MXPA94005978A - Methods for ex vivo therapy using antigen depressed cells charged with peptide for the application of - Google Patents

Abstract

The present invention relates to a method for activating cytotoxic T lymphocytes (CTL) in vitro, together with a method for using activated CTLs for in vivo therapy. In addition, we present a method to examine specific CTLs in vivo using antigen presenting cells that are modified in vitro.

Description

"METHODS FOR EX VIVO THERAPY USING ANTIGEN PRESENTATION CELLS CHARGED WITH PEPTIDE FOR LTC ACTIVATION" INVENTORS: ESTEBAN CELIS. RALPH KUBO.

NATIONALITY: NORTH AMERICAN CITIZENS.

RESIDENCE: 13644 LANDFAIR ROAD SAN DIEGO, CALIFORNIA 92130 E.U.A. 12635 FUTURA STREET SAN DIEGO, CLAIFORNIA 92130 E.U.A.

OWNER: CYTEL CORPORATION NATIONALITY: NORTH AMERICAN SOCIETY RESIDENCE: 3525 JOHN HOPKINS COURT, SAN DIEGO CALIFORNIA 92121 E.U.A.

SUMMARY OF THE INVENTION Method to activate cytotoxic T lymphocytes (CTL) in vitro are presented together with method to use activated CTLs for in vivo therapy. In addition, a method for examining specific CTLs in vivo is presented using antigen presenting cells that are modified in vitro.

BACKGROUND OF THE INVENTION The present invention relates to compositions and methods for preventing or treating a number of disease states such as viral diseases and cancer through ex vivo therapy. In particular, it provides method for inducing cytotoxic T lymphocytes (CTL) using antigen presenting cells (APCs) with a selected peptide linked to selected major histocompatibility complex (CHP) molecules. Cytotoxic T cells or CD8 cells are also known, representing the main line of defense against viral infections. CTLs specifically recognize or kill cells that are infected by a virus. The T cell receptors on the surface of the CTL can not directly recognize the foreign antigens. In contrast to antibodies, the antigen must first be presented to the T cell receptors for activation to occur. The presentation of the antigen to the T cells is achieved by the molecules of the main histocompatibility complex (CHP). The major histocompatibility complex (CHP) refers to a large genetic site that encodes an extended family of glycoproteins that have an important role in the immune response. The CHP genes, which are also referred to as the HLA complex (human leukocyte antigen), are placed on chromosome 6 in humans. The molecules encoded by the CHP genes are present on the surfaces of the cell and are largely responsible for the recognition of tissue transplants as "non-autonomous". CHP molecules are classified as either Class I, Class II, or Class III molecules. Class II CHP molecules are expressed primarily in cells involved in pa-initiation and support immune responses, such as T lymphocytes, B lymphocytes, macrophages, and so on. Class II CHP molecules are recognized by the helper T lymphocytes and induce the proliferation of helper T lymphocytes and the amplification of the immune response to the specific immunogenic peptide to be exhibited. Class I CHP molecules are expressed in almost all nucleated cells and are recognized by CTL. T cells that serve mainly as helper cells express CD4 and are restricted primarily to Class II molecules, whereas CD8 expressing cells represented by cytotoxic effector cells interact with Class I molecules. The CTL recognizes the antigen in the form of a peptide fragment linked to the CHP molecules of class I instead of the intact foreign antigen itself. The antigen must normally be synthesized endogenically by the cell, and a portion of the protein antigen is degraded into small peptide fragments in the cytoplasm. Some of these small peptides are translocated to a pre-Golgi compartment and interact with the heavy chains of class I to facilitate proper folding and association with the microglobulin of the beta2 subunit. The peptide-CHP complex of class I is then sent to the surface of the cell for expression and potential recognition by specific LPCs. Investigations of the crystal structure of the human CHP molecule of class I, ALH-A2.1, indicate that a peptide binding groove is created by folding the alfal and alpha2 domains of the heavy chain of class I (Bjor man et al., Nature, 329: 506 (1987). For many years, immunologists have wanted to elevate target viruses from specific cytotoxic cells, retroviruses and cancer cells.A possible approach is to immunize a healthy subject, isolate the CTLs from this subject and inject these cells into the sick person. The experimental protocol seems to work on congenital mouse strains, but it has not been satisfactorily tested in humans.For this approach to work, the CHP haplotype of the donor must be identical to that of the recipient.This is important because the LTC The receptor can only interact with peptides linked to one of the three to six molecules of Class I. Presently, CTLs react violently with all Class I molecules that are different. erent from those expressed in the subject from which the CD8 cells are obtained, regardless of which peptide they contain the Class I molecules. This reactivity is the basic cause of the immune rejection of the transplanted organs. Because it is difficult to find two people not related to exactly the same Class I molecules, certain therapeutic approaches take the non-specific approach to "reinforce" existing CD8 cells by incubating them in vitro with IL-2, a growth factor for T cells. However, this protocol (known as LAK cell therapy or LIT therapy [tumor infiltration lymphocytes]) will only allow the expansion of those CTLs that have already been activated. Since the immune system is always active due to one reason or another, most of the stimulated cells IL-2 will be unimportant for the purpose of fighting the disease. In fact, it has not been documented that this type of therapy activates any cells with the desired specificity. The benefits of LAK cell therapy are ambiguous at best and the side effects are often serious. [We must cite the related references that describe this approach in our IDS. We beg you to provide the quotes]. The preferred approach for the treatment of these diseases such as cancer, AIDS, hepatitis and another infectious disease would activate only those CTLs that recognize the diseased cells. Although several procedures have been applied in these diseases, few have been made available if successful attempts have been made using the cytotoxic T cells. Ex vivo activation of the CTL would be the preferred means to treat the types of disease mentioned above. However, there have been no reliable procedures available to specifically activate the CTL associated with these diseases. The present invention is directed to these and other problems.

SUMMARY OF THE INVENTION This invention is directed to the means and method for activating cytotoxic T cells (CD8 cells) in vitro or in vivo. The means and method for activating the CDS cells comprises: dissociating bound peptides from the class I CHP molecules in antigen presenting cells using a mild acid treatment; associating the selected immunogenic peptides with the class I CHP molecule in the antigen presenting cell; and incubating the antigen presenting cells with the cytotoxic T cells, thereby producing the activated cytotoxic T cells. Alternatively, CD8 cells can be generated by a means and a method comprising: generating empty CHP class I molecules by incubating the antigen presenting cells at a temperature of less than about 21 ° 0; associating the selected immunogenic peptides with the CHp molecule of class I in the antigen presenting cell; and incubating the antigen presenting cells with the cytotoxic T cells, thereby producing the activated cytotoxic T cells. The means and method for generating the class I CHP molecules empty in the antigen presenting cells, capable of inducing a CTL response was problematic. This system is capable of generating empty class I CHP molecules in the antigen presenting cells and in turn inducing the LTC and effecting the extermination of the class I matching cells. The antigen presenting cells that have molecules of Class I CHPs that are empty on their surface are able to induce cytotoxic T cells that may be useful in the treatment of chronic infectious diseases and cancer. Specifically, this invention provides the means and method for producing empty Class I CHP molecules in antigen presenting cells, loading those empty Class I CHP molecules with selected immunogenic peptides, activating the cytotoxic T cells which are specific to terminate specific antigen targets. This invention has broad therapeutic application in the treatment of cancer, certain immune diseases and viral diseases. As such, the method may further comprise: separating activated CTLs from antigen presenting cells having the class I CHP molecule empty on its surface; suspending activated CTL in an acceptable carrier or excipient as a pharmaceutical composition; and administering the pharmaceutical composition to a patient having the disease.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effects of beta2? R? Icroglo-buline and exogenous peptide 941.01 (HBc 18-27) on the CHP molecules of class 1 PHA blasts purified and acid-laden. Figure 2 shows the induction of CTL using GC43 A2.1 responders and purified autologous PBMC and PHA blasts of acid loaded with a peptide deposit of 777.03 (HBS 20-28); 924.07 (HBC 18-27); 927.32 (HBp 61-69) - Figure 3 shows the induction of CTL using X351 or X355 A2.1 responders and autologous acid-puri? Ed PBMC or PHA blasts as stimulants after loading with 1 peptide deposit 1044.04 (PAP 135-143); 1044.05 (PSA 166-175) 1044.06 (PSA 118-128). Figure 4 shows the induction of LTC using GC49 A2.1 responders and PHA Autologous blasts purified from acid as stimulants after being loaded with peptide 939.03 (PSA 49-57). Figure 5 shows the induction of CTL using GC66 Al responders and autologous acid-purified PBMCs as stimulants after loading peptide 958.01 (MAGE 1: 161-169). Figure 6 shows the induction of CTL using CG 30, ALH Al and activated PBMCS cn SAC-I responders incubated at cold autologous temperature as stimulants after loading with peptides 1044.07 MAGE-3 (161-169). Figure 7 shows a comparison of different methods for loading peptides in PBMCs such as CPA activated with SAC-I. A deposit of binding peptides MAGE-3 ALH Al (1044.07: 161-167 and 1044.01: 8-17) were tested with donor GC 164. Panel A-acid clearance; Panel B-incubation at cold temperature; Panel C- room temperature, without pre-incubation or acid purification and peptide loading of 4 hours only; - - Panel D- room temperature, without acid purification with addition of soluble peptide to the culture.

