DE102015106731A1 - Peptides for cancer immunotherapy - Google Patents

Abstract

The present invention relates to immunogenic peptides or nucleic acids encoding them, as well as their use in cancer immunotherapy, in particular in connection with B-cell lymphomas, and in particular in B-cell lymphoma patients with somatic L265P MyD88 Driver mutation.

Description

The present invention relates to immunogenic peptides for cancer immunotherapy, in particular immunogenic peptides derived from protein MyD88 carrying the somatic L265P mutation.

Cancer is the second leading cause of death in Germany after cardiovascular disease. Not all cancers are fatal if timely therapy is started or low-grade (slow-growing) cancer first occurs at such high age that the patient dies of another cause of death.

In the healthy organism, the proliferation of cells that make up the various tissues of the organs, and cell death in a species-specific balanced balance. In cancer, this balance is disturbed: cancer cells grow unhindered because inhibitory signals due to mutations or defects in genes are not recognized or performed, or mutations or defects lead to a direct activation of cell growth.

Tumors are u.a. classified according to the type of degenerated tissue. In addition to the carcinomas, which make up the majority of all cancers, the hematological neoplasms of the blood and the blood-forming organs, which can be subdivided into leukemias and lymphomas, also called "lymphoid cancer", form a large group. In turn, this can be subclassified into Hodgkin's Lymphoma and Non-Hodgkin's Lymphoma (NHL), with the latter subdivided into a B (about 80 percent of all NHL) and a T-line (20 percent), depending on whether the NHL from B lymphatic or T lymphoid cells.

The classic treatments for cancer include surgical resection, chemotherapy and radiotherapy, often using two or even all three forms of therapy simultaneously in a cancer patient. Disadvantages of the latter two methods are, in particular, the cytotoxic side effects and the fact that conventional therapies do not have a tumor-specific effect only on cancer cells, but also impair healthy body cells.

For years, therefore, research into new therapeutic methods that have the highest possible selective effect against cancer cells, with here now the cancer immunotherapy has proven to be a promising approach. A distinction is made between an active and a passive vaccination. In active immunization, the patient is given cancer vaccines that are supposed to trigger an immune response in his immune system. The immune response should ideally lead to the death of the tumor cells or at least to a delayed tumor growth or senescence (a state of growth arrest). In contrast, in passive immunization, the patient receives antibodies or antibody fragments.

These should selectively bind to tumor cells and thus lead to their demise. Finally, in adoptive immunotherapy, leukocytes are removed from the patient, cultured ex vivo, and then injected back into the patient.

Particularly involved in the defense against tumors are CD8-positive cytotoxic T lymphocytes (CTL). For such an immune response to be elicited by cytotoxic T cells, foreign peptides of class I MHC (major histocompatibility complex) receptors, also referred to as human leukocyte antigens (HLA), must be presented to cell surfaces , In addition, CD4-positive helper T lymphocytes are also involved in tumor defense, since they can, after the recognition of peptides on class II MHC receptors by the release of different cytokines, support the immune response and trigger the senescence of the tumor cells.

The recognition of MHC I or II peptide complexes by T cells is accomplished by the binding of the multimeric T cell receptor (TCR), whereby the recognition of the MHC peptide surface by a heterodimer of the so-called α- and β-chain he follows. Each naive T cell has a binding specificity determined by the gene sequence of the α and β chains. In humans, the repertoire of different combinations of TCR is> 10 ¹² . While only a few naive (unactivated) T cells have the desired MHC peptide specificity prior to vaccination, binding of specific TCRs to an appropriate MHC-peptide complex in the context of costimulatory molecules and cytokines triggers strong proliferation of the specific T cells, which can ultimately lead to a protective CD4 and / or CD8 T cell response.

The binding of a peptide to an MHC-protein complex is dependent on the allele of the MHC-protein complex and on the amino acid sequence of the peptide. MHC class I-binding peptides are usually 8-10 amino acids in length and contain in their sequence two conserved amino acids (so-called anchor amino acids) that interact with their specific side chain with the corresponding binding pocket of the MHC-protein complex. MHC class II binding peptides are usually 15-25 Amino acids long and the exact binding rules are not fully understood.

For the immune system to develop an effective CTL or T helper response against a peptide, this peptide must not only be able to bind to the particular MHC class I or II molecules expressed by the tumor cells but it also needs to be recognized by the available T-cell specificities via TCR.

The identification of tumor-associated or tumor-specific MHC-peptide complexes (antigens) opens up the possibility of specifically activating the immune system of cancer patients against the tumor cells. However, finding genes that are overexpressed in tumor tissues or that are selectively expressed in such tissues does not provide accurate information for use of the antigens transcribed from these genes in immunotherapy. This is due to the fact that only individual epitopes of these antigens are suitable for such use, since only the epitopes of the antigens - not the entire antigen - by MHC-restricted presentation cause a T cell response. Therefore, it is important to select those peptides from overexpressed, selectively or mutated-expressed proteins presented with MHC molecules, which could provide targets for specific tumor recognition by cytotoxic or helper T lymphocytes.

The currently known peptide-based immunotherapies mainly use standardized "off-the-shelf" peptide vaccines or patient-individual, personalized peptide vaccines. The former approach is based on overexpressed in the tumor peptide antigens, which are presented cross-patient especially on malignant cells. These tumor-associated antigens are characterized by broader applicability, but potentially limited tumor specificity. The approach of personalized peptide vaccines, based on patient-specific tumor-associated and tumor-specific antigens, on the one hand potentially enables a higher tumor specificity and immunogenicity of the vaccine, but on the other hand usually prevents cross-patient application. These circumstances are due to the fact that such personalized vaccines are patient / tumor individual, i. consider different mutations and expression patterns of antigens for each patient.

Against this background, it is an object of the present invention to provide immunogenic peptides which are directly or indirectly (eg via the production of cellular agents for immunotherapy) both for a tumor-specific immunotherapy and for use in a plurality of different patients and for various Cancers are suitable.

