-V ⁇ 13, while it remained susceptible to inhibition by anti-V ⁇ 8.2. Simultaneous injection of both anti-V ⁇ 8.2 and anti-V ⁇ 13 resulted in the most extreme reduction in MBP reactivity. However, a minor proliferative response to MBP remained, that was not significantly inhibited by either anti-V ⁇ 8.2 or anti-V ⁇ 13. This residual response could be the consequence of subdominant MBP-specific T-cells that might expand after the elimination of the dominant V ⁇ 8.2 and V ⁇ 13 MBP-specific T-cells.
• Anti-V ⁇ Antibody Treatment is an Effective
• mice Fourteen of 17 mice (82.4%) that were either untreated or had been injected with an IgG1 control antibody developed EAE.
• the average disease severity index of the animals in this group was 2.2 (see legend to
• mice were treated with the indicated antibodies and then immunized in the hind footpads with 150 ⁇ g of MBP .
• 75 ng of purified pertussis toxin was injected i.v. at 24 and 48 hours after immunization. Mice were then observed daily for signs of EAE. Disease severity was graded on a 5 point scale : 0 - normal ; 1 - loss of tail tone ; 2 - hind limb weakness , difficulty walking; 3 - hind limb paralysis , difficulty turning over ; 4 - severe whole body paralysis ; 5 - death.
• the average severity for each group was calculated In two different ways : (1) by averaging the maximum severity of all of the animals in the group , and (2) by averaging the maximum severity of only the diseased animals In the group .
• the arithmetic means and standard deviations are Indicated in the table .
• p ⁇ 0.001 when compared to the control group and p ⁇ 0.025 when compared to the anti-V ⁇ 8.2 treated group .
• p values for comparisons of EAE incidence were calculated using the exact probability test;
• p values for comparisons of average severity were calculated using the Student' s t test.
• mice treated with anti-V ⁇ 8.2 developed EAE.
• the average severity index of the animals in this group was 0.7. Therefore, anti-V ⁇ 8.2 treatment alone can lead to a very significant protection against EAE (p ⁇ 0.005).
• the symptoms in the five diseased animals in this group were just as severe as those seen in the control animals but were slightly delayed in onset.
• the T-cells responsible for the development of EAE in this single exception could be either V ⁇ 8.2 or V ⁇ 13 T-cells that were not eliminated by the treatment or subdominant T-cells that express as yet uncharacterized V ⁇ genes.
• MBP-induced proliferative responses of lymph node cells isolated from this animal were compared to that of control animals with EAE and double antibody treated mice without EAE. Draining lymph node cells used in this experiment were isolated 21 days after MBP immunization. A proliferative response was measured in the control animals with EAE that was inhibitable by both anti-V ⁇ 8.2 and anti-V ⁇ 13 (see Figure 13).
• mice were either untreated or treated with injections of both anti-V ⁇ 8.2 and anti-V ⁇ 13. Mice were then immunized with MBP and followed for signs of EAE. Three weeks after immunization, animals with and without EAE were sacrificed. Draining lymph node cells were collected and assayed for MBP reactivity as previously described.
• mice matched for severity of EAE symptoms were treated with IgG1 control antibody or with a combination of anti-V ⁇ 8.2 and anti-V ⁇ 13 three days after the first signs of MBP-induced paralysis.
• MBP-induced encephalomyelitis is considered to be a paradigm for T-cell mediated disease. Striking similarities in the pathology between EAE and MS in humans makes EAE an especially important model system to study.
• the dominant T-cell response to MBP in H-2 u mice is directed towards a single N-terminal determinant and involves T-cells which express a limited number of TCAR V-regions.
• V ⁇ 8.2 and V ⁇ 13-expressing T-cells led to the most dramatic loss of MBP responsiveness, however, a small proliferative response was still observed. This suggests that in the absence of V ⁇ 8.2 and V ⁇ 13-expressing T-cells, subdominant T-cells that utilize alternative V-regions might expand in response to MBP immunization.
• V ⁇ -Specific Monoclonal Antibodies Can be Used for the In Vivo Elimination of Specific T-cells
• V ⁇ -specific Monoclonal Antibodies Can be Used to Prevent and Treat EAE
• V ⁇ .3-specific antibody (KJ16) (Acha-Orbea et al. (1988) supra; Urban et al. (1988) supra, and Example 2 herein).
• F23.2 for therapy is preferential to the use of either F23.1 or KJ16, since the later reagents have broader specificities which would lead to the elimination of larger percentages of non-MBP- specific T-cells.
