Methods for developing CD3+CD8+ cells directed against multiple viral epitopes to treat viral infections, including mutants that evolve to escape previous immunity

Abstract

Disclosed herein is a method for preparing peptide-specific cytotoxic T cells (CTLs) against an emerging viral strain. The method includes providing an initial peptide composition specific to an earlier strain of the virus to which the earlier CTLs were sensitized, the earlier CTLs having reduced potency against the emerging viral strain compared to their potency against the earlier strain of the virus; identifying immunodominant peptides in the initial peptide composition against the earlier strain of the virus; decreasing the proportion of immunodominant peptides in the initial peptide composition to obtain an immunodominant peptide-diluted peptide composition; and sensitizing mononuclear cells with the immunodominant peptide-diluted peptide composition, thereby resulting in the proliferation of peptide-specific CTLs against the emerging viral strain. The virus can be SARS-COV2 (COVID-19) and the emerging strain can be delta or omicron BA.2.75.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 227,690, filed July 30, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0002] Infectious diseases were responsible for the largest global burden of premature death and disability until the end of the 20th century, a distinction that has since been passed on to noncommunicable diseases. Among infectious diseases, viral infections are a major global health problem with the potential to emerge as pandemic threats.

[0003] SARS-CoV-2 (COVID-19) has killed more than 4 million people worldwide. Vaccination has reduced mortality rates, but the need for curative therapy for active infection remains. Uncertainties regarding the duration of immunity after vaccination and how quickly this virus can mutate and evolve suggest that relying solely on preventive measures is unwise.

[0004] Humoral and cellular immunity provide selective pressure against the emergence of mutants with eliminated targeted epitopes. Elimination of immunodominant epitopes provides the strongest advantage to newly emerging strains, and as a result, immunodominant epitopes are expected to be preferentially eliminated compared to subdominant epitopes in newly emerging variants. Immunologic treatments against SARS-CoV-2 need to be continually reevaluated as new sequence information becomes available.

[0005] Although SARS-CoV-2 has captured the attention of the global health community since late 2019, the elimination of immunodominant epitopes and the emergence of new variants are phenomena present in many viral infections in humans and other animal species.

[0006] Therefore, there is a need for technologies to reevaluate immunological treatments for many viruses to better protect human and animal health from newly emerging variants of infectious viruses. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates to methods for preparing peptide-specific cytotoxic T cells (CTLs) against emerging viral strains.

[0008] The method can include providing an initial peptide composition specific to an earlier strain of the virus to which the earlier CTLs were sensitized, where the earlier CTLs have reduced potency against the emerging viral strain compared to their potency against the earlier strain of the virus.

[0009] In some embodiments, previous CTLs may be less effective against emerging viral strains due to loss of immunodominant peptide targets in the emerging strains compared to previous strains.

[0010] The method can include identifying immunodominant peptides in an initial peptide composition for earlier strains of the virus.

[0011] The method can include reducing the proportion of immunodominant peptides in the initial peptide composition to obtain an immunodominant peptide-diluted peptide composition. In some embodiments, the proportion of immunodominant peptides in the immunodominant peptide-diluted peptide composition can be at least 30% or less than the proportion of immunodominant peptides in the initial peptide composition. In some embodiments, immunodominant peptides can be essentially absent in the immunodominant peptide-diluted peptide composition.

[0012] This method may involve sensitizing mononuclear cells with a diluted peptide composition of immunodominant peptides, thereby resulting in the proliferation of peptide-specific CTL against the emerging viral strains.

[0013] This method can be used to prepare peptide-specific CTLs against any emerging strain of any virus. In some embodiments, the virus can be SARS-CoV-2 (COVID-19). In some embodiments, the emerging strain can be delta or omicron BA.2.75.

[0014] This method can be used with an initial peptide composition that is restricted to any HLA allele. In some embodiments, the initial peptide composition can include one or more peptides that are restricted to an HLA-A1 allele. In some embodiments, the initial peptide composition can include one or more peptides that are restricted to an HLA-A2 allele. In some embodiments, the initial peptide composition can include one or more peptides that are restricted to an HLA-B7 allele. In some embodiments, the initial peptide composition can include one or more peptides that are restricted to an HLA-B40 allele. In some embodiments, the initial peptide composition can include one or more peptides that are restricted to an HLA-Cw7 allele. In some embodiments, the initial peptide composition can include a combination of peptides that bind to any one or more of the HLA-A1, A2, B7, B40, Cw7 alleles.

[0015] The present disclosure also relates to a method for treating an emerging virus strain. The method can include administering to a subject in need of treatment an effective amount of peptide-specific CTLs prepared according to the methods described herein. In some embodiments, in the treatment method, the virus for which peptide-specific CTLs are prepared can be COVID-19. In some embodiments, in the treatment method, the emerging strain can be delta or omicron BA.2.75.

[0016] In some embodiments, the treatment method can further comprise administering one or more premedications prior to administering the CTLs.

[0017] In some embodiments, the method of treatment can further comprise administering one or more anti-viral agents prior to, concurrently with, or following administration of the CTLs.

[0018] The present disclosure also relates to a pharmaceutical composition comprising the peptide-specific CTL prepared according to the method described herein. In some embodiments, in the pharmaceutical composition, the virus for which the peptide-specific CTL is prepared can be COVID-19. In some embodiments, in the pharmaceutical composition, the emerging strain can be delta or omicron BA.2.75.

[0019] In some embodiments, in the pharmaceutical composition, the proportion of immunodominant peptides in the immunodominant peptide-diluted peptide composition may be at least 30% or less than the proportion of immunodominant peptides in the initial peptide composition. In some embodiments, in the pharmaceutical composition, the immunodominant peptides may be essentially absent in the immunodominant peptide-diluted peptide composition. [Brief description of the drawings]

[0020] [Figure 1A] FIG. 1 shows the results of a tetramer staining assay for a composition comprising CD8+ CTLs that recognize peptides derived from COVID-19 gene / ORF products in an HLA-restricted manner, as described in Example 1.