DESCRIPTION OF THE PREFERRED MODALITIES The term "peptide" is used interchangeably with "oligopeptide" in the present specification to designate a series of residues, typically L-amino acids, connected to one another typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids . An "immunogenic peptide" is a peptide that comprises an allele-specific motif such that the peptide will be released to the CHP allele and is capable of inducing an LTC response. Therefore, the immunogenic peptides are capable of binding to an appropriate class I CHP molecule and inducing a cytotoxic T cell response against the antigen from which the immunogenic peptide is derived. The term "residue" refers to an amino acid or a mimetic amino acid incorporated in an oligopeptide via an amide bond or a mimetic amide bond. The present invention relates to methods for improving the immune response to various diseases using ex vivo therapy. The general approach of the invention comprises the isolation of peripheral blood mononuclear cells - - (PBMCs) of a patient, loading a desired immunogenic peptide into the binding cavities of the class I CHP molecules on the surface of the antigen presenting cell (CPA), incubate the CPAs with the CTCs cursors in the sample to induce the proliferation of the CTLs recognizing the peptide, and using the CTLs to identify the antigenic epitopes and expanding their numbers introduce the activated CTLs in the patient. The methods of the present invention depend in part on the determination of epitopes recognized by the CTL capable of eliminating the target infected cells. One approach to the identification of these epitopes is the identification of the allele-specific peptide motifs associated with a disease specific for human Class I CHP allele subtypes. CHP antigens of class I are encoded by the ALH-A, B and C sites. The ALH-A and B antigens are expressed on the cell surface at approximately equal densities, while the expression of ALH-C is significantly lower, (possibly as much as 10 times lower). Each of these sites has a number of alleles. A large number of cells with defined CHP molecules are known and can easily be obtained, particularly Class I CHP molecules. These cells can be used to identify allele-specific motifs in particular, associated with target diseases.

The allele-specific motifs are then used to define the T cell epitopes of any desired antigen, particularly those associated with human viral diseases or cancer, for which, the amino acid sequence of the targets of the potential antigen is already known This general approach is described in detail in the co-pending and commonly assigned US Serial Number 07 / 926,666 and United States Serial Number 08 / 027,146, which are incorporated herein by reference. Potential potential epitopes in a number of target proteins can be identified in this way. Examples of appropriate antigens include prostate specific antigen (PSA), hepatitis B surface core and core antigens (HBVc, VLHBs), hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (v. ., MAGE-1), human immunodeficiency virus (HIV) antigens, human papilloma virus (HPV) antigens, cytomegalovirus (CMV), herpes simplex virus (HSV), and other oncogenic products (c-Erb B2 , CEA, p 53-chest / ovary). These approaches typically involve the isolation of the peptides from a specific CHP molecule and the sequence of the peptides to determine the related motif. Buus and others, Science, 242: 1065 (1988) described primkero a method for the acid removal of bound CHP peptides. Subsequently, Rammensee and his co-investigators (Fal et al., Nature, 351: 290 (1991) developed an approach to characterize naturally processed peptides linked to class I molecules. Other researchers have successfully achieved the amino acid sequence. of more abundant peptides in different HPLC fractions by conventional automated peptide sequence eluted from class I type B molecules (Jardetzky, et al., Nature, 353; 326 (1991) and type A2.1 by mass spectrometry (Hunt, et al., Science, 225: 1261 (1992)) A review of the characterization of the naturally processed peptides found in the CHP molecules of the class I is presented by Rdtzschke and Falk (Rotzschke and Falk, Immunol. Today, 12: 447 (1991).) The definition of specific motifs for different class I alleles allows the identification of potential peptide epitopes of an antigenic protein whose sequence of The amino acid is known Typically, the identification of potential peptide epitopes is carried out initially using a computer to scan the amino acid sequence of a desired antigen to determine the presence of motifs Epitope sequences are then synthesized. CHP molecules of class I are measured in a variety of different ways, using, for example, purified Class I molecules and radioiodinated peptides and / or cells expressing empty class I molecules by, for example, immunofluorescent staining and flow microfluorimetry, class I assays dependent on peptide inhibition of CTL recognition by peptide competition. Other alternatives described in the literature include inhibition of antigen presentation (Sette, et al., J. Immunol., 141: 3893 (1991), in vitro pool assays (Townsend, et al., Cell, 62: 285 (1990 ), and FACS-based assays using mutated cells, such as RA.S (Melief, et al., Eur. J. Immunol., 21: 2963 [1991]). Then, the peptides that prove to be positive in the assay of CHP binding of class I are assayed to determine the ability of the peptides to induce specific primary and secondary CTL responses in vitro.For example, antigen presenting cells that have been incubated with a peptide can be assayed for stability to induce CTL responses in responder cell populations For secondary responses, the antigen presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells (Inaba et al., J. Exp. Med., 166: 182 (1987); Boog, Eur. J. Immunol., 18: 219 [1988]). Alternatively, the mutant mammalian cell lines that are deficient in their ability to load the class I molecules with internally processed peptides, such as the RMA-S mouse cell lines (Krere, et al.

