RU2794945C2 - Antigen receptors and their applications - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, in particular to new chimeric receptors, and can be applied in medicine. The invention encompasses the treatment of diseases by targeting T cells to body cells expressing a tumor antigen on the cell surface.

EFFECT: invention discloses various combined chimeric antigen receptors (see Fig. 1). T cells modified to express such chimeric antigen receptors can be used in the treatment of diseases characterized by the expression of one or more tumor antigens, in particular in cancer therapy.

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Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to recombinant antigen receptors and their use. T cells engineered to express such antigen receptors are useful in the treatment of diseases characterized by the expression of one or more antigens associated with antigen receptors.

BACKGROUND OF THE INVENTION

T cells play a central role in cell-mediated immunity in humans and animals. Recognition and binding of a particular antigen is mediated by T cell receptors (TCRs) expressed on the surface of T-4 cells. The TCR of T cells is able to interact with immunogenic peptides (epitopes) associated with molecules of the major histocompatibility complex (MHC, MHC) and presented on the surface of target cells. Specific binding of the TCR triggers a signaling cascade within the T cell leading to proliferation and differentiation into the mature effector T cell.

TCR is part of a complex signal transduction mechanism that includes the signal transduction module, the TCR α- and β-chain heterodimer complex, the CD4 or CD8 and CD3 co-receptor. The TCR α/β heterodimer is responsible for antigen recognition and transduction of the activation signal across the cell membrane in association with CD3, while the CD3 chains themselves transmit the incoming signal to adapter proteins within the cell. Thus, the transfer of TCR α/β chains makes it possible to redirect T cells to any desired antigen.

In general terms, adoptive cell transfer immunotherapy (ACT) can be defined as a form of passive immunization with pre-sensitized T cells that are implanted in non-immune recipients or an autologous host after ex vivo cell culture from a low initial cell number to a clinically relevant cell number. . Cell types that have been used in ACT experiments include lymphokine-activated killer (LAK) cells (Mule, J. J. et al. (1984) Science 225, 1487-1489; Rosenberg, S. A. et al. (1985) N. Engl. J Med. 313, 1485-1492), tumor-infiltrating lymphocytes (TILs) (Rosenberg, S.A. et al. (1994) J. Natl. Cancer Inst. 86, 1159-1166), donor lymphocytes after hematopoietic stem cell transplantation (HSCT ), as well as tumor-specific T cell lines or clones (Dudley, M. E. et al. (2001) J. Immunother. 24, 363-373; Yee, C. et al. (2002) Proc. Natl. Acad. Sci U.S.A 99, 16168-16173). Adoptive T cell transfer has been shown to have therapeutic activity against human viral infections such as CMV. For adoptive immunotherapy for melanoma, Rosenberg and co-workers developed the ACT method, based on the infusion of in vitro cultured autologous tumor-infiltrating lymphocytes (TILs) isolated from resected tumors, in combination with non-myeloablative anti-lymphocyte chemotherapy and high-dose IL2 therapy. A clinical study yielded an objective response rate of ~50% in treated patients suffering from metastatic melanoma (Dudley, M. E. et al. (2005) J. Clin. Oncol. 23: 2346-2357).