[0021] [Figure 1B] FIG. 1 shows the results of a cytotoxicity assay for a composition comprising CD8+ CTLs that recognize peptides derived from COVID-19 gene / ORF products in an HLA-restricted manner, as described in Example 1.

[0022] [Figure 2A]FIG. 1 shows a pie chart of immunodominance measured by tetramer staining assay of CTLs generated from apheresis products from two donors for a composition comprising seven peptides that bind to HLA-A*02:01, as described in Example 1.

[0023] [Figure 2B] FIG. 1 shows a pie chart of immunodominance measured by tetramer staining assay of CTLs generated from apheresis products from one donor for a composition comprising eight peptides that bind to HLA-A*01:01, as described in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present disclosure relates to the preparation of peptide-specific cytotoxic T lymphocytes (CTLs) that can be used for immunological treatment of subjects infected with an emerging viral strain. The CTLs can be prepared by providing an initial peptide composition specific for a previous strain of the virus to which the previous CTLs were sensitized, the previous CTLs having reduced potency against the emerging viral strain compared to their potency against the previous strain of the virus, identifying immunodominant peptides in the initial peptide composition against the previous strain of the virus, decreasing the proportion of immunodominant peptides in the initial peptide composition to obtain an immunodominant peptide-diluted peptide composition, and sensitizing mononuclear cells with the immunodominant peptide-diluted peptide composition, thereby resulting in the proliferation of peptide-specific CTLs against the emerging viral strain.

[0025] All publications and patents cited in this disclosure are incorporated by reference in their entirety. In the event that any material incorporated by reference contradicts or is inconsistent with this specification, this specification takes precedence over such material. The citation of any reference herein is not an admission that such reference is prior art to this disclosure. When a range of values ​​is expressed, it includes embodiments using any specific value within that range. Furthermore, reference to values ​​stated within a range includes every value within that range. All ranges are inclusive of endpoints and combinable. When values ​​are expressed as approximations, using the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. The use of "or" means "and / or" unless the particular context of its use dictates otherwise.

[0026] Various terms are used throughout the specification and claims in connection with the embodiments of the description. Such terms shall be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms shall be interpreted consistent with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methods, such as the widely used molecular cloning methods described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions unless otherwise indicated.

[0027] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Terms such as "include," "such as," and the like are intended to mean inclusion without limitation, unless otherwise stated.

[0028] Unless otherwise indicated, the terms "at least," "less than," and "about" or similar terms preceding a series or range should be understood to refer to every element in that series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0029] The term "subject" as used herein refers to any animal, including, but not limited to, humans, non-human primates, rodents, mammals commonly kept as pets (e.g., dogs and cats, among others), farm animals (e.g., cows, sheep, goats, pigs, horses, and camels, among others), etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0030] Further description of the methods and guidance for practicing the methods is provided herein.

[0031] Viruses and strains Many viruses impose a burden on human or animal health, and new strains frequently emerge with epitopes that are sufficiently different from those of previous strains to evade the humoral and cellular immunity developed against those previous strains. Examples of such viruses include, but are not limited to, SARS-CoV-2 (COVID-19), influenza, parainfluenza, respiratory syncytial virus (RSV), metapneumovirus, hepatitis B virus (HBV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), human herpesvirus (HHV), and adenovirus.

[0032] In some embodiments, the virus can be SARS-CoV-2 (COVID-19).

[0033] As used herein, a previous strain is a virus strain that has previously undergone immunological therapy by CTL. Specifically, the initial peptide composition is derived from the previous strain, the previous CTL is sensitized to the initial peptide composition, and the previous CTL is effective in treating mammalian infection by the previous strain.

[0034] As used herein, an emerging strain is a strain of a virus that differs from an earlier strain, where the earlier CTLs have reduced efficacy against the emerging viral strain compared to their efficacy against the earlier strain of the virus.

[0035] In some embodiments, if the virus is COVID-19, the emerging strain may be Delta or Omicron BA.2.75.

[0036] Initial peptide composition and prior CTL The method can include providing an initial peptide composition specific to an earlier strain of the virus to which the earlier CTLs were sensitized, where the earlier CTLs have reduced potency against the emerging viral strain compared to their potency against the earlier strain of the virus.

[0037] The initial peptide composition may comprise about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 10 to about 20, or about 15 to about 20 peptides. The specific peptides of the initial peptide composition will vary for each virus and each previous strain. Any list of peptides described in this application is merely exemplary and not limiting, as such lists are not exhaustive, fixed, and will continue as a dynamic and open-ended list.

[0038] Peptides can be selected that bind to the HLA-restricted element of interest. The HLA-restricted element of interest can be selected from the three classical HLA-I genes, HLA-A, HLA-B, and HLA-C, which are expressed in all nucleated cells in humans. HLA-I molecules present peptides derived from intracellular proteins. The intracellular antigen presentation pathway may involve cleavage of viral proteins in the cytosol by proteasomes, translocation to the lumen of the endoplasmic reticulum (ER), trimming by ER-resident aminopeptidases, loading onto HLA, and presentation at the cell surface. HLA-II genes (HLA-DR, HLA-DP, and HLA-DQ) are constitutively expressed only in a subset of cells specialized for antigen presentation, such as dendritic cells, B cells, and macrophages, but expression can also be induced in additional cell types, for example in response to cytokine stimulation. HLA-II molecules present peptides derived from extracellular proteins that are taken up by cells via endocytosis and phagocytosis, and intracellular proteins that access the HLA-II processing pathway via autophagy.