Nature, 319: 675 (1986); Ljunggren, et al., Eur. J. Immonol., 21: 2963-2970 (1991)), and human somatic T cell inhibitome, T-2 (Cerundolo, et al., Nature, 345: 449-452 (1990) and which have been transfected with the appropriate human class I genes are conveniently used, when a peptide is added thereto to test for the ability of the peptide to induce primary CTL responses in vitro.These empty CHP cells are preferred to induce a response primary since the density of the CHP-peptide complexes on the surface of the antigen presenting cell will be higher Other eukaryotic cell lines that could be used include cell lines of various insects such as mosquito larvads (ATCC cell lines , CCL 125, 126, 1660, 1591, 6585, 6586), of sedea worm (ATTC CRL 8851), of scion worm (ATCC CRL 1711), moth cell lines (ATCC CCL 80) and Drosophila such as line of Schneider cells that have been transfected with CHP allele coding genes of human class I and human B2 microglobulin genes. Once the appropriate epitope is determined, the immunogenic peptides comprise the required motif for the binding of CHP and the epitope recognized by the CTL are synthesized. Immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or isolated from natural sources such as viruses or whole tumors. An expert person will recognize that immunogenic peptides - nicos can be of a variety of lengths, either in their neutral (uncharged) forms or in forms that are salts, and either be exempt from modifications such as glycosylation, secondary chain oxidation, or phosphorylation or containing these modifications, subject to the proviso that the modification will not destroy the biological activity of the polypeptides as described herein. Desirably, the peptide will be as small as possible while still maintaining essentially all the biological activity of the large peptide. Where possible, it may be desirable to optimally carry the peptides of the invention to a length of 9 or 10 amino acid residues, according to size with the endogenously processed viral peptides or the tumor cell peptides that are bound to the CHP of class I on the surface of the cell. Peptides having the desired activity can be modified as necessary to provide certain desired attributes, eg, improved pharmacological characteristics, while increasing or at least retaining essentially all the biological activity of the unmodified peptide to bind the CHP molecule desired and activate the appropriate T cell. For example, the peptides may be subject to various changes such as substitutions, either conservative or non-conservative, wherein these changes could provide certain advantages for their use such as improved CHP binding. By conservative substitutions it is meant to replace one amino acid residue with another which is biologically and / or chemically similar, eg, one hydrophobic residue for another, or one polar residue for another. Substitutions include combinations such as Gly, Ala; Val, lie, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of a single amino acid substitution can also be demonstrated using D-amino acids. These modifications can be made using well-known peptide synthesis methods. The peptides of the invention can be prepared in a wide variety of ways. Due to their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Several automated synthesizers are commercially available which can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, second edition, Pierce Chemical Co. (1984), supra. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence encoding an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultured under conditions suitable for expression. These methods are generally known in the art, as generally described in the Sambrook et al. Article, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), which is incorporated in the present by reference. Fusion proteins comprising one or more peptide sequences of the invention can also be used to present the epitope of the appropriate T cell. The immunogenic peptides are then used to activate the CTL ex vivo. The ex vivo therapy methods of the present invention and the pharmaceutical compositions thereof are useful for the treatment of mammals, particularly humans, to treat and / or to prevent viral infection, immune disorders and cancer. Examples of diseases that can be treated using the ex vivo therapy methods of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV, breast and ovarian cancer of the wart acuminate ( condyloma acuminatum), colon cancer, lung and VSH. For therapeutic use, therapy may begin at the first sign of viral infection or the surgical detection or removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by strengthening the LTC levels at least until the symptoms have subsided considerably and for a later period. In chronic infection, loading doses followed by booster doses may be required. The treatment of a subject infected with the methods of the invention can accelerate the resolution of infection in acutely infected subjects. For those subjects susceptible (or predisposed) to develop a chronic infection, the methods are useful to prevent the evolution from acute to chronic infection. When susceptible subjects are identified before or during infection, compositions can be directed to them, minimizing the need for administration to a larger population. The methods of the present invention can also be used for the treatment of chronic infection and for stimulating the immune system in order to eliminate in the carriers the cells infected with viruses. Ex vivo CTL responses to a specific pathogen (infectious agent or tumor antigen) are induced by incubating the tissue culture of the patient's LTC precursor cells (LTCp) together with a source of antigen presenting cells (APCs) loaded with the appropriate immunogenic peptide. After an appropriate incubation time (typically 3 to 12 weeks) [Is this the appropriate scale?], Where the LTCp are activated and mature and expanded into effector CTLs, the cells are infused back into the patient, where it will destroy your specific target cell (an infected cell or a tumor cell). Infusion of the cells in the patient may include a T cell growth factor for example interleukin 2 (IL-2). In order to optimize the in vitro conditions for the generation of specific cytotoxic T cells, the culture of the stimulating cells is maintained in a suitable serum-free medium which may include one or more growth factors such as IL-2, 11-4 , IL-7 and IL-12. Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis from normal donors or patients and are used as the sources of the responsive cell of the CTL. In one embodiment particularly for secondary CTL responses, appropriate CPAs are incubated with 10 to 100 micrometers of peptide in serum-free media for 4 hours under appropriate culture conditions. The peptide-loaded CPAs are then incubated with the populations of the responder cell in vitro for 7 to 10 days under optimal culture conditions. For the induction of primary LTC, CPAs expressing empty CHP would be used to stimulate naive LTCp. In this case, CTLs would be stimulated more frequently (1 to 2 times). Activation of positive LTC can be determined by assaying the cultures for the presence of LTC that kill the irradiated target cells, both white - driven by specific peptide as well as target cells expressing the endogenously processed form of the virus or related tumor antigen from which the peptide sequence was derived. The specificity and restriction of CHP of a patient's LTC can be determined by a number of methods known in the art. For example, the restriction of CTL can be determined by testing against different peptide-loaded blank cells expressing the human Class I CHP alleles shared with the ALH fonotype of the donor LTC. Peptides that present a positive test in CHP binding assays result in specific CTL responses are identified as immunogenic peptides. As mentioned above, induction of CTL in vitro requires the specific recognition of peptides that are linked to allele-specific CHP molecules of class I in CPA. The number of specific CHP / peptide complexes per CPA determines the level of CTL stimulation, particularly during the primary immune response. Even when small amounts of the peptide / CHP complexes are sufficient per cell to make a cell susceptible to lysis by CTL, or to stimulate a secondary CTL response, successful activation of a PCTCT during the primary response requires a significantly higher number of cells. CHP / peptide complexes.

Since the mutant cell lines capable of expressing empty CHP do not exist for each allele of human CHP, it is advantageous to use a technique to remove peptides associated with endogenous CHP from the surface of CPA, followed by loading of empty CHP molecules resulting with the immunogenic intergenic peptides. the use of uninfected, untransformed (non-tumorigenic) cells, and preferably of autologous patient cells such as CPA is desirable for the design of CTL induction protocols directed towards the de-coiling of ex vivo CTL therapies. This present invention provides novel methods that generate CHP of class I voids that can then be loaded with an appropriate immunogenic peptide by purifying the peptides associated with endogenous CHP from the surface of CPA or through incubation at a cold temperature of 37 ° C-> 0. 20 ° C) followed by loading the desired peptides. A CHP molecule of stable class I is a trimeric complex formed of the following elements: 1) a peptide usually of 8 to 10 residues, 2) a transmembrane heavy polymorphic protein chain carrying the peptide binding site in its alfal and alpha2 domains, and 3) a non-covalently associated non-polymorphic light chain, beta2 microglobulin. Removing the bound peptides and / or dissociating betya2microglobulin from the complex makes the CHP molecules of class I non-functional and unstable, resulting in rapid degradation at 37 ° C. Almost all Class I CHP oocysts isolated from PBMC have endogenous peptides bound thereto. Therefore, the first step in preparing CPA for primary CTL induction is to remove all endogenous peptides linked to class I CHP molecules in CPA without causing degradation and cell death before the exogenous peptides can be added. Two possible ways to generate free class I CHP molecules include lowering the culture temperature from 37 ° C to 26 ° C overnight to allow the class I CHP without peptide to be expressed as well as the clearance of the - endogenous peptides of the cell using a mild acid treatment. Treatment with mild acid liberates peptides previously bound in the extracellular environment allowing new exogenous peptides to bind to class molecules 1 empty Incubation overnight at cold temperature a 26 ° C that can slow down the metabolic rate of the cell allows the expression of a stable empty class I molecule that can then bind the exogenous peptides efficiently. It is also possible that cells that do not actively synthesize CHP molecules (eg., Resting PBMC) does not produce high quantities of empty surface CHP molecules by the cold temperature procedure. Peptide extraction is achieved by serious acid purification using trifluoroacetic acid, pH of 2, or acid denaturation of purified class I peptide complexes by immunoaffinity. These methods are not feasible for CTL induction since it is important to remove the endogenous peptides while maintaining the viability of CPA and an optimal metabolic state that is critical for the presentation of the antigen. Light pH solutions of 3 as pH agents of 3 such as glycine or citrate-phosphate stabilizing agents have been used to identify the endogenous peptides in order to identify the T cell epitopes associated with the tumor. The treatment is especially effective since only the CHP molecules of class I are destabilized (and the associated peptides are released), while other surface antigens remain intact including the class II CHP molecules. More importantly, the treatment of cells with mild acid solutions does not affect the cell's viability or metabolic status. Treatment with mild acid is rapid since the clearance of the endogenous peptides occurs in two minutes at 4 ° C and the CPA is functional after the appropriate peptides have been loaded. The technique is used herein to make peptides specific to the peptide for the generation of CTLs specific to the primary antigen. The resulting CPAs are efficient to induce CTCs specific for the peptide. Typically, in a primary response before the incubation of the CPAs with LTCp are activated, a quantity of the antigenic peptide is added to the CPA culture or stimulating cell, in sufficient quantity to be loaded into the human molecules of the class I that go to be expressed on the surface of the CPA. In the present invention, a sufficient amount of the peptide is an amount that allows approximately 200 or more human CHP molecules of class I to be loaded with the peptide to be expressed on the surface of each stimulating cell. Preferably, the stimulating cells are incubated with 5 to 100 micrograms per milliliter of peptide. The resting CTLs or precursors are then incubated in the culture with the appropriate CPAs for a period of time sufficient to activate the CTL. CTLs are activated in a specific manner to the antigen. The ratio of precursor CTLs to CPAs can vary from subject to subject and can also depend on variables such as the susceptibility of the subject lymphocytes to culture conditions and the nature and seriousness of the disease condition or other condition for the which method of treatment described is used. Preferably, however, the ratio of LTC: CPA (ie, responder to stimulant) is within the range of about 10: 1 to 100: 1. The LTC / CPA culture can be maintained for as long a time as necessary to stimulate a therapeutically usable or effective LTC number.