• the ideal therapeutic antibody would be reactive to clonotypic determinants, and indeed such an antibody raised against an MBP-specific rat T-cell hybridoma has been shown to partially protect rats from
• control population #1 The HLA haplotypes of these randomly selected controls, designated control population #1, were unknown and during the course of these studies it became apparent that HLA haplotypes may be important in the analysis of TCAR haplotypes (Kappler et al. (1987) Cell 49. 263-271). Consequently, an additional control population was used, consisting of 43 DR2+ unrelated normal individuals (control population #2). To assess the distribution of TCAR haplotypes, an additional 12 patients were added to the study and non-Caucasian patients were excluded for a total population of 38 Caucasian MS patients. The distribution of TCAR haplotypes of the 38 Caucasian MS patients, 84% of whom were DR2+, was compared to the Caucasian individuals comprising control populations #1 and #2.
• Genomic DNA was prepared from Epstein-Barr virustransformed lymphoblastoid B cell lines or the buffy coat from venous blood from the patient and control populations, as previously described (Beall et al. (1987) J. Immunol. 139, 1320-1325; Conconnan et al. (1987) supra). Probes cDNA probes used in the analysis corresponded to members of human V ⁇ gene segment subfamilies V ⁇ 1 through V ⁇ 14 isolated from subclones described by Concannon et al. (Concannon et al. (1986) Proc. Natl. Acad. Sci.
• DNA from each subject was digested with Bam HI, Bgl II,
• the filters were washed in 2xSSC and 0.1% SDS at 60°C and exposed to Kodak XAR-5 film for 18-76 hours. Probes were removed from blots by washing twice in boiling 0.01 x SSC for 15 min/wash on a rocker platform.
• the chi-square test was used for comparisons of allelic frequencies and haplotype frequency distributions between patients and the control populations.
• haplotype frequency distribution comparisons data for the four rarest haplotypes were combined so that small expected numbers would not degrade the accuracy of the chi-square test.
• Germline DNA from 28 MS patients was digested with four different restriction enzymes and probed with cDNAs representing each of the previously characterized human T-cell receptor V ⁇ gene subfamilies V ⁇ 1 through V314 and the C ⁇ genes (C ⁇ 1 and C ⁇ 2 ).
• the autoradiograms obtained were compared to those of the control population #1 to determine if either duplication or deletion of V ⁇ segment hybridizing bands were detected in the MS patient population. No deletions of the 53 gene segments defined by the V or C region gene families were detected (data not shown).
• V ⁇ 8 and V ⁇ 11 Two variable gene probes (V ⁇ 8 and V ⁇ 11) were hybridized to DNA digested with Bam HI and a C ⁇ probe was hybridized to DNA digested with Bgl II. These probe/enzyme combinations detect polymorphisms whose alleles occur at approximately equal frequency in the normal control population #1 (Concannon et al. (1987) supra; Robinson et al. (1985) Proc. Natl. Acad. Sci.
• haplotype 23/20/9 was over represented
• haplotype frequencies are representative of linkage to an MS susceptibility gene that maps between V ⁇ 8 and V ⁇ 11. It will be important to determine if particular V ⁇ haplotypes segregate with susceptibility to MS in families, and if additional genetic differences exist in the V ⁇ locus. Such differences may produce structural changes in the antigen binding specificity of the available TCAR repertoire. In addition, changes in a regulatory element could occur that would lead to a lack of expression of some V ⁇ genes in MS patients (with a consequent hole in the TCAR repertoire). Conversely, aberrant expression of certain V ⁇ genes could produce T-cells with autoreactive potential. Each of these alternate consequences of V ⁇ region polymorphism have implications for immune reactivity to either autoantigens or viruses that have been postulated to be involved in the pathogenesis of MS.
• the locus encoding the variable region gene segments for the ⁇ -chain of the TCAR was also investigated in a manner analogous to that presented in Example 4.
• 120 control samples from unrelated Caucasians of Northern European descent and 79 Caucasians of Northern European descent afflicted with MS were analyzed for detection of a restriction fragment length polymorphism associated with a multiple sclerosis susceptibility gene in the ⁇ -chain locus.
• approximately 30 of the individuals were the same as those used in Example 4.
• Example 4 Since there is an overlap between the MS population studied herein and that of Example 4 relating to the detection of a V ⁇ RFLP associated with an MS susceptibility gene in the V ⁇ locus, there is a substantial likelihood that the inheritance of an MS susceptibility gene in the V ⁇ locus and an MS susceptibility gene in the V ⁇ locus is required for predisposition to this disease.