An alternative method is the adoptive transfer of autologous T cells reprogrammed to express a tumor-reactive immunoreceptor of a given specificity during short-term ex vivo culture followed by reinfusion into the patient (Kershaw M.H. et al. (2013) Nature Reviews Cancer 13(8):525-41 ). This strategy makes ACT applicable to various common malignant tumors, even if tumor-reactive T cells are not present in the patient. Since the antigenic specificity of T cells is completely at rest on the heterodimeric complex of TCR α- and β-chain, the transfer of cloned TCR genes to T cells provides the ability to redirect them to any desired antigen. Therefore, TCR gene therapy provides an attractive strategy for the development of antigen-specific therapy using autologous lymphocytes as a treatment modality. The main advantages of TCR gene transfer are the production of therapeutic amounts of antigen-specific T cells in a few days and the ability to introduce specificities that are not in the patient's endogenous TCR repertoire. Several groups of investigators have demonstrated that TCR gene transfer is a tempting strategy to redirect the antigenic specificity of primary T cells (Morgan, R.A. et al. (2003) J. Immunol. 171, 3287-3295; Cooper, L.J. et al. ( 2000) J. Virol 74, 8207-8212; Fujio, K. et al. (2000) J. Immunol. 165, 528-532; Kessels, H. W. et al. (2001) Nat. Immunol. 2, 957-961 ; Dembic, Z. et al (1986) Nature 320, 232-238). The possibility of implementing gene therapy using TCR in humans was first demonstrated by Rosenberg and his group in clinical trials for the treatment of malignant melanoma. Adoptive transfer of autologous lymphocytes transduced by retroviral transduction using melanoma/melanocyte antigen-specific TCRs resulted in cancer regression in up to 30% of treated melanoma patients (Morgan, R.A. et al. (2006) Science 314, 126-129; Johnson, L.A. et al (2009) Blood 114, 535-546). Since then, TCR gene therapy clinical trials have also been extended to many different antigens targeting cancers other than melanoma (Park, T.S. et al., (2011) Trends Biotechnol. 29, 550-557).

The use of genetic engineering techniques to incorporate antigen-targeted receptors of given specificity into T cells has greatly expanded the potential of ACT. Chimeric antigen receptors (CARs) are a type of antigen-targeted receptor consisting of intracellular T-cell receptor signaling domains coupled to extracellular antigen-binding domains, most commonly single-chain variable fragments (scFv's) of monoclonal antibodies. CARs directly recognize cell surface antigens, independent of MHC-mediated presentation, allowing a single receptor construct specific for any given antigen to be used in all patients. The parent CARs linked the antigen-recognition domains to the CD3ζ activation chain of the T-cell receptor (TCR) complex. Subsequent iterations of CAR included secondary costimulatory signals along with CD3ζ, including intracellular domains from CD28 or various TNF receptor family molecules such as 4-1BB (CD137) and OX40 (CD134). Further, third generation receptors include two co-stimulatory signals besides CD3ζ, typically from CD28 and 4-1BB. Second and third generation CARs dramatically increased in vitro and in vivo antitumor efficacy (Zhao et al., (2009) J. Immunol., (183) 5563-5574), in some cases inducing complete remission in patients with advanced cancer (Porter et al., (2011) N. Engl. J. Med., (365) 725-733).

The classic CAR consists of an antigen-specific single chain antibody (scFv) fragment associated with a transmembrane and signaling domain such as CD3ζ. After introduction into T cells, it is expressed as a membrane-bound protein and induces immune responses when bound to its recognized antigen (Eshhar et al., (1993) PNAS, (90) 720-724). The induced antigen-specific immune response causes the activation of cytotoxic CD8+ T cells, which in turn leads to the eradication of cells expressing the specific antigen, such as tumor cells or virus-infected cells expressing the specific antigen. However, these classical CAR constructs do not activate/stimulate T cells through the use of their endogenous CD3 complex, which is usually essential for T cell activation. By fusion of the antigen-binding domain to CD3ζ, T cell activation is induced by biochemical slippage (Aggen et al., (2012) Gene Therapy, (19) 365-374). This non-physiological T cell activation poses a risk to the patient treated in this manner, as over-activation of T cells can lead to unwanted side effects. For example, due to the expression of CAR, a prolonged basal activation of 'recombinant' T cells ("tonic signaling") was observed in vitro, which led to an increased accumulation of inhibitory molecules such as LAG-3, TIM-3 and PD-1 on the surface of expressing recombinant CAR T cells, which in turn resulted in prematurely depleted T cells, and this further had a strong negative impact on the response to tumor cells in vivo (Long et al., (2015) Nat. Med., (21) 581-590). This side reaction is associated with irregular clustering of scFv fragments with the frame residues of this antibody. Furthermore, while classic CAR constructs of this type have been successfully tested against various neoplasias such as leukemia (Porter et al., (2011) N. Engl. J. Med., (365) 725-733), they have also resulted in fatal autoimmune diseases due to basal expression of a target antigen (tumor target antigen) in healthy tissues (on-target/off-tumor-reaction; Morgan et al., (2010) Mol Ther., (18) 843-51) .