According to the invention, this object is achieved by providing one or more immunogenic peptide / peptides having a length of 8 to 25 amino acids, which comprise at least 8 consecutive amino acids from the amino acid sequence with SEQ ID NO: 1 of the attached sequence listing, as well as those occurring only in cancer cells Amino acid No. 25 of SEQ ID NO: 1, wherein the immunogenic peptide has the ability to bind to a molecule of the human major histocompatibility complex (MHC) of class I or class II.

The amino acid sequence having SEQ ID NO: 1 represents a portion of the MyD88 protein carrying the L256P mutation. The inventors of the present application have been able to demonstrate in their own experiments the immunogenicity for peptides derived from this sequence and the mutation against the corresponding wild-type peptide sequences.

The MyD88 protein is an adapter for many toll-like receptors (TLRs) and interleukin-1 receptors (IL1Rs).

So-called "driver mutations" are cancer-associated mutations that are essential for the malignant transformation of cells, whereas "passenger mutations" are functionally less relevant. The driver mutations, which are much rarer, give the tumor clone a growth advantage over normal cells, as well as spreading the clone into other tissues.

The somatic L265P driver mutation in MyD88 is a relatively widespread mutation, the occurrence of which has been described in a variety of neoplasms derived from B lymphocytes, e.g. but not exclusively in chronic lymphocytic leukemia (CLL), diffuse large cell B-cell lymphoma, Waldenström macroglobulinemia.

The amino acid position "Leucine 265" refers to the Publication of Ngo et al, "Oncogenically active MYD88 mutations in human lymphoma", Nature, 470, 115-119, 2011 , However, in the MyD88 protein reference sequence listed under UniProt ID Q99836 - MYD88_HUMAN, the mutated leucine is found at position 252. Since despite this deviation in the literature cited since always leucine 265 is mentioned, the following nomenclature is used as in Ngo et al., Supra, which in the sequence context with 1A covers. In the in 1A this position carries the number 25.

The broad occurrence of the well-defined L265P mutation in various neoplasms and different patients results in a broad scope of the newly identified immunogenic peptides: Since the immune response is mutagenic and thus tumor-specific, these are peptides that bind to a molecule of the human major histocompatibility Compound (MHC) Class I or Class II binding, therapeutically applicable in all cancer patients with somatic L265P MyD88 Driver mutation, and particularly in positivity for the class I HLA B * 07, the HLA B * 15 or the B * 35 allele , and more particularly in mutated B-cell lymphoma, and more particularly B * 07, B * 35 or B * 15 patients with MyD88 mutant B-cell lymphoma.

The immunogenic peptides presented here for the first time provide virtually ideal tumor antigens (fragments of proteins produced in tumors) since they: (i) are expressed only by cancer cells on the cell membrane and are absent in healthy cells (tumor specific antigens (TSA)) , By contrast, the tumor antigens hitherto known for B-cell lymphomas and leukemias are only tumor-associated and accordingly are also expressed by healthy cells. Therefore, tumor-associated antigens are only conditionally applicable in overexpression for immunotherapy; (ii) derived from a clonal driver mutation, and thus the vast majority of tumor cells carry this mutation that present mutation-derived peptides on MHC molecules on the cell surface and are recognized and controlled by an induced immune response; (iii) by the limited number of peptides containing the L265P mutation are principally applicable in a plurality of different patients of the same class I or class II HLA type.

The tumor antigens provided herein have high immunogenicity and thus provide an outstanding means of immunization. For example, naive CD8 ⁺ T cells can be activated into effector cells (CTL) by targeted priming (in vivo, in vitro or ex vivo) with the immunogenic peptides according to the invention. Priming refers to the peptide-specific stimulation of naive (previously unactivated) T cells, which stimulates peptide-specific T cells to differentiate into effector cells and to proliferate.

Upon activation, the CTLs release perforins and granzymes which drive the MHC-I-bearing antigen-presenting cell into programmed cell death. In addition, activated CTL also produce and secrete proteins such as interferon-γ, which promotes the production of MHC-I proteins in neighboring cells. This in turn leads to a stronger presentation of the peptides.

Helper T lymphocytes may release tumor necrosis factor (TNF) and interferon-gamma (IFNγ) after peptide-specific activation, which may induce senescence of tumor cells.

As used herein, a "peptide" refers to a chain of amino acids linked together by peptide bonds, Generally, a longer peptide is termed a "polypeptide." A "protein" is then composed of one or more polypeptides.

As used herein, as well as generally known in the art, a tumor-associated antigen polypeptide or "TAA polypeptide" is understood to be a full-length non-fragmented polypeptide of a tumor-associated antigen, while a tumor-associated antigen peptide or a TAA peptide is a fragment As mentioned above, TAA peptides are not tumor-specific peptides, but are peptides that are more highly expressed in a tumor than in other tissues, for example.

In contrast, tumor-specific polypeptides or tumor-specific antigens (TSA) are proteins that are quite specific for tumor cells. Similarly, a tumor-specific antigenic peptide is a fragment of such a TSA polypeptide. The immunogenic peptides described herein are tumor-specific peptides.

These peptides may be of any length up to just below the full length of a TSA polypeptide, however, the immunogenic peptides for use in the invention have a relatively short length, such as eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acids.

Peptides containing amino acid substitutions can also be used as immunogenic peptides in the context of the present invention. For example, an immunogenic peptide may include a region of at least nine amino acids, six or more of which are identical to the amino acids within a nine amino acid strand in the given TSA, here SEQ ID NO: 1. Preferably, at least seven, more preferably at least eight, and most preferably all nine of the amino acids in an immunogenic peptide-nine amino acid region are identical to a nine amino acid region in the TSA.

Also included are peptides which have post-translational modifications, such as, for example, phosphorylations, in particular of the amino acids serine, threonine or tyrosine, and, for example, Hydroxylations, in particular of lysine residues, glycosylations, acetylations and methylations, in particular on lysines, succinations on cysteines, sulfations, etc.

As discussed further below, it is preferred that the immunogenic peptides of the invention include regions that bind to major histocompatibility complex (MHC) class I or class II antigens.