The activated LTC can be effectively separated from the CPA using one of a variety of known methods. For example, monoclonal antibodies specific for CPA, for peptides loaded in stimulating cells, or for LTC (or a segment thereof) can be used to link their appropriate complementary coordinator branch. The labeled-antibody cells can then be extracted from the mixture through appropriate means, e.g., by well-known immunoprecipitation or immunoassay methods. The effective cytotoxic amounts of activated CTLs can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the final target of these killing cells. The amount will also vary depending on the patient's condition and should be determined by taking into account all the appropriate factors by the practitioner. Preferably, however, from about 1 X 106 to about 1 X 10 12, more preferably from about 1 X 108 to about 1 X 10 11, and even especially preferred, from about 1 X 109 to about 1 X 10 10 are used. Activated CTLs for adult humans, compared to approximately 5 X 106 - 5 X 107 cells used in mice. As Cmose has discussed above, activated CTLs can be harvested from a cell culture prior to administration of the cells to the subject being treated. It is important to note, however, that unlike other present treatment modalities, the present method uses a cell culture system that does not contain transformed cells or tumor cells. Therefore, if complete separation of antigen presenting cells and activated CTLs is not achieved, there is no inherent danger known to be associated with the administration of a small number of stimulating cells, while the administration of cells that activate the mammalian tumor can be extremely dangerous. One embodiment of the present invention uses the CPA generated by the in vitro techniques of this application for therapy against LTC in vivo. In this embodiment, the CPAs are a patient's cell (e.g., peripheral blood cells) that are cleared of their natural antigenic peptides and loaded with a selected peptide which is conjugated to an immunotoxin. The CPAs are then re-introduced into the patient, where they will be bound by the endogenous CTLs that are specific for the antigenic peptide. The coupled immunotoxin will kill the LTC after it binds to the CPA. This targeted LTC extermination is widely useful to treat the rejection of tissue transplantation and the auto-immune alterations, which are mediated through the LTC. Typically, the CPAs carrying the selection antigen will be used in a first round of treatment, followed by retreatment using CPA at intervals of days. The treatment regimen will vary depending on the specific disorder to be treated and the judgment of the treating doctor. [This mode is described in the Scripps application. Should we make it known here?]. Methods for re-introducing cellular components are known in the art and include methods such as those exemplified in U.S. Patent Number 4,844,893 issued to Honsik, et al., And in U.S. Patent No. 4,690,915 issued to Rosenberg, which are incorporated herein by reference. present by reference. For example, the administration of activated CTLs through intravenous infusion is appropriate. [We ask them to provide more details about how the activated cells are re-introduced into the patient. For example, pharmaceutical carriers to be used, preferred methods of administration and the like]. The following examples are offered by way of illustration, and not by way of limitation.

Example 1 Ex vivo induction of cytotoxic T lymphocytes (CTL) Peripheral blood mononuclear cells (PBMCs) are isolated from an ALH patient either by venipuncture or leukapheresis (depending on the initial amount of the required LTCp), and purified by gradient centrifugation using Ficoll-Paque (Pharmacia). Typically, one million PBMC can be obtained per milliliter of peripheral blood, or alternatively, a typical leukapheresis procedure that can yield up to a total of 1-10 X 10 10 PBMC. The isolated and purified PBMCs are co-cultured with an appropriate number of CPA expressing the empty CHP molecules previously incubated ("boosted") with an appropriate amount of synthetic peptide (containing the binding motif of the HLA and the antigen sequence). in question) . PBMCs are usually incubated at 1 to 3 X 106 cells per milliliter in the culture medium such as RPMI-1640 (with autologous serum or plasma) or the serum-free medium AIM-V (Gibco). CPAs are usually used at concentrations ranging from 1X104 to 1X106 cells per milliliter, depending on the type of cell used. Possible sources of CPA include: Autologous PBMC, PBMC activated with SAC-I, PHA blasts; autologous dendritic cells (DC) that are isolated from PBMC and purified as described (Inaba, et al., J. Exp. Med., 166: 182 (1987)); and unchanging genetically modified mammalian cells such as the RMA-S mouse cell line or the human T2 cell line transfected with the appropriate CHP genes that express the "empty" ALH molecules that are syngeneic to the ALH allelic form of the patient). CPAs containing empty HLA molecules are known to be potent inducers of CTL responses, possibly because the peptide can more readily associate with empty CHP molecules than CHP molecules that are occupied by other peptides (DeBruijn, et al. others, Eur. J. Immunol., 21: 2963-2970 (1991)). The CPA is irradiated with gamma irradiation at an appropriate dose (using, e.g., radioactive cesium or cobalt) to prevent its proliferation and to facilitate the expansion of the LTCp. The cultures of the mixture containing PBMC, CPA and peptides are kept in a suitable culture vessel such as plastic "T" jars, gas permeable plastic bags or bottles, at 37 ° C in a humid air incubator / C02 After the culture activation phase, which usually occurs during the first 3 to 5 days, the resulting effector CTL can be further expanded by the addition of recombinant growth factors such as interleukin-2) IL-2, interleukin-4 ( IL-4), or interleukin-7 (IL-7) to the cultures. An expansion culture can be maintained for an additional 5 to 12 days, depending on the effector LTC numbers required for a specific patient. In addition, expansion cultures can be carried out using artificial hollow fiber (Cellic) capillary systems, where larger numbers of cells can be maintained (up to 1X1011). In order to obtain the numbers of cells required for treatment, it may be necessary to restimulate the cultures 2 to 4 times with adherent PBMC driven by irradiated autologous peptides. Before the cells are infused into the patient, they are tested to determine their activity, viability, toxicity and sterility. The resulting cytotoxic activity of the CTL can be determined by a normal 5ICr release assay (WE Biddison, 1991, Current Protocols in Immunology, p7, 17.1-7.17.5, Editor J. Coligan et al., J. Wiley and Sons, New York ), using target cells expressing the appropriate ALH molecule in the presence and absence of the immunogenic peptide. Viability is determined by the exclusion of a trypan blue dye by the active cells. The cells are tested for the presence of endotoxin by conventional techniques. Finally, the presence of bacterial or fungal contamination is determined by appropriate microbiological methods (chocolate agar, etc). Once the cells have passed all quality control and safety tests, they are washed and placed in an appropriate infusion solution (Ringer / glucose lactate / human serum albumin) which may include a cell growth factor T such as IL-2 and are infused intravenously into the patient.

Example 2 Preparation of allele-specific antigen-presenting cells, effective ALHs by acid clearance followed by peptide loading.