An alternative method, in which T cell activation follows a more physiological mechanism, is to present a similar single-stranded TCR (scTv) fragment fused to the Cβ constant domain derived from the T cell receptor (TCR) and co-express it with the Cα constant TCR-derived domain (Voss et al., (2010) Blood, (115) 5154-5163), the last to be recruited into the essential endogenous CD3ζ homodimer (Call et al., (2002) Cell, (111) 967-79 .). However, for these constructs to act as immune system activators, it was critical that their constant domains be derived from murine TCRs or that murine sequence residues be introduced (Cohen et al., (2006) Cancer Res., (66) 8878- 86; Bialer et al., (2010) J. Immunol.(184) 6232-41) to effect chain pairing between scTCR and Cα. The fact that these constructs must have xenogeneic sequences to be functional increases the risk that the immune system will antagonize them when administered and reduce or nullify their therapeutic efficacy.

Therefore, there is a need for alternative recombinant antigen receptors, wherein, for example, the receptor, after antigen binding, has a sufficient ability to activate a T cell in which it is expressed in a normal physiological way using the endogenous CD3 complex and, optionally, without the need for the presence of any or amino acid sequences of non-human origin, at least in the signal transduction domain of the antigen receptor, which could elicit an undesirable immune response against the recombinant antigen receptor itself.

BRIEF DESCRIPTION OF THE INVENTION

The essence of the invention

The present invention relates to recombinant antigen receptors having at least two binding sites. Antigenic receptors contain two peptide chains. Each peptide chain contains at least two domains, in addition to the T-cell receptor chain variable region, or portion thereof, and the immunoreceptor signal transduction domain, with each of the two domains on one peptide chain forming an antigen-binding site with one of the domains on the other peptide chain. chains. According to one embodiment, the antigen receptor of the invention has the structure of a T-cell receptor, wherein its chains each contain said at least two domains forming antigen-binding sites, preferably at the N-terminus of the T-cell receptor chains.

In one aspect, the present invention relates to an antigen receptor that comprises a first peptide chain and a second peptide chain, the first peptide chain comprising a first domain, a second domain, a T cell receptor chain variable region or a portion thereof, and an immunoreceptor signal transduction domain; the second peptide chain contains the first domain, the second domain, the variable region of the T-cell receptor chain or part thereof, and the immunoreceptor signal transduction domain; wherein the first domain from the first peptide chain, together with one of the domains from the second peptide chain, forms the first antigen-binding site, and the second domain from the first peptide chain, together with another domain from the second peptide chain, forms the second antigen-binding site. In an antigen receptor according to this aspect, the domains forming the respective antigen-binding sites are preferably located on different peptide chains. Thus, antigen-binding sites are formed as a result of intermolecular interaction of domains.

In one embodiment, the first and/or second domains each comprise an immunoglobulin chain variable region, or a T cell receptor chain variable region, or a portion of a variable region.

In one embodiment, one of the domains forming the first antigen binding site comprises an immunoglobulin heavy chain variable region with antigen specificity or a portion thereof, and the other domain forming the first antigen binding site comprises an immunoglobulin heavy chain variable region with antigen specificity or a portion thereof. In one embodiment, one of the domains forming the second antigen binding site comprises an immunoglobulin heavy chain variable region with antigen specificity or a portion thereof, and the other domain forming the second antigen binding site comprises an immunoglobulin light chain variable region with antigen specificity or a portion thereof.