An immunogenic peptide according to the present invention may be linked to amino acid sequences that are not naturally occurring in the TSA described with SEQ ID NO: 1. In addition, an immunogenic peptide may be attached to the surface of a cell or to a molecule or macromolecule (eg, a histocompatibility antigen), or an immunogenic peptide may be immunohistochemically or adjuvanted to those skilled in the art, for example, limpet hemocyanin ( KLH) are conjugated for the purpose of eliciting a TSA-specific immune response.

By "nucleic acid molecule" or "nucleic acid" herein is meant a DNA or RNA (e.g., mRNA) molecule. This can, for example, code for an immunogenic peptide or protein as defined above, in which case the vaccine is not bound to a specific HLA type, since the molecule encoded by the nucleic acid can be processed and presented in vivo in accordance with HLA, and thus can be used for a wider range of patients. A nucleic acid or nucleic acid sequences according to the invention can furthermore code for α and / or β chains of T cell receptors which can be used for the transduction or transfection of T cells for the purpose of transmitting a new specificity (see also below).

"Isolated" in this case means ex vivo isolated from a sample by conventional methods, or produced recombinantly or synthetically and present in isolated form.

By "vaccine" or vaccine is meant herein the administration of an immunogenic preparation which includes one or more immunogenic peptides according to the present invention, nucleic acid molecules, or a cell presenting the immunogenic peptide, or mixtures of these. The vaccine is administered to a person having a tumor, a history of a tumor or tumors, or likely to develop a tumor, or to a subject in which peptide-specific immune cells (such as CTLs) are transmitted a patient to be generated performed. The vaccine stimulates a specific immune response in the person. In addition, the vaccine may provide prophylaxis against the development of new tumors.

Accordingly, a "vaccine" as used herein is an immunogenic composition that can be administered in the vaccination method described above Thus, for example, a vaccine will include one or more immunogenic peptides according to the present invention, nucleic acids, or cells presenting or responding to the immunogenic peptides Alternatively, a vaccine composition may include adjuvants that stimulate an immune response to the vaccine antigen or agent co-administered therewith, such as, but not limited to, immunostimulatory adjuvants such as imiquimod or GM-CSF; Inhibiting the immune response as a whole cellularly or molecularly, such as, but not limited to, anti-regulatory T cell directed cyclophosphamide or immunocheckpoint inhibitors such as but not limited to ipilimumab.

A "sample" herein means a tumor or tissue biopsy, lymph node biopsy, bone marrow, cells, blood, serum, urine, stool, sputum, saliva, cerebrospinal fluid, or another sample obtained from a patient to analyze the presence of CTLs or helper T lymphocytes specific for the immunogenic peptides, the level of antibodies specific to the immunogenic peptides, or the level of any other indicator of immune response (eg, cytokine level ) in the patient from whom the sample was taken by methods known in the art.

For example, an ELISA can be used to measure the levels of specific antibodies, and ELISPOT or intracellular cytokine staining can be used to measure cytokine levels. Similarly, Cr ⁵¹ release (T cell cytotoxicity) assays and assays that test the binding of CTLs or T helper lymphocytes to tetrameric peptide / MHC complexes as described herein can be used to assess levels of to measure specific CTLs or T helper lymphocytes.

According to one aspect of the present invention, the immunogenic peptide has an amino acid sequence having one of SEQ ID Nos. 2 to 93, and according to another embodiment, SEQ ID Nos. 17, 20, 23 or 27.

In a preferred embodiment, the peptide is capable of a T helper cell response stimulate or produce a cytotoxic T-lymphocyte (CTL) response.

According to one aspect of the present invention, the immunogenic peptide is 8, 9, 10, 11 or 12, or 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 25 amino acids, preferably, 8 , 9, 10, 11, 12, 13, 14, or 15 amino acids in length, and more preferably 8, 9, 10, 11 or 12 amino acids in length.

As mentioned above, therefore, the immunogenic peptides of the invention can be used in cancer therapy, e.g. to induce an immune response against tumor cells, particularly B cell lymphomas, expressing the corresponding antigens from which the peptides are derived.

Such an immune response e.g. in the form of induction of CTL, on the one hand, can be achieved in vivo. For this purpose, a patient who suffers from a tumor disease associated with the tumor-specific antigen, the peptide, eg. Also in the form of a pharmaceutical composition administered.

On the other hand, a T cell response to a tumor that expresses the antigens from which the peptides are derived may also be triggered ex vivo. For this, naive T-progenitor cells are incubated together with antigen-presenting cells and the peptides, or uses so-called "artificial antigen-presenting cells" (streptavidin beads, on which recombinant MHC protein loaded with a peptide according to the invention, as well as anti-CD28 antibodies to "Prime" of T cells are anchored in vitro). Subsequently, the peptide-specific CTL or T helper lymphocytes stimulated thereby are cultivated and expanded, and the activated CTL or T helper lymphocytes are administered to the patient.

Furthermore, it is possible to load APCs (antigen-presenting cells) with the peptides ex vivo and to administer these loaded APCs to the patient, who in the tumor tissue expresses the antigen from which the peptide is derived. The APCs may then in turn present the peptide to the T lymphocytes in vivo and activate them.

However, the immunogenic peptides according to the invention can also be used as diagnostic reagents.

Thus it can be found with the immunogenic peptides whether in a T-cell population specifically against a peptide directed CTLs or T helper lymphocytes are present or induced by a therapy.

In addition, with the immunogenic peptides, the increase of precursor T cells can be tested, which have a reactivity against the defined peptide.

Further, the immunogenic peptide can be used as a marker to track the disease process of a tumor expressing the antigen from which the immunogenic peptide is derived.

For application of tumor-specific antigens in a tumor vaccine several application forms are possible in principle. So described Tighe et al., 1998, "Gene vaccination: plasmid DNA is more than just a blueprint", Immunol. Today 19 (2): 89-97 in that the antigen can be administered either as a recombinant protein with suitable adjuvants or carrier systems or as the cDNA coding for the antigen in plasmid vectors. RNA-based tumor vaccines have also been described (see Weide et al., "Direct injection of protamine-protected mRNA: results of a phase 1/2 vaccination trial in metastatic melanoma patients", Journal of Immunotherapy 32.5 (2009): 498-507 ). In the case of cDNA or RNA, the antigen in the patient's body must be processed and presented by antigen presenting cells (APCs) to elicit an immune response.