This example demonstrates the use of cold temperature incubation and acid clearance for generation of empty Class I CHP molecules to allow the peptide loading method in order to prepare the antigen-specific display cells to the effective ALH allele. (CPA) for use in diagnosis or ex vivo therapy applications. The CPAs in this example were used to sensitize precursor cytotoxic T lymphocytes to the development of antigen-specific cytotoxic cells. This was achieved using either PBMC activated with SAC I, cowan I, staphylococcus aureus, phytohemagglutinin (PHA) T-cell blasts or peripheral blood mononuclear cells (PBMC) as the CPA in the ALH-A2.1 or ALH systems. -A1. The results are applicable to other CPAs and other CHP alleles. Cul tive Medium. PHA blasts and CTL inductions were carried out in RPMI 1640 + Hepes + glutamine (Gibco) supplemented with 2 mM L-glutamine (Irvine Scientific), 50 micrograms per milliliter of gentamicin (Gibco), and 5% of human Type AB serum collected inactivated by heating (Gemini Bioproducts) [RPMI / 5 percent HS]. The lymphoblastoid cell lines transformed with EBV (LCL) were maintained in RPMI 1640 + Hepes + glutmin (BioWhittaker) supplemented with L-glutamine and gentamicin as above and 10 percent inactivated fetal calf serum by heating (Irvine Scientific) [RPMI / 10 percent FCS]. Chromko release assays were performed in RPMI / 10 percent FCS. Ci tocinas. Recombinant human interleukin-2 (rIL-2) (Sandoz) was used at a final concentration of 10 units per milliliter. Recombinant human interleukin-7 (rIL-7) (Genzyme) was used at a final concentration of 10 ng / milliliter. Cell Lines Cul tivated. JY, an ALH A2.1 expressing the B cell line transformed by human EBV was grown in RPMI / 10 percent FCS. K562, an NK cell-sensitive erythroblastoma line was grown in RPMI / 10 percent FCS. K562 was used to reduce the background killing of NK cells and LAK in the chromium release assays. Peptides The immunogenic peptides used in these studies were synthesized as described above using motifs for the ALH alleles for specific target antigens as described in detail in the commonly assigned and co-pending applications of United States Serial Number 07 / 926,666 and from the United States. United Serial Number 08 / 027,146 and their sequences are shown in Table 1. Peptides were routinely dissolved in 100 percent DMSO at 20 milligrams per milliliter, formed into aliquots, and stored at -20 ° C. C. Isolation of Peripheral Blood Mononuclear Cells (PBMC). Whole blood was collected in heparin (10 units per milliliter) containing syringes and centrifuged in conical centrifuge tubes with a capacity of 50 cubic centimeters (Falco) at 1600 revolutions per minute (Beckman GS-6KR) for 15 minutes. The plasma layer was then removed and 10 milliliters of the layer was collected with a 10 milliliter pipette using a circular motion. The layer was mixed vigorously and was diluted with a volume of RPMI 1640 free of serum. The diluted layer was then layered through 20 milliliters of Ficoll-Paque (Pharmacia) in a 50 cubic centimeter conical tube and centrifuged 400xg for 20 minutes at room temperature without a brake. The interface containing the PBMC was collected using a transfer pipette (two interfaces per tube of 50 cubic centimeters) and washed three times with 50 ml-liter of serum-free RPMI (1700, 1500 and 1300 revolutions per minute for 10 minutes ). Freezing and Thawing of PBMC. The PBMC were frozen at 30 x 106 cells per milliliter of 90 percent FCS + 10 percent DMSO (Sigma) in 1 milliliter aliquots using small cryocharas (Nalge). The small criofrascos were placed in cryogenic freezing containers of 1 ° C (Nalge) containing isopropanol (Fisher) and placed at a temperature of -70 ° C for 4 hours (minimum) until overnight (maximum). Isopropanol was changed after every 5 uses. The small cryochages were transferred to a liquid nitrogen for long-term storage. The PBMC were thawed by continuous stirring in a 37 ° C water bath until the last crystal had almost thawed. The cells were immediately diluted in serum-free RPMI medium containing 30 micrograms per milliliter of DNase (to prevent agglomeration by dead cells) (Calbiochem) and washed twice. Preparation of the depleted cell population of the CD4 + cell. Depletion of the CD4 + lymphocyte was carried out using antibody-coated flasks: MicroCELLector T-150 flasks for selection of CD4 + cells (Applied Immune Sciences) which were washed according to the manufacturer's instructions with 25 milliliters of PBS CMF (magnesium-free) of calcium) + mM EDTA (Sigma) by shaking the flasks for 30 seconds followed by incubation for 1 hour at room temperature on a flat surface. The stabilizing agent was aspirated and the flasks were washed an additional 2 times by shaking the flasks for 30 seconds and maintaining the coverage of the binding surface. To each washing bottle, 25 milliliters of the culture medium were added and - incubated for 20 minutes at room temperature on a flat surface. The media was left in the flask until they were ready to receive the cells. The PBMC were thawed in the culture medium containing 30 micrograms per milliliter of DNAse and washed twice. For a flask, a maximum of 12 x 10 7 cells were resuspended in 25 milliliters of the culture medium. The culture medium was aspirated from the flask and then the cell suspension was added gently to the MicroCeLLector. Flasks containing cells were incubated for 1 hour at room temperature on a flat surface. At the end of the incubation, the flask was gently oscillated from side to side for 10 seconds, to resuspend the non-adherent cells. Cells depleted of non-adherent CD4 + T-cell cells were harvested and then the flasks were washed twice with PBS CMF to collect the non-adherent cells. The harvested T cell + CD4 + cells were pelleted by centrifugation and resuspended in the culture medium. PHA Blast Generation. The PBMC were isolated using the normal Ficoll-Paque protocol. The frozen cells were washed twice before use. The cells were cultured at 2 x 106 x milliliter in RPMI / 5 percent HS containing one microgram per milliliter of PHA (Wellcome) and 10 units per milliliter of rIL-2. The PHA blasts were maintained in the culture medium containing 10 units per milliliter of rIL-2 with feed and division as necessary. PHA blasts were used as CPAs on day 6 of culture. The generation of empty class I molecules and peptide loading was only carried out by the acid clearance method when PBMCs were used as CPAs. Acid Purification / Peptide Loading of PBMC and PHA Blasts. PBMC were isolated using the Ficoll-Paque protocol. When frozen cells are used, the PBMCs were washed twice before use. PHA blasts were prepared as described above and washed twice before use. Once the cells were prepared, they were washed once in cold sterile 0.9 percent NaCl (J.T.Baker) + 1 percent BSA. In a conical centrifuge tube with a capacity of 50 cubic centimeters, the cells were resuspended at 107 per milliliter in a sterile cold citrate-phosphate stabilizer [0 to 13 M citric acid (JT Baker), 0.06 M sodium phosphate) monobasic (Sigma) pH of 3.1 percent BSA, 3 micrograms per milliliter of beta2microglobulin (Scripps Labs)] and incubated for 2 minutes on ice. Immediately, 5 volumes of cold sterile neutralization buffer No. 1 were added [0.15 M sodium phosphate monobasic pH 7.5, 1 percent BSA, 3 micrograms per milliliter of beta2microglobulin, 10 micrograms per milliliter of peptide] and the cells were granulated at 1500 revolutions per minute, for 5 minutes at 4 ° C. Cells were resuspended in 1 volume of cold sterile neutralizer # 2 stabilizer [PBS CMF, 1 percent BSA, 30 lig / milliliter of DNase, 3 microgram per milliliter of beta2microglobulin, 40 microgram per milliliter of peptide] and incubated for 4 hours at 20 ° C. The cells were diluted with the culture medium to approximately 5 x 106 / milliliter and irradiated with 6000 rads. The cells were then centrifuged at 1500 revolutions per minute for 5 minutes at room temperature and resuspended in the culture medium. Acid-purified / peptide-loaded cells were used immediately in the LTC induction cultures (below). Ligand Assays Using Intact Cells and Irradiated Peptide. The JY cells were either purified from the acid (i.e., treated with the citrate-phosphate stabilizer and the neutralization number 1 stabilizer as described above) or incubated at a reduced temperature. The JY control cells were left untreated in the tissue culture medium. After the treatment, both cell populations were washed twice with serum-free RPMI and loaded with peptide 941.01 (HBc 18-27) irradiated with 1251 (normal chloramine T-iodination). To determine the binding specificity, 2 x 106 cells were resuspended in 200 microliters of neutralization number 2 stabilizer (described above) containing 125I-941.01 (105 cpms) +/- 100 micrograms of 941.01 unirradiated. The cells were incubated for 4 hours at 20 ° C and washed twice with serum-free RPMI to remove the free peptide. The cells were resuspended in 200 microliters of serum-free RPMI. In a micro-fugo tube the cell suspension was layered through 800 microliters of FCS and granulated by centrifugation for 5 seconds. The supernatants were aspirated and the remaining radioactivity in the granule was measured (micrometric automatic gamma counter, 1 minute per tube). Ligament of Irradiated Peptides to Empty CHP Molecules. To determine the efficiency of peptide loading using incubation at cold temperature or peptide loading acid purification protocol by JY cells (a B cell line transformed with EBV of ALH-A2.1) they were pre-incubated at 26 ° C. ° C overnight or acidic clearance to remove the peptides associated with the endogenous CHP and the exogenous peptide loading was determined using the ligation peptide of ALH-A2.1 irradiated with 125I. The specificity of this reaction was determined by measuring the inhibition of the binding of the irradiated peptide using a cold peptide of the same sequence. The results presented in Table 2 demonstrate that the acid treatment of cells significantly (approximately 10 times) increased the amount of binding of the irradiated peptide to the JY cells. In addition, the binding of the irradiated peptide was completely blocked by the addition of the peptide, demonstrating specific binding (data not shown).