[0039] HLA-I molecules typically bind peptides that are 8–12 amino acids (aa) in length. The HLA-1 peptide-binding cleft is closed at both the N- and C-termini, and the optimal length preference is often biased towards binding approximately 9-mer peptides. For most HLA-I alleles, the length preference differs between alleles. High-affinity ligands for a given HLA allele usually share a common amino acid motif with relatively strict selectivity at anchor positions (usually the second (P2) and last (PΩ) for HLA-I, and -P1, P4, P6, and P9 for HLA-II) that form specific interactions with residues in the corresponding HLA-binding pocket. The HLA locus is the most polymorphic in the human genome, with tens of thousands of alleles reported to date. HLA variants that differ in peptide contact residues differ in the repertoire of peptides that they present. The diversity of HLA alleles in a population is an important evolutionary mechanism for defense against diverse pathogens, e.g., rapidly mutating viruses, newly emerging viruses and virus strains, etc. HLA alleles may be associated with the severity and outcome of viral infections: for example, the HLA-C*15:02 allele is associated with protection against SARS-CoV-1, and HLA-B57 is highly associated with efficient HIV-1 control and long-term non-progressive infection in the absence of antiretroviral therapy.

[0040] The peptides of the initial peptide composition can be derived from any virus. Peptides can be found empirically, by bioinformatics techniques, or from publicly available sources. For example, T cell epitopes have been identified, collected, and reported for a wide range of viruses in the Immune Epitope Database and Analysis Resource (IEDB).

[0041] In some embodiments, the initial peptide composition may include one or more peptides that are restricted to an HLA-A1 allele.

[0042] Additionally or alternatively, in some embodiments, the initial peptide composition may include one or more peptides restricted to HLA-A2 alleles.

[0043] Alternatively or additionally, in some embodiments, the initial peptide composition may include one or more peptides restricted to HLA-B7 alleles.

[0044] Additionally or alternatively, in some embodiments, the initial peptide composition may include one or more peptides restricted to HLA-B40 alleles.

[0045] Alternatively or additionally, in some embodiments, the initial peptide composition may comprise one or more peptides restricted to the HLA-Cw7 allele.

[0046] As will be apparent, in some embodiments, the initial peptide composition may comprise a combination of peptides, each peptide binding to any one or more of the HLA-A1, -A2, -B7, -B40, or -Cw7 alleles.

[0047] Identification of immunodominant peptides The method can also include identifying immunodominant peptides in the initial peptide composition for previous strains of the virus, for example, by tetramer assays, interferon production assays, cytotoxicity assays, or other known techniques that can find T cells within a T cell population that have specificity for individual peptides.

[0048] After assaying each peptide in the initial peptide composition individually against the T cell population, including the prior CTL, the immunodominant peptide(s) can be identified based on one or more criteria. The peptides with the highest scores in the tetramer assay, interferon production assay, cytotoxicity assay, or other assay can be identified as the immunodominant peptide. This approach can be modified by requiring that the highest score be greater than a threshold level, such as about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, or about 10%. In this modified approach, the initial peptide composition may be deemed to be free of immunodominant peptides if the highest score among all peptides is below a threshold level.

[0049] In another approach, any score above a threshold level can be considered sufficient to identify a peptide as an immunodominant peptide. In this approach, an initial peptide composition may be considered to have 0, 1, 2, or more immunodominant peptides.

[0050] Desirably, considering that the previous CTLs sensitized to the initial peptide composition have been found to have reduced efficacy against the emerging virus strains compared to the efficacy against the previous strains of the virus, the conditions for identifying immunodominant peptides in the initial peptide composition should be selected so that at least one peptide is identified as an immunodominant peptide.Without being bound by theory, the previous CTLs may have reduced efficacy due to the unbalanced recognition of the immunodominant peptides that are lost in the emerging strains.Therefore, modification of the initial peptide composition is necessary.

[0051] If more than one peptide in the initial peptide composition is identified as immunodominant, the following further steps can be performed on one, some, or all of the immunodominant peptides.

[0052] Reduction in the proportion of immunodominant peptides in a peptide composition The method can also include reducing the proportion of immunodominant peptides in the initial peptide composition to obtain an immunodominant peptide-diluted peptide composition.

[0053] Depletion can involve removing peptide molecules from the initial peptide composition, or producing a composition with a controlled proportion of the same peptides contained in the initial peptide composition.

[0054] The percentage of immunodominant peptides in the immunodominant peptide diluted peptide composition can be at any desired level, for example, about 50% or less, about 49% or less, about 48% or less, about 47% or less, about 46% or less, about 45% or less, about 44% or less, about 43% or less, about 42% or less, about 41% or less, about 40% or less, about 39% or less, about 38% or less, about 37% or less, about 36% or less, about 35% or less, about 34% or less, about 33% or less, about 32% or less, about 31% or less, about 30% or less, about 29% or less, about 28% or less, about 27% or less, about The immunodominant peptide content can be reduced to 26% or less, about 25% or less, about 24% or less, about 23% or less, about 22% or less, about 21% or less, about 20% or less, about 19% or less, about 18% or less, about 17% or less, about 16% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1%, or less than the proportion of immunodominant peptides in the initial peptide composition.

[0055] In some embodiments, the proportion of immunodominant peptides in the immunodominant peptide-diluted peptide composition can be reduced by at least 30% or less than the proportion of immunodominant peptides in the initial peptide composition.

[0056] In some embodiments, the immunodominant peptide may be essentially absent in the immunodominant peptide-diluted peptide composition (i.e., if present, it is present at a level that is undetectable by the same assay used to identify the immunodominant peptide in the initial peptide composition).

[0057] Generation of peptide-specific CTL against emerging virus strains The method may also include sensitizing mononuclear cells with a diluted peptide composition of immunodominant peptides, thereby resulting in the proliferation of peptide-specific CTL against the emerging viral strains.

[0058] In one exemplary approach, lymphocytes (a type of mononuclear cell) undergo three in vitro stimulation-expansion cycles to produce the final CTL product used in the therapeutic methods described herein. Each of these three stimulation-expansion cycles follows a different procedure, as each has a different purpose in the overall production process. Optionally, a fourth restimulation may be performed. The fourth restimulation may be performed (1) for products that fall just short of meeting the release criteria, when it is expected that an additional round of stimulation and expansion will enable the product to meet these criteria, or (2) when additional cell expansion is desired and it is believed that an additional round of stimulation and expansion may significantly increase the number of therapeutic doses obtained from the batch. Such an optional fourth restimulation may be performed following the same process as the third stimulation.