Melief et al., 1996, Peptide-based cancer vaccines, Curr. Opin. Immunol. 8: 651-657 are among the first to demonstrate the use of synthetic peptides as vaccines; By way of example, the publication of Arens et al., 2013, Prospects of combinatorial synthetic peptide vaccine-based immunotherapy against cancer, Seminars in Immunology, 25: 182-190 , whose contents, uses and possibilities are expressly referred to in connection with a peptide-based immunotherapy.

The peptide can be used in a preferred embodiment with the addition of adjuvants, or even in isolation.

Various adjuvants are known in the art in the meantime, and reference is made to the publications mentioned above in this respect. As an adjuvant, for example, the granulocyte-macrophage-colony stimulating factor (GM-CSF) can be used. Other examples of such adjuvants are aluminum hydroxide, emulsions of mineral oils such as Freund's adjuvant, saponins or silicon compounds, imiquimod or monophosphoryl lipid A. Use with adjuvants has the advantage that the immune response elicited by the peptide can be enhanced and / or that the peptide is stabilized.

In another preferred embodiment, the immunogenic peptide is bound to an antigen presenting cell or the so-called "artificial antigen presenting cells".

This measure has the advantage that the immunogenic peptides can be presented to the immune system, in particular the cytotoxic T lymphocytes (CTL). This allows the CTL to recognize and specifically kill the tumor cells. As antigen-presenting cells, for example, dendritic cells, monocytes or B-lymphocytes are suitable for such use.

For example, dendritic cells (DCs) are capable of accepting and processing antigens and presenting them on their cell surface, activating antigen-specific T cells and thereby stimulating maturation / differentiation and division. The T cells are thereby enabled to recognize and eliminate the antigen-bearing tumor cells. Likewise, dendritic cells can activate natural killer cells (= NK cells) to attack tumor cells. Accordingly, the dendritic cells can be provided with different tumor antigens to treat different types of tumors ("DC vaccination").

The antigen-presenting cells carrying the peptide can either be used directly or activated with the heat-shock protein gp96 prior to use, for example.

In another preferred embodiment, the peptides are used to label leukocytes, especially T lymphocytes.

This use is advantageous when the peptides are to be used to find out whether CTLs or helper T cells directed against an immunogenic peptide are present in a T cell population.

Furthermore, the immunogenic peptide can be used as a marker for evaluating a course of therapy in a cancer, in particular a B-cell lymphoma.

The immunogenic peptide according to the invention can also be used for monitoring the therapy in other immunizations or therapies. Thus, the peptide is not only therapeutically but also diagnostically useful.

In another embodiment, the peptides are used to produce an antibody which can be prepared by methods well known in the art. "Antibody" in the following also includes all state-of-the-art "antibody" principles in the broadest sense based applications such as Fab fragments, single-chain variable fragments, scFv, single domain antibodies, V _H H, V _NAR or nanobodies, one. Thus, polyclonal antibodies can be obtained in a conventional manner by immunizing animals by injection of the peptides and subsequent purification of the immunoglobulin. Monoclonal antibodies can be prepared according to standard protocols, such as in Methods Enzymol. (1986), 121, "Hybridoma Technology and Monoclonal Antibodies" , described. In this case, the peptides can be used with or without complex with recombinant MHC I or MHC II of a specific HLA type for the preparation of a therapeutic or diagnostic antibody.

In a further embodiment, the peptides are used to produce T cells which are specific for the peptides of the invention in the context of MHC I or II molecules which can be prepared by methods well known in the art. These T cells can be isolated after vaccination of the patient and re-administered to the patient after amplification, or generated in vitro by stimulation of naive T cells of the patient and administered for cancer therapy.

In yet another embodiment, the peptides are used to produce T cells whose peptide-MHC-specific T cell receptor (TCR) clones and sequences α and / or β chains to generate nucleic acids, which in turn can be used to generate MHC-peptide-specific T cells from T cells of other specificity by transfer of these nucleic acids. This can be accomplished, for example, by transduction or transfection of autologous PBMC (peripheral blood mononuclear cells) or isolated T cells of the patient. These autologous T cells can be re-administered to the patient for cancer therapy.

This embodiment has the advantage over the use of peptide-specific T-cell populations that the often problematic diversity of MHC molecules between donor T-cells and the patient can be overcome. Advantageously, in this embodiment, instead of the whole T cells, the specificity of a characterized tumor antigen-specific T cell colon, thus the specific T cell receptor, can be introduced into the patient to be treated or into autologous T cells of the patient or into suitable donor cells. Thus, the difficulty in transferring fresh T cells can be overcome that they are usually active only limited in time.

The ability to transfer the reactivity and specificity of a T cell clone to secondary cells by transferring the sequences of the α and β chains of a T cell receptor has been generally demonstrated in the prior art. In this regard, for example, on Dembic et al., "Transfer of specificity by murine alpha and beta T-cell receptor genes", Nature, 1986, 320: 232-238 , and Morgan et al., Cancer regression in patients after transfer of genetically engineered lymphocytes, Science, 2006, 314: 126, 129 , the disclosures of which are made subject matter of the present invention with regard to the methods and techniques used in the present case.

For this so-called T cell receptor therapy, a T cell clone is first identified and isolated which has a high affinity for the immunogenic peptide, which can be done, for example, by isolating tumor-specific T cells from patients, followed by a subsequent in vitro multiplication and cloning. Subsequently, the α and / or β chain of the T cell receptor are identified and the genes coding for them are isolated by molecular cloning.

The nucleic acids encoding the α and / or β chain of the T cell receptor specific for the immunogenic peptide are then transduced, for example, into peripheral T cells by means of a retroviral or lentiviral vector (see, e.g. Schmitt et al., "T-cell receptor gene therapy for cancer", Hum Gene Ther, 2009,20: 1240-1248 ,

The invention also relates in a further aspect to a pharmaceutical composition containing one or more of the peptides.