FACS analysis. About 106 cells were used for each antibody to be tested. Cells were elevated twice with PBS CMF + 0.1 percent BSA. To each sample, 100 microliters of PBS CMF + 0.1 percent BSA + primary antibody at 2 micrograms per milliliter (BB7.2, ATCC) or (9.12.1, Univ. Of Wisconsin) or (LB3.1 was added, Children's Hospital, Pittsburgh). A negative control was always included. The cells were incubated on ice for 20 minutes and washed twice with PBS CMF + 0.1 percent BSA. The cells were resuspended in 100 microliters of the anti-mouse IgG FITC conjugate (Sigma), diluted 1:50 in PBS CMF + 0.1 percent BSA, and incubated for 20 minutes on ice. Cells were washed twice with PBS CMF + 0.1 percent BSA, and resuspended in PBS for FACScan analysis (Becton Dicki). When it was necessary to postpone the analysis until the subsequent days, the cells were fixed with PBS / 1 percent paraformaldehyde (Fisher) and analyzed within a week. Measures through the FACS Analysis. The PHA-induced T cell blasts were acid-purified / loaded with peptide according to the methods described above. The resulting cells were stained for FACS analysis using alpha chain specific monoclonal antibodies of anti-ALH (9.12.1) and anti-ALH-A2 (BB7.2). Controls for this experiment included the same population of cells that were not treated at a pH of 3 (but treated with a PBS stabilizer at a pH of 7.2), and cells treated with the citrate-phosphate stabilizer ( to purify CHP) but neutralized in the absence of beta2microglobulin and peptide. The results presented in Figure 1 indicate that the treatment of these cells with the citrate-phosphate stabilizer (pH 3) significantly (10 times) reduced the reactivity of the cells towards both antibodies of the anti-ALH class alone ( anti-ALH-A2 and alpha chain specific) but not towards the monoclonal antibody specific for class II CHP molecules (anti-ALH-DR). Most importantly, neutralization of treated cells acid in the presence of beta2microglobulina and peptide resulted in preservation of a significant amount of the reactive sites of the antibody CHP class I, with only decreased 2.5 times in fluorescence intensity. Acid-treated cells remained viable, as measured by trypan blue exclusion analysis and forward / lateral FACS scattering analysis. Similar results were obtained using B cell lines transformed by EBV, fresh (or frozen) PBMCs and other peptides (which bind to either ALH-A2.1 or ALH-A1) (data not shown). Induction of Primary LTC Using autologous PBMC or PHA Blasts and Acid Purified Stimulants / Peptide Loaded. Acid depletion / peptide loading of PBMC or PHA blasts is described above. During the 4 hour incubation of stimulating cells with the peptide, the population of responder cells was prepared: the responders were PNMC and were depleted of the CD4 + T cells (described above). Responder cells were resuspended in culture medium at 3 x 106 per milliliter and 1 milliliter of cell suspension was distributed to responding each well of a tissue culture plate 24 well (Falcon, Becton Dicki). Plates were placed in the incubator at 37 ° C. 5 percent C02 until the population of the stimulator cell was ready. Once irradiated, the stimulating CPA resuspended in culture medium containing 20 ng per ml of rIL-7-106 / ml for the PBMC, or 3 x 105 / ml for the PHA blasts, was added 1 milliliter of the suspension of the stimulating cell per well to the plates containing the responders. On day 7 after induction, 100 microliters of the culture medium containing 200 ng / milliliter of rIL-7 was added to each well (lOng / milliliter of final rIL-7). On day 10 after induction, 100 microliters of the culture medium containing 200 units per milliliter of rIL-2 was added to each well (10 units / milliliter of final rIL-2). Restimulated with LTC Antigen. On days 12 to 14 after induction, the primary CTLs were restimulated with peptide using the autologous adherent APCs. Autologous PBMC were thawed and washed as described above. The cells were irradiated at 6000 rads. The cells were pelleted and resuspended in the culture medium at 4 x 106 per milliliter and 1 milliliter of the cell suspension was added to each well of a 24-well tissue culture plate and incubated for 2 hours at 37 °. C, 5 percent of C02. The non-adherent cells were removed by washing each well three times with RPMI free of serum. After this step, 0.5 milliliter of the culture medium containing 3 micrograms per milliliter of beta2microglobulin and 20 micrograms per milliliter of total peptide was added to each well. The CPAs were incubated for 2 hours at 37 ° C, under 5 percent C02 and with the peptide and beta2microglobulin. The wells were aspirated and one milliliter of responder cells was added to each well at 1.5 x 106 per milliliter in the culture medium. After 2 days, 1 milliliter of the culture medium containing 20 units per milliliter of rIL-2 was added to each well. The cultures were supplemented with 10 units per milliliter of rIL-2 (final) every three days thereafter. Chromium Release Test of Ci totoxicity. Seven days after the resuscitation of the primary induction, the cytotoxic activity of the cultures was evaluated. to. Preparation of the Effector Cell: The responders were centrifuged and resuspended at 107 / milliliter in RPMI / 10 percent FCS. The three-fold serial dilutions of the effectors were carried out to yield effector to target ratios of 100: 1, 33: 1, 11: 1 and 3: 1. The effector cells were taken by aliquots at 100 microliters per well in plates of "U" bottom pools of 96 wells (Costar), in duplicate. b. White Cell Preparation: Approximately 16 to 20 hours before the assay, the target cells were resuspended at 3 x 10 5 / milliliter in RPMI / 10 percent of FCS in the presence or absence of 3 micrograms per milliliter of beta2microglobulin and 10 micrograms per milliliter of total peptide. After preincubation, the target cells were centrifuged and the pellets were resuspended in 200 microliters (300 microCi) sodium chromate (51 Cr) (NEN). The cells were incubated at 37 ° C. for 1 hour with agitation. The irradiated target cells were washed 3 times with RPMI / 10 percent FCS. c. Test Set-up: The concentration of the target cell was adjusted to 105 / milliliter in RPMI / 10 percent FCS and aliquots were added to 100 microliters to each well containing the responders. K562 cells (cold-blooded, to block NK, and LAK activity) were washed and resuspended in RPMI / 10 percent FCS at 107 per milliliter. The aliquots of 20 microliters were added per well, yielding a ratio of 20: 1 of the white K562 to the irradiated target. For the determination of spontaneous 51 Cr release, 100 micro-liters per RPMI well were added per 10 percent FCS at 100 microliters per well of the irradiated target cell and 20 microliters per well of K562. For maximum 51 Cr release, 100 microliters of 1 percent Triton X-100 (Sigma) in PBS CMF was added at 100 microliters per well of irradiated target cells and 20 microliters per well of K562. The plates were centrifuged for 2 minutes at 1200 revolutions per minute to accelerate the formation of the cell conjugate. The assays were incubated for 5 hours at 37 ° C, 5 percent C02. The tests were harvested by centrifuging plates for 5 minutes at 1200 revolutions per minute and collecting 100 milliliters per well of supernatant liquid. Gamma-normal count techniques were used to determine the percentage of specific lysis (automatic micrometer gamma counter, 0.5 minute per tube). Perfect specific lysis was determined by the following formula: experimental release of cpm - spontaneous release of cpm / maximum release of cpm-spontaneous release of cpm x 100. Induction In Vi tro of LTC Specific to the Primary Antigen Using Acid Purified CPAs / Loaded with Peptide. The fc - - Additional critical parameters for the induction of primary CTLs are: 1) enrichment of CD8 + T cells in the poolation of responder cells (by depletion of CD4 + T cells), 2) addition of rIL-7 to the induction cultures of LTC from on day 0 and 3) restimulation of the cultures with antigen on days 12 to 14 using autologous adherent cells driven with peptide. Figure 7 shows a comparison of the acid-charge clearance technique (panel a) to the cold temperature incubation technique (panel 6).