In one embodiment, the first domain of the first peptide chain comprises an immunoglobulin heavy chain variable region with antigen specificity, or a portion thereof, and the domain of the second peptide chain, forming an antigen-binding site with the first domain of the first peptide chain, comprises an immunoglobulin light chain variable region with antigen specificity, or part of it. In one embodiment, the second domain from the first peptide chain comprises an immunoglobulin heavy chain variable region with antigen specificity or a portion thereof, and the domain from the second peptide chain forming an antigen-binding site with the second domain from the first peptide chain comprises an immunoglobulin light chain variable region with specificity for antigen or part of it.

In one embodiment, the first and second domains of the first peptide chain each comprise an immunoglobulin heavy chain variable region or a portion thereof; and the first and second domains of the second peptide chain each contain an immunoglobulin light chain variable region or a portion thereof.

In one embodiment, the N-terminal domain from the first peptide chain, together with the N-terminal domain from the second peptide chain, forms an antigen-binding site; and the C-terminal domain from the first peptide chain together with the C-terminal domain from the second peptide chain forms an antigen-binding site.

In one embodiment, the N-terminal domain from the first peptide chain, together with the C-terminal domain from the second peptide chain, forms an antigen-binding site; and the C-terminal domain from the first peptide chain together with the N-terminal domain from the second peptide chain forms an antigen-binding site.

In one embodiment of the antigen receptors of the invention, the immunoreceptor signaling domain comprises a T cell receptor chain constant or conserved region, or an immune receptor chain constant or conserved region, or a portion of a constant or conserved region. In one embodiment of the antigen receptors of the invention, (i) the first peptide chain comprises the T cell receptor alpha chain variable region or a portion thereof and the T cell receptor alpha chain constant region or portion thereof, and the second peptide chain comprises the T cell receptor beta chain variable region. or a portion thereof and a T cell receptor beta chain constant region or portion thereof, or (ii) the first peptide chain comprises a T cell receptor beta chain variable region or portion thereof and a T cell receptor beta chain constant region or portion thereof, and the second peptide chain comprises a T cell receptor alpha chain variable region or a portion thereof and a T cell receptor alpha chain constant region or a portion thereof. According to this variant, the variable region of the alpha chain of the T cell receptor or part thereof and the constant region of the alpha chain of the T cell receptor or part of it corresponds or essentially corresponds to the alpha chain of the T cell receptor, and the variable region of the beta chain of the T cell receptor or its part and constant the T cell receptor beta chain region or portion thereof corresponds or substantially corresponds to the T cell receptor beta chain, preferably the same T cell receptor from which the T cell receptor alpha chain is derived.

The domains forming the antigen-binding sites are preferably confluent at the N-terminus of the chains, optionally separated by a linker.

In one embodiment of the antigen receptors of the invention, the T cell receptor chain variable region, or portion thereof, and/or the immunoreceptor signal transduction domain, such as the T cell receptor constant region, or portion thereof, are sequences of human origin. Thus, the T-cell receptor chain that the T-cell receptor chain variable region or portion thereof and the T-cell receptor constant region or portion thereof may correspond or substantially correspond may be of human origin.

In one embodiment, the antigen receptor of the invention comprises (a) linker(s) that bind(s) domains of the antigen receptor. In one embodiment, the antigen receptor of the invention comprises one or more linkers between antigen-binding site-forming domains and/or between antigen-binding site-forming domains and variable regions of the T-cell receptor chain or a portion thereof. The linker may be an arbitrary amino acid sequence of any length, provided that it does not interfere with the functions of the antigen receptor, such as the ability of the antigen receptor to bind antigen or associate with the endogenous CD3 complex, or interfere with the ability of the antigen receptor to stimulate an immune response to antigen binding.

According to one embodiment of the antigen receptors of the invention, the first and second antigen binding sites bind to the same antigen or to different antigens. In one embodiment of the antigen receptors of the invention, the first and second antigen binding sites bind to different epitopes on the same antigen. Accordingly, although the domains forming the first antigen binding site are preferably derived from the same immunoglobulin and the domains forming the second antigen binding site are preferably derived from the same immunoglobulin, the domains forming the first antigen binding site and the domains forming the second antigen binding site , are derived from the same immunoglobulin or from different immunoglobulins, wherein said different immunoglobulins bind to the same antigen or to different antigens.