This composition serves, for example, parenteral administration, for example, subcutaneously, intradermally or intramuscularly, or oral administration. The peptides are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. In addition, the composition may contain adjuvants, such as buffers, binders, diluents, etc. The peptides may also be administered together with immunostimulating substances, eg cytokines. In addition, the peptides can also be administered together with substances that negate a negative effect on the immune response (eg depletion of regulatory T cells or immuncheckpoint blockade). A comprehensive list of excipients, as they can be used in such a composition is, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients, 3rd ed., 2000, American Pharmaceutical Association and pharmaceutical press represented.

The agent can be used for the prevention, prophylaxis and / or therapy of tumor diseases.

The amount of the peptide present in the pharmaceutical composition is present in a therapeutically effective amount.

The immunogenic peptides bind according to a preferred embodiment of the HLA type B * 07, B * 35, B * 15. It goes without saying that the specifically listed HLA types are given by way of example only, and all other HLA types are also suitable.

In a further aspect, the present invention relates to nucleic acid molecules which comprise or consist of the sequence which is suitable for the immunogenic peptides according to the invention having the sequence ID no. 2 to 93 encode.

The nucleic acid molecules may be DNA or RNA molecules and also be used for the immunotherapy of cancers. In this case, the immunogenic peptide expressed by the nucleic acid molecule induces an immune response against tumor cells expressing the peptide.

According to the invention, the nucleic acid molecules can also be present in a vector.

Accordingly, the present invention also relates to a vaccine comprising an immunogenic peptide or a nucleic acid according to the invention. This vaccine can be administered to patients previously detected for the somatic L265P MyD88 Driver mutation. This triggers the formation of an immune response in these patients, which can specifically target the tumor.

Further, the present invention also relates to an isolated T cell specific for an immunogenic peptide of the invention, or produced by stimulating peripheral blood mononuclear cells (PBMCs) with an immunogenic peptide of the invention or a nucleic acid of the invention.

Furthermore, the present invention relates to the nucleic acid sequence of one of the α and / or β chain of a T cell receptor (TCR), which is specific for an immunogenic peptide according to the invention, for example, but not exclusively, for the production of peptide-specific T cells by transduction or Transfection can be used from PBMCs or isolated T cells of a donor. The nucleic acid sequence of such a TCR which is specific for an immunogenic peptide according to the invention, can be identified and generated by methods and methods known in the art. In this regard, reference is made to the comments above on the use of the peptides of the invention for the production of T cells whose peptide-MHC-specific TCR clones α and / or β chains and are sequenced to produce nucleic acids that can serve this purpose to generate MHC-peptide-specific T cells from T cells of a different specificity by transferring these nucleic acids.

Furthermore, the present invention also relates to isolated APCs (for example dendritic cells) which specifically present the immunogenic peptide of the invention after treatment with immunogenic peptide or a nucleic acid according to the invention on MHC I or MHC II, e.g. in the form of a so-called "DC vaccine".

• (a) ein erfindungsgemäßes immunogenes Peptid und einen pharmazeutisch akzeptablen Träger oder Hilfsstoff;
• (b) eine erfindungsgemäße Nukleinsäure und einen pharmazeutisch akzeptablen Träger oder Hilfsstoff; oder
• (c) zwei oder mehrere eines erfindungsgemäßen immunogenen Peptids, oder einer erfindungsgemäßen Nukleinsäure, zur gleichzeitigen, sequenziellen oder getrennten Verabreichung.

The present invention accordingly also relates to a pharmaceutical composition comprising:

• (a) an immunogenic peptide of the invention and a pharmaceutically acceptable carrier or excipient;
• (b) a nucleic acid of the invention and a pharmaceutically acceptable carrier or excipient; or
• (c) two or more of an immunogenic peptide of the invention, or a nucleic acid of the invention, for simultaneous, sequential or separate administration.

The present invention furthermore relates to an immunogenic peptide according to the invention, a nucleic acid according to the invention, a vaccine according to the invention, an isolated T cell according to the invention, a peptide-specific T cell receptor sequence-encoding nucleic acid according to the invention, an APC according to the invention or a pharmaceutical composition according to the invention for use in the Prophylaxis, therapy or treatment of B-cell lymphomas and leukemias.

In particular, the present invention relates to an immunogenic peptide according to the invention, a nucleic acid according to the invention, a vaccine according to the invention, an isolated T cell according to the invention, an APC according to the invention or a pharmaceutical composition according to the invention for use for the in vitro stimulation of T cells in adoptive immunotherapy of humans ,

The present invention therefore also relates to the use of the immunogenic peptide according to the invention for the production of a therapeutic or diagnostic antibody.

As stated above, the present invention also relates to an immunogenic peptide according to the invention, a nucleic acid according to the invention, a vaccine according to the invention, an isolated T cell according to the invention, an APC according to the invention or a pharmaceutical composition according to the invention for use in the prophylaxis, therapy or treatment of B cells. Cell lymphomas and leukemias, and particularly in patients with somatic L265P MyD88 Driver mutation, and in one embodiment in patients with B * 07, B * 35 or B * 15 HLA type.

In a preferred embodiment, the B-cell lymphomas and leukemias are selected from the group comprising: diffuse large B-cell lymphoma (DLBCL), mucosa-associated lymphoid tissue lymphoma (MALT), chronic lymphocytic leukemia (CLL), Waldenstrom's macroglobulinemia (WM), primary cutaneous large cell B-cell lymphoma (PCLBCL), primary central nervous B-cell lymphoma (PCNBL), extranodal marginal zone lymphoma in ocular adnex (OAEMZL), and splenic marginal zone lymphoma (SMZL), Burkitt's lymphoma (BL) , Monoclonal gammopathy of unclear significance (MGUS), intra-ocular B-cell lymphoma (IOL).