Example 3 Selection of Peptides to identify the LTC epitopes In order to identify the CTL epitopes, the CTLs were stimulated by PBMC activated with SAC-I as the CPA. The cold temperature improvement of the empty CHP expression was used in addition to the acid clearance to the charge with the antigenic peptide and generate CPA of PBMC activated with SAC-1. This method presents an alternative protocol to the methods described above for the generation of CPAs that were used to stimulate CTL. This example also presents an alternative protocol for the stimulation of CTLs by CPA. Full Cul tive Medium. The tissue culture medium used in this study consisted of RPMI 1640 with Hepes and L-glutamine (Gibco) (Biowhittaker) supplemented with 2 mM L-glutamine - - (Irvine Scientific), 0.5 mM sodium pyruvate (Gibco), 100 units per 100 micrograms per milliliter of penicillin / streptomycin (Irvine), and 5 percent heat-inactivated Type AB Human Serum (RPMI / 5 percent HS; Gemini Bioproducts). The culture medium used in the growth of the lines transformed with EBV contained 10 percent heat inactivated fetal calf serum (RPMI / 10 percent FCS, Irvine) instead of human serum. Ci tocinas. Recombinant human Interleukin-2 (rIL-2) and Interleukin-4 (rIL-4) were obtained from Sandoz and used at a final concentration of 10 units per milliliter and 10 ng per milliliter, respectively. Human interferon-gamma (IFN-gamma) and recombinant human Interleukin-7 (rIL-7) were obtained from Genzyme and used at 20 units per milliliter and 10 ng per milliliter, respectively. Peptides The peptides were synthesized as described above and as described in Table 1. The peptides were routinely dissolved in 100 percent DMSO at 10 milligrams per milliliter, aliquoted, and stored at -70 temperature. ° C until they were used. Cell lines. JY. Steinlin, EHM, BVR, and KT3 are B cell lines transformed with homozygous human EBV that express the ALH A2-1, Al r A3, A13_, and A2, respectively. They were grown in RPMI / 10 percent FCS and used as targets in the LCT assays. K562, a sensitive erythroblastoma line - - the NK cell, which was grown in RPMI / 10 percent FCS, was used for the reduction of background kill in the LTC assays. The melanoma and A1 + cell ALH lines expressing either MAGE antigen, mel 397 and mel 938 are those that do not express the MAGE antigen, mel 888, were also grown in RPMI / 10 percent FCS. Isolation of Peripheral Blood Mononuclear Cells (PBMC). The whole blood was collected in syringes containing heparin and centrifuged in tubes of 50 cubic centimeters capacity at 1600 revolutions per minute (Beckman GS-6KR) for 15 minutes. The plasma layer was then removed and 10 milliliters of the layer was collected with a pipette using a circular motion. The layer was mixed well and diluted with an equal volume of RPMI. The layer (30 milliliters) was then layered on 20 milliliters of Ficoll-Paque (Pharmacia) and centrifuged at 1850 revolutions per minute (400xg) for 20 minutes, at 25 ° C, with braking. The interface between the ficoll and the plasma containing the PBMCs was recovered with a transfer pipette (two interfaces per tube of 50 milliliters) and was also washed with 50 milliliters of RPMI (1700, 1500, and 1300 revolutions per minute for 10 minutes ). The cells were resuspended in 10 to 20 milliliters of culture medium, counted, and adjusted to the appropriate concentration. Freezing of PBMCs. 30 million cells were inserted - per tube (90 percent FCS / 10 percent DMSO; Sigma) in the 1 ° C Cryogenic Freezing Container Nalgene containing Isopropanol (Fisher) and placed at -70 ° C from 4 hours (minimum) a throughout the night (maximum). Isopropanol was changed every five times. The tubes were transferred to a storage of liquid nitrogen for a long term. To defrost, PBMCs were continuously stirred in a water bath at 37 ° C until the last crystal had almost thawed (the tubes were not allowed to settle in the water bath or at room temperature for any period of time). The cells were diluted in serum-free RPMI containing 30 micrograms per milliliter of DNAse to prevent agglomeration by DNA from the dead cell and washed twice. Induction of Primary LTC Using Activated PBMCs with SAC-I as CPA. to. Preparation of PBMCs activated with SAC-I as the CPAs: the PBMCs were purified using the normal Ficoll-Paque protocol and resuspended at 1 x 10β per milliliter in RPMI / 5 percent FCS containing 0.005 percent Pansorbin cells (cells SAC-I expressing Protein A; Calbiochem), 20 micrograms per milliliter of Immunocuentas (Rabbit Human IgM, Biorad), and 20 ng per milliliter of human rIL-4. Two milliliters of the cells per well were plated in a 24 well plate (Falco, Becto - Dickinson) and were cultured at 37 ° C. After 3 days, the medium was removed and the cells were washed three times followed by the addition of RPMI / 10 percent HS. The cells were used after being cultured for 2 additional days in RPMI / 10 percent HS. b. Expression of empty Class I molecules on the surfaces of the CPA and peptide loading of the CPAs 1. Incubation at cold temperature: a. Expression of empty CHP in the CPAs: The CPAs were adjusted to a concentration of 2 x 106 per milliliter in a complete culture medium containing 10 ng per milliliter of rIL-4, 20 units per milliliter of human IFN-gamma, and 3 micrograms per milliliter of beta2-microgobulin (beta2m, Scripps Lab). The cells were then incubated overnight at 26 ° C in the presence of 5 percent C02. It should be noted that these cells only express a fraction of the Class I molecules in the empty state ("10 percent." B) Peptide loading of the CPA stimulating cells: Class I Empty expressing the CPAs were washed 1 to 2-fold with serum-free RPMI (+ L-glutamine and Hepes) and re-suspended at 1 X 107 in serum-free RPMI containing 50 micrograms per milliliter total peptide pool (i.e., 16.7 micrograms per milliliter of each peptide in a deposit of three, 25 micrograms per milliliter of each peptide in a deposit of two, 50 micrograms per milliliter of the individual peptide), 30 micrograms per milliliter of DNAse and 3 micrograms per milliliter of betam. an incubation period of 4 hours at 20 ° C, the cells were irradiated at 6100 rads (5 x 106 per milliliter, 25 million cells per tube), washed and adjusted to the appropriate concentration to be added to the induction culture ( see below 2. Purification of Acid: This was used as an alternative method to generate empty CHP on the surface of the CPA. PBMC activated with SAC-I were washed once in cold 0.9% sodi chloride (J.T. Baker) containing 1 percent BSA. The cells were resuspended at 107 / milliliter in a cold citrate-phosphate stabilizer (0.13M citric acid [JT Baker], 0.06M sodium phosphate monobasic [Sigma], pH 3) containing 1 percent of BSA and 3 micro-grams per milliliter of beta2m and incubated on ice. After 2 minutes, 5 volumes of the cold sodium phosphate stabilizer of 0.15M, pH 7.5, containing 1 percent BSA, 3 micrograms per milliliter of Beta2m, and 10 micrograms per milliliter of peptide [stabilizer number 1 of neutralization] and the cells were centrifuged at 1500 revolutions per minute for 5 minutes at 4 ° C. Cells were resuspended in one milliliter of cold PBS containing 1 percent BSA, 30 micrograms per milliliter of DNase, 3 micrograms per milliliter of beta2-nicotoglobulin, and 50 micrograms per milliliter of peptide [neutralizer No. 2] and incubated for 4 hours. hours at 20 ° C. As above, subsequent to incubation for four hours at 20 ° C, the cells were irradiated at 6100 rads (5 x 106 per milliliter, 25 million cells per tube), washed, and then adjusted to the appropriate concentration to be added. to the induction culture (see below). c. Preparation of CDMC-depleted PBMC-responsive cell population (depletion of lymphocyte subpopulations using AIS flasks). AIS MicroCellector T-150 flasks (specific for the depletion of CD4 + T cells, Menlo Park, CA) were primed by adding 25 milliliters of PBS / l mM EDTA, with turbulence for 30 seconds so that all surfaces were wetted and then incubated with the bonding surface down at room temperature for 1 hour. After this incubation, the flasks were shaken vigorously for 30 seconds, washed 1 time with PBS / EDTA, 2 additional times with PBS and then incubated with 25 milliliters of the culture medium for 15 minutes. PBMC were thawed in serum-free RPMI (+ L-glutamine + Hepes) containing 30 micrograms per milliliter of DNAse, washed once, and incubated for 15 minutes in the culture medium. After aspiration of the culture medium from the flasks, up to 180 million PBMC were added in 25 milliliters of the culture medium containing 30 micrograms per milliliter of DNAse. After 1 hour at room temperature, the flasks were gently oscillated for 10 seconds to resuspend the non-adherent cells. The non-adherent cell suspension containing the CD8 + T cells was collected and the flasks were washed 2 times with PBS. The depleted CD4 + T cell PBMCs were centrifuged and counted for addition to the induction culture. The cD4 + and CD8 + phenotype of the population of CD4 + depleted cells was determined by FACS analysis (see below). Typically, this technique resulted in a two-fold enrichment for CD8 + T cells with an average of approximately 40 percent to 50 percent CD8 + T cells and 15 percent to 20 percent CD4 + T cells remaining followed by depletion of CD4 + T cells. Depletion of CD4 + T cells can also be achieved using antibody and complementary methods or magnetic beads coated with antibody (Dynabeads). The depletion of CD4 + T cells enriched with the LTCp and removed cells that competed for the cell nutrients. d. Induction of primary LTC. During peptide loading for four hours of CPA stimulants, the depleted CD4 + PBMC to be used as the responder population were prepared using AIS flasks for CD8 + cell selection through CD4 + T cell depletion (above).