[0059] In some embodiments, mononuclear cells from healthy volunteer donors can be separated into lymphocyte and monocyte fractions by elutriation. The lymphocytes can be stimulated with immunodominant peptide dilution peptide compositions. It is not necessary to use virus-derived products.

[0060] In the first stimulation, a subset of collected monocytes can be treated to induce maturation into dendritic cells. Dendritic cells can be pulsed with one or more virus-specific peptides and co-cultured with lymphocytes for 7 days. In the second and third stimulation, monocytes can be used to present peptides and stimulated lymphocytes can be allowed to proliferate / expand for 7-12 days. The second stimulation can also include an enrichment step, which serves to select peptide-specific CTLs (as T cells that recognize the pulsed peptides will preferentially adhere to the adherent monocyte layer) and to reduce the content of other non-specific "bystander" lymphocytes or other immune cells from the donor. Monocytes and peptides can also be used in the third stimulation, but this selection step does not need to be repeated.

[0061] Most of the procedures described herein can be carried out in RPMI-1640 with 10% heat inactivated AB serum. The amount of AB serum in the medium can be reduced to about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%. Serum-free medium and autologous plasma may be used as an alternative. This is called "complete medium" or CM. Any suitable medium can be used, such as AIM V or other serum-free medium preparations alone with a concentration of pooled serum or autologous serum or plasma of about 10% or less, RPMI-1640 with serum replacement with or without a lower concentration of pooled serum or autologous serum or plasma, as determined by one of skill in the art.

[0062] In some embodiments, in a first stimulation, referred to as the initial in vitro sensitization, lymphocytes can be stimulated with a diluted peptide composition of immunodominant peptides as a pool rather than as individual peptides.

[0063] For initial in vitro sensitization, dendritic cells can be used as antigen-presenting cells. These cells can be prepared from elutriated monocytes. Fresh or freshly thawed monocytes can be enriched by adherence to plastic. An appropriate number of monocytes can be resuspended in medium, after which the cells can be transferred to tissue culture plates. The cells can then be incubated for an appropriate time (e.g., at least 60 minutes, at least 90 minutes, at least 120 minutes) to allow the monocytes to adhere to the culture plate. After incubation, the supernatant can be removed from the culture plate. The adherent cells can then be cultured with GM-CSF and IL-4 for an appropriate time (e.g., 24 hours), at which point maturation cytokines (e.g., TNF-α, IL-1 β, IL-6, and / or prostaglandin E2) can be added. After an additional 24 hours of culture, the dendritic cells are detached and ready to be harvested by aspiration of the medium and centrifugation. The culture period in mature cytokines can be extended beyond 24 hours, to about 30 hours, to about 36 hours, to about 42 hours, or up to about 48 hours, if desired.

[0064] After harvesting, dendritic cells are pulsed with peptides (e.g., 2 μg / ml each) for an appropriate time (e.g., about 60, 75, 90, or 120 minutes) and then plated in tissue culture flasks (e.g., 75 cm 2 The lymphocytes can be co-cultured with dendritic cells in medium (e.g., CM) in a culture of about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, or about 25:1. The lymphocytes (e.g., a total of 80×10 6 , 100×10 6 , 120×10 6 ) can be added to each flask. This can be considered day 0 of the CTL culture process. After this stimulation, the cultures can be left undisturbed for 7 days.

[0065] The second stimulation results in the enrichment and subsequent proliferation of virus-specific CTLs. Seven days after the first sensitization, CTLs can be restimulated as part of an enrichment step, which helps to select for peptide-specific CTLs and reduces the content of other non-specific "bystander" lymphocytes or other immune cells from the donor. Enrichment can be based on preferential adhesion of peptide-specific CTLs to a monocyte layer pulsed with the peptide used for the first sensitization. CTLs that recognize any of the peptides presented by the appropriate HLA alleles will preferentially adhere to the monocyte + peptide layer through the formation of an immunological synapse, in contrast to "bystander" lymphocytes that can be gently washed away. Although some "bystander" lymphocytes may adhere non-specifically, this usually results in an enrichment of peptide-specific CTLs of about 10-fold or more compared to the starting material. To carry out this enrichment step, monocytes (e.g., 10x10 6 ) can be added to tissue culture plates. Peptides can be added (e.g., at a final concentration of 2 μg / ml each) and incubated with monocytes (e.g., for about 90 minutes). Lymphocytes (e.g., about 60×10 6 , about 70×10 6 , about 80×10 6 , about 90×10 6 , about 100×10 6 , about 110×10 6 , or about 120 × 10 6 , typically 75 cm 2The contents of one of the tissue culture flasks) can be added to the well. The "bystander" lymphocytes can be removed from the well by gently washing with PBS after an appropriate time (e.g., about 5, about 7.5, about 10, or about 12 minutes). The adherent lymphocytes can be contacted with the peptide-pulsed monocytes overnight to complete the activation / restimulation process. The following day, the lymphocytes can be removed from the monocyte layer. The removed adherent lymphocytes can be transferred to a tissue culture flask in medium containing recombinant human IL-2 (e.g., at a concentration of 50 U / ml). IL-2 can be added over time (e.g., 50 U / ml every 48 hours). If the flask changes color to more orange / yellow, the medium can be replaced. After the second stimulation, the cells can be cultured for a total of 7 days. Enrichment of the monocyte layer as part of this second stimulation is desirable and can provide an advantage over conventional methods. Without wishing to be bound by theory, it is believed that the enrichment of the monocyte layer is responsible for the significantly higher purity levels in total T cells that have not been achieved prior to the present invention.