In one embodiment, the antigen is a disease specific antigen, preferably a tumor antigen. In one embodiment, the antigen is expressed on the cell surface.

In one aspect, the present invention provides an antigen receptor peptide chain of the invention. In one embodiment, the present invention provides a peptide chain comprising a first and a second domain, each comprising an immunoglobulin heavy chain variable region or a portion thereof, or each comprising an immunoglobulin light chain variable region or a portion thereof, and wherein the peptide chain further comprises an immunoglobulin heavy chain variable region a T cell receptor, or a portion thereof; and an immunoreceptor signal transduction domain, such as a T cell receptor chain constant region, or a portion thereof. Additional variants of the peptide chains of the invention are described herein for the antigen receptors of the invention.

In one aspect, the present invention provides a cell, in particular an immune effector cell, such as a T cell, genetically modified to express an antigen receptor of the invention. In one aspect, the present invention relates to a cell, in particular, to an immune effector cell, such as a T cell expressing the first peptide chain, the second peptide chain, or both the first and second peptide chains of the antigen receptor of the invention, or expressing the peptide chain according to invention. Additional variants of the cell or 'recombinant' cell of the invention are described herein for the antigen receptors of the invention or the peptide chains of the invention.

In one aspect, the present invention relates to a method for obtaining a cell expressing an antigen receptor of the invention, comprising: (a) providing the cell; (b) providing a first genetic construct encoding a first antigen receptor peptide chain of the invention; (c) providing a second genetic construct encoding a second antigen receptor peptide chain of the invention; (d) introducing the first and second genetic constructs into the cell; and (e) enabling expression of the constructs in the cell. According to one embodiment, the present invention relates to a method for obtaining a cell expressing an antigen receptor, the receptor comprising a first peptide chain and a second peptide chain, comprising: (a) providing the cell; (b) providing a first genetic construct encoding a first peptide chain comprising a first domain, a second domain, a T cell receptor chain variable region or a portion thereof, and an immunosuppressant signal transduction domain; (c) providing a second genetic construct encoding a second peptide chain comprising at least a first domain, a second domain, a T cell receptor chain variable region or portion thereof, and an immunoreceptor signal transduction domain; (d) introducing the first and second genetic constructs into the cell; and (e) enabling expression of the constructs in the cell; moreover, the first domain from the first peptide chain is able to form, together with one of the domains from the second peptide chain, the first antigen-binding site, and the second domain from the first peptide chain is able to form, together with another domain from the second peptide chain, the second antigen-binding site. In one embodiment of the methods of the invention, expression of the antigen receptor occurs on the cell surface. In one embodiment of the methods of the invention, the first peptide chain and the second peptide chain are provided in a single genetic construct. In one embodiment of the methods of the invention, the cell is a human cell. In one embodiment of the methods of the invention, the genetic constructs comprise DNA and/or RNA. Other variants of the methods according to the invention correspond to the methods described in this application for antigen receptors according to the invention.

In one aspect, the present invention relates to a 'recombinant' cell, in particular an immune effector cell, such as a T cell, obtained by the methods of the invention, to obtain a cell expressing an antigen receptor. Other variants of the 'recombinant' cell of the invention are as described herein for the antigen receptors of the invention or methods for producing a cell expressing the antigen receptor of the invention.

In one aspect, the present invention provides a nucleic acid, such as DNA or RNA, encoding a first peptide chain, a second peptide chain, or both the first and second peptide chains of an antigen receptor of the invention, or encoding a peptide chain of the invention. Other nucleic acid variants of the invention correspond to the nucleic acid variants of the invention for antigen receptors of the invention or peptide chains of the invention.