- The responder cells were plated at 3 x 10 7mi-liliter in a volume of 1 milliliter (24-well plate) and placed at 37 ° C until the stimulatory CPA loaded with peptide was prepared. The irradiated peptide-loaded CPAs were washed once in serum-free RPMI (+ L-glutamine and Hepes), adjusted to the appropriate concentration in the complete medium, and plated in a 24-well plate to one milliliter per milliliter. plate: For PBMC and PBMC activated with SAC-I as CPA 1 x 10 6 stimulant cells (volume of 1 milliliter) were plated into the wells containing the responder cells; For PHA blasts such as CPA, one milliliter of 3 x 105 / milliliter of stimulating cells was plated in each well. A final concentration of 10 ng / milliliter of rIL-7 (total volume of 2 milliliters) was added. The cells were cultured for 12 days. (For "pulse-only" induction protocol, 10 micrograms per milliliter of the soluble peptide was not added to the cultures). On day 12, the cultures were restimulated with adherent cells driven by peptide and tested for cytolytic activity 7 days later (continued). Protocol for Re-stimulation of Primary LTC Using Autologous Adherent CPAs. Autologous PBMC were thawed in serum-free RPMI (+ L-glutamine and Hepes) containing 30 micrograms per milliliter of DNase, washed 2 times, and adjusted to 5 x 10 6 / milliliter in the culture medium contained therein. DNase. The PBMC (25 million cells per tube in 5 milliliters) were irradiated at 6100 R. After one wash, the PBMC were resuspended in the culture medium and adjusted to 4 x 10 6 / milliliter and 1 milliliter of the irradiated PBMC were They added per well to a 24-well plate. The PBMC were incubated for 2 hours at 37 ° C, washed 3 times to remove the non-adherent celias, and cultured in the medium containing 20 micrograms per milliliter of total peptide and 3 micrograms per milliliter of beta2microglobulin were added in one volume from 0. 5 milliliter and again incubated for two hours at 37 ° C. The peptide was aspirated and 1.5 x 10 6 responder cells were re-suspended in the culture medium and added in a volume of 1 milliliter. After 2 days, 1 milliliter of the culture medium containing 20 units per milliliter of rIL-2 was added. FACS analysis. One million cells were centrifuged per tube resuspended in 100 microliters per PBS tube / 0.1 percent BSA / 0.1 percent sodium azide (Sigma) plus 10 microoiters per tube of directly conjugated antibody (Becton Dickinson), and incubated on ice for 15 to 20 minutes. The cells were then washed twice with PBS / 0.1 percent BSA / 0.1 percent sodium azide and resuspended in PBS for analysis on FACS (ecton Dickinson) scan. When it was not possible to analyze the samples within 1 to 2 days, the cells were fixed with PBS containing 1 percent paraformaldehyde (Fisher) and analyzed within one week. Cytotoxicity test a. Preparation of target cells. Approximately 16 to 20 days prior to the CTL assay, the target cells (lines matched with matching Class I EBVs) were washed once and resuspended in a volume of 10 milliliters at 3 x 10 5 per milliliter in RPMI / 5 percent. of FCS in the presence or absence of 10 micrograms per milliliter of total peptide. b. Irradiation of target cells: The target cells were centrifuged and resuspended in 200 microliters per 51Cr sodium chromate tube (NEN), and then incubated at 37 ° C for 1 hour on a shaker. The target cells were washed 3 times (10 milliliters per wash) with RPMI / 10 percent FCS and resuspended in 10 milliliters (to determine the irradiation efficiency, 50 micro-liters were counted per cell of blank in the counter automatic gamma Micromedic). c. LTC test. The target cells were adjusted to 2 x 105 / milliliter and 50 microliters of the cell culture were added to each well of a 96-well "U" bottom plate (Costar Corp.) for a final concentration of 1 X 104 / water well. K562 cells were washed once, resuspended at 4 x 106 / milliliter, and 50 microliters were added per well to - - a final concentration of 2 x 105 / well (ratio of cold K562 to the target cell was 20: 1). The responder cells were washed once, resuspended at 9x106 / milliliter, and serial three-fold dilutions were performed for effector cell to target cell ratios of 90: 1, 30: 1, 10: 1 and 3: 1. The responder cells were added in a volume of 100 microliters in duplicate wells. For spontaneous release, 50 microliters per well of the irradiated target cells, 50 microliters per well of the K562 cell, and 100 microliters per well of the. medium were then added. For maximum release, 50 microliters per well of the target cell, 50 microliters per well of the K562 cell, and 100 microliters per well of 0.1 percent Triton-XlOO (Sigma) were added. The plates were centrifuged for 5 minutes at 1200 revolutions per minute. After an incubation of 5 hours at 37 ° C, the plates were again centrifuged for 5 minutes at 1200 revolutions per minute, and 100 microliters were collected per well of the supernatant. The normal gamma-count techniques (automatic micrometer gamma counter, 0.5 minute per tube) were used to determine the percentage of specific lysis according to the formula: percentage of specific lysis = experimental release of cpm - spontaneous release of cpm / maximum release of cpm - spontaneous release of cpm x 100. d. Results Of the peptides that bind to the indicated alleles, XX of the 62 MAGE, XX peptides of the HIV peptides 52, XX of the VCH 30 and XX peptides of the HBV 35 peptides tested to date induced primary CTL in vitro . Representative graphs illustrating the CTL responses to various immunogenic peptides are shown for MAGE (Figure XX), HIV (Figure XX), VCH (Figure XX) and HBV (Figure XX). The CTL induction data is summarized in Table 24 which lists the immunogenic peptides that bind to the appropriate CHP and induce primary CTL in vitro. The peptide sequence corresponding to the antigen and the ALH allele to which it is linked is indicated. The results presented in Figures 2, 3 and 5 correspond to the experiments carried out using PBMC as the CPA. The results presented in Figure 4 presents the results obtained using PHA-induced T-cell blasts such as CPA. The results shown in Figure 6 illustrate lysis and sensitized target cells of peptide and endogenous target cells after stimulation with PBMCs activated with SAC-I loaded with the immunogenic peptide] [AGE-3 1044.07 and which had been loaded using cold temperature incubation. While the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Table 1 Synthesized Peptides for Charging in PBMC and Blasts PHA Acid Purified Autologous Peptide ID Number Antigen Sequence 777. 03 HBs 20-28 FLLTRILTI 924. 07 HBc 18-27 FLPSDFFPSV 927. 32 HBp 61-69 GLYSSTVPV 938. 01 MAGE 1 161-169 EADPTGHSY 939.03 PSA 49-57 VLVHPQWVL 941. 01 HBc 18-27 analog FLPSDYFPSV 1044. 04 PAP 135-143 ILLWDPIPV 1044. 05 PSA 166-175 KLQCVDLVHI 1044. 06 PSA 118-128 MLLRLSEPAEL 1044. 07 MAGE 3 161-169 EVDPIGHLY 1044. 01 MAGE 3 8-17 ASSLPTTMNY

Claims (19)

Acid Purification, Peptide Loading of JY Cells with 941.01 Irradiated Table 2 Population of Cells Irradiated Peptide CPMS I25j + / _ peptide +/- dev. Normal cold JY purified from acid - cold peptide 3553 + 157 n = 3 JY purified from acid + peptide fried 13 n = 1 JY control - cold peptide 370 + 37 n = 3 JY control + cold peptide 50 n = 1 N O V E D A D I N V E N C L I N Having described the invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property:

1. A method for activating cytotoxic cells in vitro which comprises: dissociating bound peptides from the class I CHP molecules in antigen presenting cells, using a mild acid treatment; associating the desired immunogenic peptides with the class I CHP molecules in the antigen presenting cells; and incubating the antigen presenting cells with the cytotoxic T cells, thereby producing activated cytotoxic T cells.

The method according to claim 1, wherein the step of dissociating the bound peptides is carried out by incubating the antigen presenting cells in a glycine or citrate-phosphate stabilizing solution at a pH of 3.

3. The The method according to claim 1, wherein the step of associating the desired immunogenic peptides with the CHP molecules is carried out by incubating the antigen presenting cells with about 10 to 50 micrograms per milliliter of immunogenic peptide.

The method according to claim 1, wherein the step of incubating the antigen presenting cells with the cytotoxic T cells for from about 7 to about 10 days.

5. The method according to claim 1, wherein the antigen presenting cells are peripheral blood mononuclear cells isolated from a patient.

6. The method according to claim 5, wherein the peripheral blood mononuclear cells are activated with SAC-I.

The method according to claim 1, further comprising: contacting the activated cytotoxic T cells with an acceptable carrier, thereby forming a pharmaceutical composition; and administering the pharmaceutical composition to a patient.

The method according to claim 7, further comprising separating activated cytotoxic T cells from antigen presenting cells.

The method according to claim 7, wherein the cytotoxic T cells are useful in the treatment of cancer, AIDS, hepatitis, bacterial infection, fungal infection, malaria or tuberculosis.

A method for specifically killing target cells in a human patient, comprising: obtaining a fluid sample containing patient's cytotoxic T cells; contacting cytotoxic T cells with antigen presenting cells comprising CHP molecules of class I having selected immunogenic peptides - two associated with them, thus producing activated cytotoxic T cells; contacting activated cytotoxic T cells with an acceptable carrier, thereby forming a pharmaceutical composition; and administering the pharmaceutical composition to a patient.

The method according to claim 10, further comprising the step of dissociating the bound peptides from the antigen presenting cells by incubating the antigen presenting cells in a glycine or citrate-phosphate stabilizing solution at a pH of 3.

12. The method according to claim 9, further comprising the step of associating desired immunogenic peptides with the HCP molecules in the antigen presenting cells by incubating the antigen presenting cells with from about 10 to 50 micrograms per milliliter. of the immunogenic peptide.

The method according to claim 9, wherein the antigen-presenting cells are peripheral blood mononuclear cells isolated from a patient.

The method according to claim 9, wherein the step of incubating the antigen presenting cells with the cytotoxic T cells for from about 7 to about 10 days. . • *

15. A method for activating cytotoxic T cells in vitro which comprises: incubating the antigen presenting cells at a temperature of less than about 27 ° C in order to generate empty class I CHP molecules in the antigen presenting cells.

16. The method according to claim 15, wherein the step of associating the desired immunogenic peptides with the CHP molecules is carried out by incubating the antigen presenting cells with from about 10 to 50 micrograms per milliliter of immunogenic peptide. .

The method according to claim 15, wherein the step of incubating the antigen presenting cells with the cytotoxic T cells for from about 7 to about 10 days.

18. The method according to claim 15, wherein the antigen presenting cells are peripheral blood mononuclear cells isolated from a patient.

19. The method according to claim 18, wherein the peripheral blood mononuclear cells are activated with SAC-I. IN WITNESS WHEREOVER, I have signed the above description and claims of novelty of the invention, as attorney of CYTEL CORPORAT in Mexico City, Republic of Mexico on August 5, 1994.