[0066] A third stimulation can be performed to further expand the viral peptide-specific CTLs. The enrichment step performed as part of the second stimulation does not need to be repeated as part of the third stimulation. However, if the evaluation of the percentage of viral-peptide-reactive lymphocytes (measured by intracellular cytokine assay or tetramer assay) falls below a certain limit (e.g., about 12-18%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17% or about 18%) within one day of the planned third stimulation, the second stimulation procedure can be repeated instead of the usual procedure of the third stimulation. Of the entire CTL production process, the enrichment step involves the most manipulations and may be the most prone point for contamination to enter, so it may be desirable to avoid repeating this step multiple times whenever possible. Even without repeating the enrichment step, it is expected that the percentage of viral peptide-specific CTLs will be further enriched after the third stimulation. This reflects the fact that stimulated cells will grow in IL-2-containing medium, whereas unstimulated "bystander" cells will not, and over time, the unstimulated "bystander" cells will die in culture, resulting in a more concentrated product. By setting the above threshold for when the enrichment step from the second stimulation can be repeated, it is expected that it will not be repeated frequently, and will only be repeated when essential to the manufacturing process.

[0067] After 7 days of culture in tissue culture flasks (day 14 of CTL stimulation / total culture), cells can be counted and restimulated with monocytes and peptides in G-Rex flasks. The lymphocyte:monocyte ratio can be about 4:1 to about 5:1, about 4:1, about 4.1:1, about 4.2:1, about 4.3:1, about 4.4:1, about 4.5:1, about 4.6:1, about 4.7:1, about 4.8:1, about 4.9:1, or about 5:1. Each peptide can be added again at a concentration of about 2 μg / ml. For this secondary restimulation, lymphocytes (e.g., 15x10 6 ) can be added to each G-Rex10 flask. Sensitization can be performed in medium (e.g., 40 ml complete medium) containing IL-2 (e.g., 50 U / ml).

[0068] After the third stimulation, the CTLs can be cultured again (e.g., for 7 days). The replacement of medium and IL-2 can be performed every 3-4 days depending on when a color change in the medium is observed.

[0069] After the end of this 21-day stimulation and expansion period, the CTLs can be evaluated for whether they meet the required criteria, and if so, can be harvested (e.g., within the next 24 hours) for cryopreservation. Optionally, in some circumstances, a fourth stimulation can be performed following the guidelines for the third stimulation. This is typically performed when further cell expansion is deemed desirable to increase the number of doses of CTLs generated, or when the product is slightly below the release criteria and an additional round of stimulation and expansion would allow the product to meet all criteria. Re-stimulation steps can be performed at 6-10 day intervals (e.g., 6, 7, 8, 9, 10 day intervals), although these steps can be performed up to 1 day earlier or later than this typical 7 day interval, if necessary.

[0070] After completion of three in vitro stimulation-growth cycles, the products can be screened for proper cell content, function, viability, and sterility.

[0071] Suitable cell content refers to at least 20% (e.g., at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and the like) of cells based on intracellular cytokine (ICC) staining or tetramer binding. means that about 100%) respond to the viral peptides and the content of naive T cells, monocytes, and NK cells in the product can be less than about 2.5% (e.g., about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%).

[0072] Function can be based on approximately 40% cytolytic activity of CTLs against peptide-pulsed targets at an effector:target ratio of approximately 40:1.

[0073] The viability should be greater than 70%, for example a viability of about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% may be appropriate.

[0074] Sterility can be assessed through routine and fungal cultures, as well as mycoplasma and endotoxin assays. CTLs can be cultured at any desired concentration, e.g., about 2 x 10 per milliliter. 6 and stored frozen in freezer bags at a concentration of 0.01% and stored for later use in the methods disclosed herein.

[0075] Treatment of emerging virus strains with peptide-specific CTLs This method can further comprise administering to a subject an effective amount of the peptide-specific CTLs prepared as described above.

[0076] In some embodiments, the method can include administering an effective amount of peptide-specific CTLs to a subject in need of treatment by intravenous infusion. The peptide-specific CTLs can be administered to a subject by intravenous delivery, for example, by administration via a central line, midline, or peripheral IV.

[0077] In some embodiments, prior to administration of peptide-specific CTLs, subjects may undergo complete HLA typing and then be treated with the appropriate CTLs.

[0078] In some embodiments, prior to administration of peptide-specific CTLs, subjects can be blood tested for rapid and low-resolution human leukocyte antigen (HLA) typing with high-resolution PCR SSP supplementation to determine whether they have potentially suitable HLA antigens for treatment (HLA-A1, -A2, -B7, -B40, -Cw7). High-resolution supplementation can provide evidence that the subject is HLA-A*01:01, -A*02:01, -B*07:02, -B*40:01, -C*07:02, and thus a match for CTLs of one or more alleles.

[0079] Prior to administration of peptide-specific CTL, premedication can be administered. In some embodiments, subjects can receive premedication such as diphenhydramine and acetaminophen. The dose of diphenhydramine can be about 15, about 20, about 25, or about 30 mg. The dose of acetaminophen can be about 500, about 550, about 600, about 650, about 700, or about 750 mg.

[0080] The subject can also be treated with one or more antiviral agents (eg, agents containing remdesivir or other standard of care drugs) prior to, concurrently with, or following administration of the peptide-specific CTLs.