Patients suitable for therapy with the peptides of the present invention, the vaccine described herein, or other use in the present invention can be identified by simple PCR-based methods for the presence of the L265P MyD88 mutation and HLA typing For example, see Capaldi et al., Exp Mol Pathol 2014; 87 (1) 57-65 ; Xu et al., Blood 2013; 121 (11) 2051-8 , or Argentou et al., Leukemia 2013; 28 (2): 447-9 ,

Accordingly, the present invention also relates to a method of treatment in which patients with L265P MyD88 mutation are first identified and subsequently treated with the immunogenic peptides / vaccine / T cells / pharmaceutical composition (s) or in general according to the present invention become. According to one embodiment, in particular patients with L265P MyD88 mutation and with B * 07, B * 35, B * 15 HLA type are identified.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the respectively specified combination but also alone, without departing from the scope of the present invention.

Further features will become apparent from the following figures and the description. Show it:

1 the representation of the SEQ ID No. 1 (A), as well as a review of 92 exemplary immunogenic peptides or their amino acid sequences according to the invention with the SEQ ID Nos. 2 to 93, which are derived from the SEQ ID No. 1 according to the invention (B); the peptides with the SEQ ID Nos. 2 to 9 are 8-mers, the peptides with the SEQ ID Nos. 10 to 18 9-mers, the peptides with the SEQ ID Nos. 19 to 28 10-mers, the peptides with the SEQ ID Nos. 29 to 39 are 11-mers, the peptides having SEQ ID Nos. 40 to 51 are 12-mers, the peptides having SEQ ID Nos. 52 to 64 are 13-mers, the peptides have SEQ ID Nos. 65 to 78 14-mers, and the peptides having SEQ ID Nos. 79 to 93, 15-mers; the amino acid position 25 (proline) of SEQ ID No. 1 ( 1A ) corresponds to L265P in the sense of Ngo et al., Supra, and leucine 252 with reference to the reference UniProt reference Q99836 ; in the 1A and 1B the mutation to proline in the peptides according to the invention is in each case marked in bold;

2 various flow cytometry graphs demonstrating in vitro priming of naive CD8 ⁺ cells with the B * 07 restrictive peptide RPIPIKYKAM (SEQ ID NO: 27) in six healthy donors (A: donors 1 to 4, B: donors 5 and 6);

3 various flow cytometry graphs for detecting in vitro priming of naive CD8 ⁺ cells with the B * 07 restrictive peptide SPGAHQKRPI (SEQ ID NO: 20) in three healthy donors;

4 various flow cytometry graphs for detecting in vitro priming of naive CD8 ⁺ cells with the B * 15 restrictive peptide HQKRPIPIKY (SEQ ID NO: 23) in a healthy donor;

5 various flow cytometry graphs for detecting in vitro priming of naive CD8 ⁺ cells with the B * 07 restrictive peptide RPIPIKYKA (SEQ ID NO: 17) in a healthy donor; and

6 the results of detection of interferon-gamma production by CD8 ⁺ cells after stimulation with the peptide RPIPIKYKAM (SEQ ID NO: 27): A and C show IFN-γ production after stimulation with the mutant peptide in donor no. Figure 3, B and D show flow cytometry graphs showing identification of tetramer-positive CD8 ⁺ T cells after priming; E shows IFN-γ production after stimulation with the mutated peptide in donor No. 4, and F a flow cytometry graph showing the identification of the tetramer-positive CD8 ⁺ T cells after priming. Special mention should be made of the mutation-specific reaction since the CD8 ⁺ T cells react only to the mutation-derived peptide but not to the wild-type peptide with IFN-γ production.

7 Flow cytometric graphs demonstrating the detection of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) production by CD8 ⁺ T cells after stimulation with the L265P mutant peptide RPIPIKYKAM (SEQ ID NO: 27, left column of the graphs in FIG 7A and 7B ) in two donors (7A and 7B) against which they were primed; the right column of the graphs each represent the results of stimulation with the wild-type peptide RLIPIKYKAM (SEQ ID NO: 94) (unmutated).

material and methods

The Institute for Clinical and Experimental Transfusion Medicine at the University Hospital Tübingen transfers whole blood preserves of HLA-typed healthy blood donors anonymously to the Department of Immunology after informed consent and written consent of the donors. Here an in vitro priming with artificial antigen-presenting cells (aAPCs, see above) of the contained CD8 ⁺ T-cells took place. The processing and isolation of the contained peripheral mononuclear cells (PBMCs) is carried out by Ficoll density gradient centrifugation. The isolation of the cytotoxic CD8 ⁺ T cells takes place via MACS enrichment (magnetically activated cell sorting) by means of positive selection by CD8 microbeads. The aAPCs used consist of streptavidin-coated polystyrene particles loaded with biotinylated anti-CD28 antibody and the desired biotinylated HLA-peptide complexes. The latter are produced in-house after recombinant expression in bacteria by refolding with the desired peptide. Per each approach, one million CD8 ⁺ T cells are stimulated with the same amount of aAPCs every 7 days for a total period of 3-4 weeks. The expansion of activated T cells is also promoted by the addition of recombinant IL-2 every 2 days after aAPC stimulation. Subsequently, the successful expansion of specific T cells by HLA multimer staining is detected. Here, the same biotinylated HLA-peptide complexes are used, which were also used for the production of aAPCs. However, these are tetramerized with phycoerythrin-coupled streptavidin to HLA multimers. The latter can then be used together with other antibodies to identify tetramer-positive T cells in flow cytometry.

For the detection of the functionality and specific interferon-gamma (IFN-γ) production of peptide-specific T cells after stimulation with the Mutation-derived peptide is an ELISPOT (Enzyme Linked Immuno Spot Assay) performed. For this, the in vitro primed cells, which were identified as tetramer-positive, are seeded in a previously coated with IFN-γ antibodies plate and stimulated again with the respective peptide. After 24 h, the secondary staining of the secreted IFN-γ can then take place. Here, each IFN-γ-producing T cell is displayed as a spot. To confirm the mutation specificity, the same analyzes are also performed with the appropriate wild-type peptide.