• markers will also be employed to determine whether there are hot spots of recombination in the V ⁇ or V ⁇ loci.
• the polymorphic markers for the V ⁇ and V ⁇ loci of humans are determined, we will be able to screen populations that have particular diseases for haplotype associations. Such associations can then be used to screen populations for their risk in developing the disease in question, e.g. their predisposition to disease.
• OLA OLA assays
• OLA assays can currently be performed at the rate of 1200 ligation assays per day by one technician. Multiple analyses can be done on the cells obtained from a sample of saliva. Saliva contains more than sufficient cells such that, with PCR techniques, 5 to 10 analyses can easily be done.
• Highly Informative Polymorphic Markers Biallelic markers are not intrinsically informative. However, we have made the startling discovery that clusters of biallelic markers can be highly informative. For example, we have identified four biallelic markers in the second intron of the human C ⁇ gene, for example, see Table XI.
• markers are within 400 base pairs of one another, yet strikingly, they appear to be in partial linkage disequilibria with one another. This implies either hot spots of recombination have separated these markers or that gene conversion has occurred. In either case, the important point is, a vast array of different C ⁇ haplotypes has been created in the human population. For just three of these markers, the heterozygosity index exceeds 70% - an extremely informative marker. The C ⁇ locus results are even more striking, for example. See Table XI. Here we have identified 7 biallelic markers, most of which are in partial linkage disequilbria with one another.
• DNA primers were synthesized in order to amplify, using PCR, genes from every known human TCR ⁇ and ⁇ subfamily (see Table X).
• the PCR primers were selected from the V or C gene sequence by analyzing the percentage of G/C content and calculating the melting temperatures.
• a primer that was fairly A/T rich was generally longer (about 24 nucleotides) than one that was quite G/C rich (18 nucleotides).
• Each primer was selected to have a melting point within the range of 58 - 62°C.
• the melting point was determined by assigning a value of 4°C for each G or C residue in the primer sequence and a value of 2°C for each A or T residue and then summing up the assigned values. After PCR amplification using the specific primers, the PCR products from several unrelated individuals were then electrophoresed through a denaturing gradient gel. This gel makes possible the discrimination of DNA sequence differences on the basis of altered melting properties of allelic forms of the same gene (Meyers et al., 1987, Meth. Enzymol. 155: 501 527). In this way, several TCR genes were found to be polymorphic. These include the genes for V ⁇ 2, V ⁇ 4.1, V ⁇ 10 and V ⁇ 15.
• sequence polymorphism in these genes is now being determined by standard sequencing techniques. Polymorphisms can also be detected by the Southern blot/RFLP procedure described in example 1 as well as by sequencing the same gene from multiple individuals to determine the existance of sequence polymorphisms. Once a polymorphism has been detected, many individuals from a population are screened to determine the frequency of the polymorphism. In general, very rare polymorphisms that do not occur very often in the population are less valuable for defining haplotypes containing multiple polymorphic markers, since they do not occur often enough to be useful.
• each allelic form is then sequenced in order to determine the specific DNA base(s) which differ between the allelic forms (see Table XI).
• different strategies can be employed which allow the discrimination of the allelic forms in any individual.
• restriction enzyme digestion if any DNA change alters a restriction site, e.g. RFLP analysis, see example 1 and Charmley et al., 1990, Proc. Natl. Acad. Sci. USA 87: 4823-4827; Concannon et al., 1990, Am. J. Hum. Genet., 47: 45-52
• OVA oligonucleotide ligase assay
• the DNA sequence identified by the use of the DRS18 primers was derived from a non-coding region contained in a cosmid which had been initially isolated by screening a total human cosmid library with various V region gene segments. This polymorphism is an example of sequence polymorphisms which can be detected in "anonymous" regions in and around the TCR gene complexes. From sequence information of the cosmid, PCR primers were synthesized and product amplified from multiple unrelated individuals. The amplified products were then scanned for polymorphisms by direct sequencing.
• V ⁇ 18 polymorphism was initially discovered by sequencing the V ⁇ 18 gene from multiple individuals. This is an extremely interesting polymorphism, because the polymorphic sequence results from an altered sequence that generates an amino acid stop codon. Thus, in Caucasians, the 10-20% of individuals who are homozygous for this stop codon are unable to express the V ⁇ 18 protein product. This is an example of a polymorphism that can alter function.

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