[0081] The effective dose of peptide-specific CTL is approximately 1 × 10 5 Total cells / kg ~ approx. 3 x 10 6Total cells / kg can be about 1×10 5 Total cells / kg, approximately 2 x 10 5 Total cells / kg, approximately 3 × 10 5 Total cells / kg, approximately 4 × 10 5 Total cells / kg, approximately 5 × 10 5 Total cells / kg, approximately 6 × 10 5 Total cells / kg, approximately 7 × 10 5 Total cells / kg, approximately 8 × 10 5 Total cells / kg, approximately 9 × 10 5 Total cells / kg, approximately 1 x 10 6 Total cells / kg, approximately 2 x 10 6 Total cells / kg, approximately 3 × 10 6 Total cells / kg, approximately 4 × 10 6 Total cells / kg, approximately 5 × 10 6 Total cells / kg, approximately 6 × 10 6 Total cells / kg, approximately 7 × 10 6 Total cells / kg, approximately 8 × 10 6 Total cells / kg, or approximately 9 x 10 6 A dose of total cells / kg can be administered. In some embodiments, the dose can be measured by the number of virus-reactive cells instead of the total amount. For example, an effective dose can be about 1×10 5 Virus-reactive cells / kg ~ approx. 3 x 10 6 It can be about 1×10 virus-reactive cells / kg. 5 Virus-reactive cells / kg, approximately 2 × 10 5 Virus-reactive cells / kg, approximately 3 × 10 5 Virus-reactive cells / kg, approximately 4 × 10 5 Virus-reactive cells / kg, approximately 5 × 10 5 Virus-reactive cells / kg, approximately 6 × 10 5 Virus-reactive cells / kg, approximately 7 × 10 5 Virus-reactive cells / kg, approximately 8 × 10 5 Virus-reactive cells / kg, approximately 9 × 10 5 Virus-reactive cells / kg, approximately 1 × 10 6 Virus-reactive cells / kg, approximately 2 × 10 6 Virus-reactive cells / kg, approximately 3 × 10 6 Virus-reactive cells / kg, approximately 4 × 10 6Virus-reactive cells / kg, approximately 5 × 10 6 Virus-reactive cells / kg, approximately 6 × 10 6 Virus-reactive cells / kg, approximately 7 × 10 6 Virus-reactive cells / kg, approximately 8 × 10 6 Virus-reactive cells / kg, or approximately 9 × 10 6 A dose of virus reactive cells / kg may be administered. In some embodiments 、 Effective doses can be based on actual body weight. In certain embodiments where actual body weight is higher than ideal body weight, doses can be based on corrected body weight (ideal body weight + 40% of the difference between actual body weight and ideal body weight). Ideal body weight for height can be calculated from the formula of BJ Devine (1974): Male: 50.0 kg + 2.3 kg per inch above 5 feet, Female: 45.5 kg + 2.3 kg per inch above 5 feet.

[0082] An effective amount of a pharmaceutical composition comprising peptide-specific CTLs can be administered to an individual in need thereof, for example, an individual suffering from a viral infection, suffering from a viral-like disease, experiencing viral-like symptoms, or at risk of infection with an emerging viral strain. An effective amount is an amount sufficient to achieve a desired therapeutic or prophylactic effect, for example, an amount sufficient to reduce viral infection, viral-like disease, or viral-like symptoms, shorten the duration of illness, reduce viral titer, reduce the number of days an infected individual experiences viral-like symptoms, reduce the number of subjects who develop viral-associated cytokine release syndrome, and / or reduce the incidence or rate of viral infection. A skilled clinician can determine the appropriate dose and, if necessary, anti-drug, based on, for example, the age, susceptibility, resistance, and overall health of the individual. Peptide-specific CTLs can be administered in a single dose or multiple doses as indicated.

[0083] Intravenous delivery of CTLs (e.g., infusion via a peripheral, central, or midline) can take less than 10 minutes, in some embodiments, the time to infuse CTLs can be about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute.

[0084] In some embodiments, the method can include administering an effective amount of the pharmaceutical composition to an individual suspected of, confirmed to have, or at risk for a viral infection. The method can include administering an effective amount of the pharmaceutical composition to an individual suffering from a viral-like disease.

[0085] The pharmaceutical composition may be intended for administration into the blood of a subject and may be administered in any suitable form, such as intravenously.

[0086] In some embodiments, the method can include administering an effective amount of a pharmaceutical composition of the present invention to an individual suspected of having or at risk of having a viral infection. For example, in some embodiments, the individual can be suspected of having COVID-19 and / or can have one or more symptoms of COVID-19. Symptoms of COVID-19 are well known and include fever, cough, and shortness of breath. Other symptoms of COVID-19 include difficulty breathing, persistent pain or pressure in the chest, confusion, impaired consciousness, and blue lips or face.

[0087] In some embodiments, the method may be for treating an infectious disease and comprises administering to an individual in need of treatment an effective amount of a pharmaceutical composition of the invention. In other embodiments, the method may be for preventing an infection and comprises administering to an individual at risk of infection with an emerging viral strain an effective amount of a pharmaceutical composition of the invention. In other embodiments, the method may be for reducing the spread of an infection and comprises administering to an individual infected with or at risk of infection with an emerging viral strain an effective amount of a pharmaceutical composition as described herein.

[0088] The appropriate dosing interval to provide the desired therapeutic effect can be determined based on the severity of the disease (e.g., infection), the overall health of the subject, the subject's tolerance to the pharmaceutical composition, and other considerations. Based on these and other considerations, the clinician can determine the appropriate dosing interval. Generally, the pharmaceutical composition can be administered once, but can also be administered every 1-4 days or once a week, if necessary.

[0089] After administration, the success of the treatment can be determined by testing the subject's blood and / or nasal or nasopharyngeal swab specimens for viral diagnosis, CTL persistence, endogenous CTL formation, and antibody responses to emerging viral strains. Response to treatment can be tested, for example, at about 4 days, about 7 days, about 14 days, about 28 days, about 2 months, about 3 months, and about 6 months after infusion.

[0090] In some embodiments, the virus can be COVID-19. In some further embodiments, the emerging strain of COVID-19 can be Delta or Omicron BA.2.75.

[0091] Pharmaceutical Compositions The pharmaceutical composition can include peptide-specific CTLs prepared according to the methods disclosed herein.

[0092] The pharmaceutical composition can be formulated for intravenous delivery to an individual in need thereof, for example, by infusion via a peripheral IV, central line, or midline catheter. The pharmaceutical composition can also include one or more carriers or excipients suitable for delivery of the cryopreserved CTLs, such as DMSO.