Results

 For the identification of potential mutation-derived peptides of the MyD88 L265P mutation, 8 to 12mer peptides comprising the mutation at every possible position within the peptide were generated. The peptides were then predicted with the SYFPEITHI and NetMHC databases as potential ligands for various MHC class I alleles. Such peptides, which were predicted to be good ligands, were synthesized to perform experimental binding and immunogenicity testing.

1 Figure A shows in A a protein portion (SEQ ID NO: 1) from the L265P mutant MyD88 in which the prolactin mutated in comparison to the wild-type (leucine) is bold. "L265P" thus indicates the mutation of the wild-type leucine at amino acid position 265 of the wild-type protein MyD88 to proline. B shows an overview of 92 possible peptides which are prepared by means of the databases SYFPEITHI (FIG. http://www.syfpeithi.de/ ) and NetMHC ( http://www.cbs.dtu.dk/services/NetMHC/ ) were predicted as possible ligands for different MHC class I molecules, in which the mutant proline was also labeled in bold. In further experiments, the predicted peptides were examined for their immunogenicity

2 Figure 3 shows the graphical results of flow cytometry demonstrating in vitro priming of naive CD8 ⁺ T cells with the B * 07 restrictive peptide RPIPIKYKAM (SEQ ID NO: 27). The first column shows each a representative example of an approach that could be successfully primed in the naive CD8 ⁺ T cells for six out of six tested donors. The peptide-specific primed T cells are recognizable as tetramer-positive populations. In the middle column are the associated negative controls, right shows the appropriate ex vivo controls. For the negative controls, cells primed with another peptide were stained with the RPIPIKYKAM tetramer (SEQ ID NO: 27). In none of the donors could a significant number of tetramer-positive cells be detected in the negative controls. This confirms the exclusively specific binding of the tetramer. For the ex vivo controls, unprimed cells were stained with the RPIPIKYKAM tetramer (SEQ ID NO: 27). All four donors did not show any antigen-specific cells before priming.

3 Figure 3 shows the graphical results of flow cytometry demonstrating in vitro priming of naive CD8 ⁺ T cells with the B * 07 restrictive peptide SPGAHQKRPI (SEQ ID NO: 20). The first column shows for each of three donors tested a representative example of an approach in which naive CD8 ⁺ T cells could be successfully primed. The peptide-specific primed T cells are recognizable as tetramer-positive populations. In the middle column are the associated negative controls, right shows the appropriate ex vivo controls. None of the donors could detect a significant number of tetramer-positive cells in the negative controls or in the ex vivo controls.

4 Figure 3 shows the graphical results of flow cytometry demonstrating in vitro priming of naive CD8 ⁺ T cells with the B * 15 restrictive peptide HQKRPIPIKY (SEQ ID NO: 23). The first column shows for a donor tested a representative example of an approach in which naive CD8 ⁺ T cells could be successfully primed. The peptide-specific primed T cells are recognizable as tetramer-positive populations. In the middle column are the associated negative controls, right shows the appropriate ex vivo controls. In the donor, no significant number of tetramer-positive cells could be detected in the negative controls or in the ex vivo controls.

5 Figure 3 shows the graphical results of flow cytometry demonstrating in vitro priming of naive CD8 ⁺ T cells with the B * 07 restrictive peptide RPIPIKYKA (SEQ ID NO: 17). The first column shows the approach for one of four tested donors, in which naive CD8 ⁺ T cells could be successfully primed. In the middle column is the associated negative control, right shows the appropriate ex vivo control. The donor did not show any antigen-specific cells in the negative control and in the ex vivo control.

The following gating strategy was used in the flow cytometric analysis of tetramerpositiven CD8 ⁺ T cells after in vitro priming: Primed CD8 ⁺ T-cells were stained with the dye reactive aqua fluorescent dye, a monoclonal antibody against the human CD8 as well as a specific tetramer. For this purpose, the lymphocytes were first gated using the forward light scatter (FSC) and the side light scatter (SSC). Around To eliminate doublets, the forward light scatter area (FSC-A) was used against the forward light scatter height (FSC-H). Dead cells were excluded using the aqua fluorescent reactive dye. The CD8 ⁺ T cells with a specific T cell receptor for the antigen were identified with staining for CD8 and the specific tetramer (data not shown).

In 6 For example, data showing interferon-γ production by CD8 ⁺ T cells after stimulation with the L265P mutant peptide RPIPIKYKAM (SEQ ID NO: 27) against which they were primed but not after stimulation with the corresponding WT (unmutated ) Peptide RLIPIKYKAM (SEQ ID NO: 94). This experiment was performed in two donors. For donor # 3, three positive primed approaches were tested in the ELISpot. In two of three studies, IFN-γ production was detected after stimulation with the mutated peptide (see 5A , C). The flow cytometry graphs each show the identification of the tetramer-positive CD8 ⁺ T cells after priming (see 5B , D). For donor # 4, seven positive-primed approaches were tested in the ELISpot. In one approach, IFN-γ production after stimulation with the mutated peptide could be detected (see 5E ). The graph of flow cytometry shows the identification of tetramer-positive CD8 ⁺ T cells after priming (See 5F ). PHA (phytohemagglutinin) served as a positive control.

In further experiments, interferon gamma and tumor necrosis factor alpha production by CD8 ⁺ T cells after stimulation with a peptide according to the invention for the detection of the functionality of the primed T cells was investigated (see in 7 presented results).

 In addition to the ELISPOT, intracellular cytokine staining (ICS) and staining of the degranulation marker CD107a were also performed. For this, the primed cells were stained for inhibition of cytokine release by blocking the Golgi apparatus with fluorescence-coupled antibodies to CD8, CD107a and after permeabilization with antibodies to interferon-gamma and tumor necrosis factor-alpha and analyzed by flow cytometry.