The present invention generally encompasses the treatment of diseases by targeting cells expressing one or more antigens on the cell surface, e.g., diseased cells expressing one or more disease-specific antigens on the cell surface, in particular cancer cells expressing one or more tumor antigens. on the surface of cells, using antigen receptors according to the invention. The methods involve selective eradication of cells that express one or more antigens on their surface, thereby minimizing side effects on normal cells that do not express the antigen(s). In one embodiment, T cells modified to express an antigen receptor of the invention targeting cells by binding to antigen(s) are administered. T cells are able to recognize diseased cells expressing antigen(s) on the cell surface, resulting in the eradication of diseased cells. In one embodiment, the target cell population or target tissue is tumor cells or tumor tissue.

In one aspect, the present invention provides a pharmaceutical composition comprising an antigen receptor of the invention, a recombinant cell of the invention, or a nucleic acid of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the invention can be used as a medicine, in particular for the treatment of a disease, such as cancer, characterized by the expression of one or more antigens that bind to an antigen receptor of the invention, such as, for example, one or more tumor antigens.

In one aspect, the present invention relates to a method of treating a disease, such as cancer, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition of the invention, wherein the disease is characterized by the expression of at least one antigen, such as a tumor antigen that binds to an antigen receptor.

In one aspect, the present invention relates to a method of treating a subject who has a disease, disorder or pathological condition associated with the expression or overexpression of at least one antigen, the method comprising administering to the subject T cells genetically modified to express an antigen receptor at of the invention targeting at least one antigen. In one embodiment, the disease, disorder or condition is cancer. In one embodiment, the T cells may be autologous, homologous, or syngeneic to the subject.

In one embodiment of the present invention, the antigen receptor binds to only one antigen (for example, being monospecific and recognizing the same epitope, or being bispecific or multispecific and recognizing different epitopes on the same antigen) or binds to different antigens, in particular to two different antigens.

In one embodiment of all aspects of the present invention, the method of treatment also includes taking a sample of cells from a subject, wherein the sample contains T cells or T cell precursors, and transfecting the cells using a nucleic acid encoding an antigen receptor of the invention to provide T cells genetically modified to express the antigen receptor. In one embodiment of all aspects of the invention, T cells genetically modified to express an antigen receptor are stably or transiently transfected with a nucleic acid encoding the antigen receptor. Thus, the nucleic acid encoding the antigen receptor is integrated or non-integrated into the T cell genome. In one embodiment of all aspects of the invention, the T cells and/or cell sample are from a subject receiving T cells genetically modified to express an antigen receptor. In one embodiment of all aspects of the invention, the T cells and/or cell sample are from a non-mammalian to which T cells genetically modified to express the antigen receptor are administered.

In one embodiment of all aspects of the invention, T cells genetically modified to express the antigen receptor are inactivated to express the endogenous T cell receptor and/or endogenous HLA.

In one embodiment of all aspects of the invention, the antigen is expressed in a diseased cell, such as, for example, a cancer cell. In one embodiment, the antigen is expressed on the surface of a diseased cell, such as, for example, a cancer cell. In one embodiment, the antigen receptor binds to the extracellular domain or to an epitope in the extracellular domain of the antigen. In one embodiment, the antigen receptor binds to native antigen epitopes present on the surface of viable cells. In one embodiment of all aspects of the invention, the antigen is a tumor antigen. In one embodiment of all aspects of the invention, the antigen is selected from the group consisting of claudins, such as claudin 6 and claudin 18.2, CD19, CD20, CD22, CD33, CD123, mesothelin, CEA, c-Met, PSMA, GD-2, and NY -ESO-1. In one embodiment of all aspects of the invention, the antigen is an antigen from a pathogen. The pathogen may be a fungal, viral or bacterial pathogen. According to one variant of all aspects of the invention, the expression of the antigen is carried out on the surface of the cell. According to one variant, the antigen is claudin (Claudin), in particular Claudin (claudin) 6 or Claudin (claudin) 18.2, and said antigen receptor binds to the first extracellular loop of said claudin. In one embodiment, binding of said antigen receptor expressed by T cells and/or present on T cells to an antigen present on cells causes effector functions of said T cells of the immune system, such as release of cytokines. In one embodiment, binding of said antigen receptor expressed by T cells and/or present on T cells, such as antigen presenting cells, to an antigen present on cells, such as antigen presenting cells, causes stimulation, priming, and/or expansion of said T cells. . In one embodiment, binding of said antigen receptor expressed by T cells and/or present on T cells to an antigen present on diseased cells results in cytolysis and/or apoptosis of diseased cells, wherein said T cells preferentially release cytotoxic factors, e.g. , perforins and granzymes.