[0093] In some embodiments, the pharmaceutical composition comprises specific CTLs sensitized to peptides that bind to specific HLA-A1 alleles.

[0094] In some embodiments, the pharmaceutical composition comprises specific CTLs sensitized to peptides that bind to specific HLA-A2 alleles.

[0095] In some embodiments, the pharmaceutical composition comprises specific CTLs sensitized to peptides that bind to specific HLA-B7 alleles.

[0096] In some embodiments, the pharmaceutical composition comprises specific CTLs sensitized to peptides that bind to specific HLA-B40 alleles.

[0097] In some embodiments, the pharmaceutical composition comprises specific CTLs sensitized to peptides that bind to specific HLA-Cw7 alleles.

[0098] In some embodiments, the pharmaceutical composition comprises specific CTLs sensitized to one or more peptides that bind to any one or combination of HLA-A1, -A2, -B7, -B40, and -Cw7 alleles, hi another embodiment, the pharmaceutical composition comprises specific CTLs sensitized to a combination of peptides that bind to a combination of alleles (e.g., a half dose of -A2 CTLs combined with a half dose of -B7 CTLs).

[0099] In some embodiments, the pharmaceutical composition comprises CTLs cryopreserved in DMSO, RPMI-1640, albumin, or a combination thereof.

[0100] Optionally, the pharmaceutical compositions described herein can also include one or more additional antiviral agents (such as remdesivir).

[0101] The pharmaceutical composition may be in any form suitable for administration by the desired route, such as intravenous administration.

[0102] In some embodiments, the virus of interest for preparation of peptide-specific CTLs is COVID-19 and the emerged strain can be Delta or Omicron BA.2.75.

[0103] equivalent Other suitable modifications and variations of the methods of the present invention described herein will be apparent and may be made using appropriate equivalents without departing from the scope of the present disclosure or embodiments, as will be readily apparent to those skilled in the art. Although certain compositions and methods have been described in detail above, they will be more clearly understood by reference to the following examples, which are presented for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0104] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments can be practiced in light of the general description provided herein.

[0105] Example 1. Loss of immunodominant HLA-A*01:01-restricted epitopes on CD8+ cytotoxic T lymphocytes (CTLs) in the delta variant of COVID-19: An example of immune evasion and implications for immunological treatment TVGN-489 is a clinical grade product consisting of highly enriched and highly potent CD8+ CTLs that recognize peptides derived from COVID-19 gene / ORF products in an HLA-restricted manner. CTLs were generated from apheresis products from individuals who had recovered from COVID-19 infection. Lymphocytes were sequentially primed and selected using APCs from these donors pulsed with a small number of peptides encoded by the COVID-19 genome predicted or demonstrated to bind specific HLA class I alleles. The resulting product was typically >95% CD3+ / CD8+, showed >60% positivity by tetramer staining, and exhibited potent cytolytic activity with >60% lysis of peptide-pulsed targets at an effector-to-target ratio of typically 3:1 (see Figure 1A and Figure 1B).

[0106] Given the immunological pressure to lose dominant target epitopes, we assessed whether peptides derived from the genomic sequences of early SARS-CoV-2 strains, which were successfully used to generate CTLs from donors infected with these early strains, are still present in the more recently evolved delta variants.

[0107] Seven peptides were used to generate CTL products restricted by HLA-A*02:01, the most common allele worldwide. These peptides were derived from the spike (S) and nucleocapsid (N) proteins, as well as ORF3a and ORF1ab. The contribution of the seven peptides to overall cytotoxicity and tetramer staining ranged from 2% to 18%, with no apparent immunodominance by one of these peptides (Tables 1 and 2). Although these sequences were identified in early virus strains, they continued to be present in 97.5 to 100% of the more than 120 delta variant sequences present in the NIH database.

[0108] A tetramer assay was used to quantitate viral peptide-specific CD8+ T cells within the T cell population (in this example, CD8+ T cells primed against the pre-delta strain of COVID-19).

[0109] Functional analysis of virus-specific CTLs was measured according to CTL cytotoxicity against viral peptide-pulsed targets. Effector cells were pulsed with 2 μg / mL of viral peptide and incubated at 37 °C for the appropriate time. After incubation with viral peptide, the medium was aspirated, effector cells were rinsed with warm PBS and replaced with fresh complete medium. CTLs were titrated into wells seeded with effector cells at ratios of 30:1, 10:1, 3:1, and 1:1 (non-viral peptide-pulsed effector cell condition was kept as control). Cytotoxicity was measured according to the release of a radioactive tracer (51Cr) from cells that underwent lysis (compared to a control in which all cells were chemically lysed). CTL-mediated cytotoxicity was usually observed to be greater than 80% at E:T ratios of 30:1 to 3:1. Cytotoxicity was approximately 60% at an effector-to-target ratio of 1:1.

[0110] For HLA-A*01:01, eight peptides derived from the matrix (M) protein, as well as ORF1ab and ORF3a, were utilized to generate CTLs. Seven of the eight peptides showed binding similar to that seen with the HLA-A*02:01 peptides (1%-18%) (Table 3). However, in contrast to HLA-A*02:01, an immunodominant peptide (TTDPSFLGRY, ORF1ab 1637-1646, SEQ ID NO:1) was noted that was responsible for half of the observed tetramer binding (Table 3, peptide 3). This region of ORF1ab was mutated in the delta variant, resulting in the loss of this immunodominant epitope from nearly 93% of the delta genome sequences in the NIH database. All remaining subdominant peptides were conserved in 100% of the sequences.

[0111] Similar results for peptide 3 were seen with the omicron mutant BA.2.75 (data not shown).