In 7 The results of these experiments, which show interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) production by CD8 ⁺ T cells after stimulation with the L265P mutant peptide RPIPIKYKAM (SEQ ID NO: 27) in two donors show against which they were primed (in 7A and 7B left column), but not after stimulation with the corresponding WT (unmutated) peptide RLIPIKYKAM (SEQ ID NO: 94) (right column in each case) 7A and 7B ). Furthermore, for a donor ( 7A ) an upregulation of the degranulation marker CD107a after stimulation with the L265P mutant peptide RPIPIKYKAM (SEQ ID NO: 27) is shown ( 7A bottom left), but not after stimulation with the corresponding WT (unmutated) peptide RLIPIKYKAM (SEQ ID NO: 94) ( 7A bottom right). The cells tested are pooled tetramer-positive donor approaches. In addition, the cells were stained with the L265P mutant RPIPIKYKAM tetramer ( 7A and 7B top left) to again detect peptide-specific T cells.

 Thus, the peptide-specific functionality of the primed T cells could be detected.

The above data shows impressively that in healthy donors in vitro priming of naïve CD8 ⁺ T cells is possible.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

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Claims (20)

An immunogenic peptide of 8 to 25 amino acids in length, comprising at least 8 consecutive amino acids of the amino acid sequence of SEQ ID NO: 1, and amino acid no. 25 of SEQ ID NO: 1 of the attached Sequence Listing, wherein the immunogenic peptide has the ability has to bind to a molecule of the human major histocompatibility complex (MHC) class I or class II. Immunogenic peptide according to claim 1, characterized in that it is 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long. Immunogenic peptide according to claim 1 or 2, characterized in that it has an amino acid sequence with one of SEQ ID Nos. 2 to 93. An immunogenic peptide according to claim 1, characterized in that the peptide is capable of producing a cytotoxic T lymphocyte (CTL) response or T helper lymphocyte response. Immunogenic peptide according to one of claims 1 to 4, characterized in that the peptide has an amino acid sequence with one of SEQ ID Nos. 17, 20, 23 or 27. A nucleic acid encoding or consisting of the sequence for the immunogenic peptide of any one of claims 1 to 5. A vaccine comprising an immunogenic peptide according to any one of claims 1 to 5, or a nucleic acid according to claim 6. An isolated T cell specific for an immunogenic peptide according to any one of claims 1 to 5, or produced by stimulating peripheral blood mononuclear cells (PBMCs) with an immunogenic peptide according to any one of claims 1 to 5. An isolated nucleic acid encoding the α and / or β chain of a T cell receptor of a T cell, which is a complex of (i) an immunogenic peptide according to any one of claims 1 to 5 and (ii) an MHC molecule specific recognizes.  An isolated T cell generated by transferring a nucleic acid according to claim 9 in vitro. An isolated antigen-presenting cell (APC) which presents an immunogenic peptide according to any one of claims 1 to 5 or has been treated with a nucleic acid according to claim 6. A pharmaceutical composition comprising: (a) an immunogenic peptide according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient; (b) a nucleic acid according to claim 6 and a pharmaceutically acceptable carrier or excipient; or (c) two or more of an immunogenic peptide according to any one of claims 1 to 5, or a nucleic acid according to claim 6, for simultaneous, sequential or separate administration. The immunogenic peptide of any one of claims 1 to 5, the nucleic acid of claim 6, the vaccine of claim 7, the isolated T cell of claim 8 or 10, isolated nucleic acids of claim 9, the isolated APC of claim 11 or the pharmaceutical composition of claim 12 for use in therapy or for use in the treatment of tumors. The immunogenic peptide of any one of claims 1 to 5, the nucleic acid of claim 6, the vaccine of claim 7, the isolated T cell of claim 8 or 10, isolated nucleic acids of claim 9, the isolated APC of claim 11 or the pharmaceutical composition of claim 12 for use in therapy or for use in the treatment of B-cell lymphomas and leukemias. The immunogenic peptide of any one of claims 1 to 5, the nucleic acid of claim 6, the vaccine of claim 7, the isolated T cell of claim 8 or 10, the isolated nucleic acids of claim 9, the isolated APC of claim 11 or the pharmaceutical composition of claim 12, for use for the in vitro stimulation of T cells in adoptive immunotherapy. The immunogenic peptide of any one of claims 1 to 5, the nucleic acid of claim 6, the vaccine of claim 7, the isolated T cell of claim 8 or 10, the isolated nucleic acids of claim 9, the isolated APC of claim 11 or the pharmaceutical composition of claim 12, for use for the DC vaccination. Use of the immunogenic peptide according to any one of claims 1 to 5 with or without complex with recombinant MHC I or MHC II of a particular HLA type for the production of a therapeutic or diagnostic antibody or for the production of T cells or DC. Use of the nucleic acids of claim 9 for use in in vitro stimulation of T cells in adoptive immunotherapy. The immunogenic peptide of any one of claims 1 to 5, the nucleic acid of claim 6, the vaccine of claim 7, the isolated T cell of claim 8 or 10, isolated nucleic acids of claim 9, the isolated APC of claim 11 or the pharmaceutical composition of claim 12 for use in the prophylaxis, treatment or treatment of B-cell lymphomas and leukemias in patients with somatic L265P MyD88 Driver mutation. The immunogenic peptide of any one of claims 1 to 5, the nucleic acid of claim 6, the vaccine of claim 7, the isolated T cell of claim 8 or 10, isolated nucleic acids of claim 9, the isolated APC of claim 11 or the pharmaceutical composition of claim 12 for use in the therapy or for use in the treatment of B-cell lymphomas and leukemias, wherein the B-cell lymphomas and leukemias are selected from the group comprising: diffuse large B-cell lymphoma (DLBCL), mucosa-associated lymphoid Tissue Lymphoma (MALT), Chronic Lymphocytic Leukemia (CLL), Waldenstrom's Macroglobulinemia (WM), Primary Cutaneous Large Cell B-cell Lymphoma (PCLBCL), Primary Central Nerve B-Cell Lymphoma (PCNBL), Extranodal Peripheral Zone Lymphoma in the Ocular Adnexus (PCLBL) OAEMZL), and splenic marginal zone lymphoma (SMZL), Burkitt's lymphoma (BL), monoclonal gammopathy of unclear significance (MGUS), intra-ocular B-cell lymphoma.

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