[0112] Given the increasing number of Delta cases, it becomes essential to remove this peptide from the HLA-A*01:01 peptide pool used to stimulate SARS-CoV-2-specific CD8+ CTLs to avoid promoting the proliferation of cells that recognize early strains of the virus but not the Delta variant. The remaining CTLs generated in the absence of TTDPSFLGRY (SEQ ID NO: 1) should be able to eradicate Delta as well as early prototype strains of COVID-19.

[0113] A similar prediction holds for the efficacy of CTL generated in the absence of TTDPSFLGRY (SEQ ID NO:1) against the omicron mutant BA.2.75.

[0114] In a virus with a high frequency of mutations, such as SARS-CoV-2, the loss of immunodominant epitopes is not surprising. This provides an example of immune evasion similar to that described for delta variants in the case of HLA-A24. These data are consistent with the hypothesis that immunodominant epitopes are preferentially eliminated as the virus continues to evolve. They further indicate the need to monitor viral sequences and tailor CTL production to ensure that we can continue to recognize and effectively treat newly emerging variants of COVID-19. [Table 1] [Table 2] [Table 3]

[0115] Example 2. Dilution of immunodominant HLA-A*01:01 restricted epitopes The composition containing the eight HLA-A*01:01 peptides used in Example 1 was modified by excluding peptide 8. Peptide 8 had negligible immunogenicity and was essentially the same as background, as determined by tetramer staining assay results. Based on Example 1, peptide 3 was identified as the immunodominant peptide.

[0116] Six samples were prepared, each containing the same concentration of the six peptides, i.e., peptides 1-2 and 4-7, and various concentrations of peptide 3 expressed as a percentage of the peptide 3 concentration (2 μg / ml) in the composition of Example 1. Thus, the sample containing 0% peptide 3 contained six peptides, and all samples containing 6.25% to 100% peptide 3 contained seven peptides.

[0117] As shown in Table 4, the sample containing 0% peptide 3 yielded 64.1% tetramer staining. The peptide 3 tetramer staining value of 0.3% reflected background staining and was consistent with the sample containing 0% peptide 3.

[0118] At 100% peptide 3 (normal concentration, approximately 2 μg / ml), the total tetramer staining was nearly the same as in the 0% sample (66.8%-64.1%), but this time peptide 3 accounted for 25.7%. The other peptides were suppressed so that the total remained similar.

[0119] Tetramer staining ranged from 74.3% to 83.1% across four 50% dilutions of peptide 3. The contribution of peptide 3 to total tetramer staining generally decreased as the proportion of peptide 3 in the sample decreased.

[0120] In conclusion, the tetramer staining results shown in Table 4 demonstrate that it is possible to reduce the proportion of immunodominant peptides in a peptide composition while maintaining a desirable level of potency of the peptide composition. [Table 4]

Claims

1. A method for preparing peptide-specific cytotoxic T cells (CTLs) against an emerging virus strain, comprising: a. providing an initial peptide composition specific to an earlier strain of the virus to which earlier CTLs were primed, wherein the earlier CTLs have reduced efficacy against the emerging virus strain compared to their efficacy against earlier strains of the virus; b. Identifying immunodominant peptides in the initial peptide composition for the previous strain of the virus; c. reducing the proportion of said immunodominant peptides in said initial peptide composition to obtain an immunodominant peptide-diluted peptide composition; d. sensitizing mononuclear cells with said immunodominant peptide diluted peptide composition, thereby resulting in the proliferation of peptide-specific CTL against said emerged viral strain.

2. 2. The method of claim 1, wherein the proportion of said immunodominant peptides in said immunodominant peptide-diluted peptide composition is at least 30% or less than the proportion of said immunodominant peptides in said initial peptide composition.

3. The method of claim 1 , wherein the immunodominant peptide is essentially absent from the immunodominant peptide-diluted peptide composition.

4. 2. The method of claim 1, wherein the virus is SARS-CoV-2 (COVID-19).

5. 5. The method of claim 4, wherein the emerged strain is Delta or Omicron BA.2.

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6. 10. The method of claim 1, wherein the initial peptide composition comprises one or more peptides restricted to an HLA-A1 allele.

7. 10. The method of claim 1, wherein the initial peptide composition comprises one or more peptides restricted to an HLA-A2 allele.

8. 10. The method of claim 1, wherein the initial peptide composition comprises one or more peptides restricted to an HLA-B7 allele.

9. 10. The method of claim 1, wherein the initial peptide composition comprises one or more peptides restricted to an HLA-B40 allele.

10. The method of claim 1, wherein the initial peptide composition comprises one or more peptides restricted to the HLA-Cw7 allele.

11. The method of claim 1, wherein the initial peptide composition comprises a combination of peptides that bind to any one or more of HLA-A1, -A2, -B7, -B40, and -Cw7 alleles.

12. A composition for treating an emerging viral strain in a subject in need of treatment, said composition comprising peptide-specific CTLs prepared according to the method of any one of claims 1 to 11.

13. The composition according to claim 12, wherein the virus for which the peptide-specific CTL is prepared is COVID-19.

14. 14. The composition of claim 13, wherein the emerged strain is Delta or Omicron BA.2.

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15. The composition described in claim 12, characterized in that it is used in combination with one or more premedications before administering the composition.

16. The composition described in claim 12, characterized in that it is used in combination with one or more antiviral preparations before, simultaneously with, or after administration of the composition.

17. A pharmaceutical composition comprising peptide-specific CTLs prepared according to the method of any one of claims 1 to 11.

18. The pharmaceutical composition according to claim 17, wherein the virus for which the peptide-specific CTL is prepared is COVID-19.

19. 19. The pharmaceutical composition of claim 18, wherein the emerged strain is Delta or Omicron BA.2.

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20. 18. The pharmaceutical composition of claim 17, wherein the proportion of the immunodominant peptides in the immunodominant peptide-diluted peptide composition is at least 30% or less than the proportion of the immunodominant peptides in the initial peptide composition.

21. 21. The pharmaceutical composition of claim 20, wherein the immunodominant peptide is essentially absent from the immunodominant peptide-diluted peptide composition.

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