In one embodiment of all aspects of the invention, the antigen receptor domains forming the antigen-binding sites are composed of an antigen receptor ectodomain. In one embodiment of all aspects of the invention, the antigen receptor of the invention comprises a transmembrane domain. In one embodiment, the transmembrane domain is a hydrophobic alpha helix that spans the membrane.

In one embodiment of all aspects of the invention, the antigen receptor of the invention comprises a signal peptide that directs the nascent protein to the endoplasmic reticulum. In one embodiment, the signal peptide precedes the domain that forms the antigen-binding sites.

In one embodiment of all aspects of the invention, the antigen receptor of the invention is preferably specific for the antigen to which it is targeted, in particular on the surface of a cell, such as a diseased cell or an antigen presenting cell.

In one embodiment of all aspects of the invention, the antigen receptor of the invention may be surface-expressed and/or present on the surface of an immunoreactive cell, such as, for example, a T cell, preferably a cytotoxic T cell. In one embodiment, the T cell is capable of reacting with the antigen(s) targeted by the antigen receptor of the invention.

According to another aspect of the invention, the agents and compositions described herein are provided for use in the methods of this invention.

Other features and advantages of the present invention will be apparent from the following detailed description and claims.

Detailed description of the invention

Although the present invention is described in detail below, it should be understood that this invention is not limited to the specific methods, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used in this application is given for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless specifically stated otherwise, all technical and scientific terms used in this specification have the same meanings as those commonly understood by those of ordinary skill in the art.

The elements of the present invention are described below. These elements are given with specific options, however, it should be understood that they can be combined in any other way and in any number, creating additional options. The various examples and preferred embodiments described are not to be construed as limiting the present invention to the expressly described embodiments. It should be understood that this description protects and covers options that combine the explicitly described options with any number of disclosed and/or preferred elements. Also, any permutations and combinations of all elements described in this application should be considered as disclosed in the description of this application, unless the context indicates otherwise.

, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.Preferably, the terms used in this application are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H.

, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

In the practice of the present invention, unless specifically indicated otherwise, conventional methods of biochemistry, cell biology, immunology and recombinant DNA methods are used, which are explained in the literature related to this field of technology (cf., for example, Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, J. Sambrook et al.eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this description and in the claims that follow, unless the context otherwise requires, it should be understood that the word "comprise" and its variants, such as "comprises" and "comprising", is intended to include the specified element, the whole number or stage or group of elements, integers or stages, although in some embodiments such other element, integer or stage or group of elements, integers or stages may be excluded, i. the subject matter of the invention is the inclusion of said element, integer or step or group of elements, integers or steps. Singular terms and similar indications used in the context of the description of the invention (in particular in relation to the claims) should be considered as covering both the singular and the plural, unless otherwise indicated in this description or unless this clearly contradicts the context. . It is assumed that the listing of ranges of values in this description is only for brevity when referring individually to each individual value within the interval. Unless otherwise indicated in the present description, each individual value is included in the description as if it were listed individually.

All methods described in this application can be performed in any suitable order, unless otherwise indicated, or if it is otherwise clearly contrary to the context. The use of any examples or introductory words before an example (eg, "such as") in the present description is only intended to more clearly illustrate the present invention and does not limit the scope of the invention as claimed otherwise. None of the terms in the description should be construed as indicating any unclaimed element essential to the practice of the present invention.

Some documents are cited in the text of this description. Each of the documents cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), either supra or infra, is incorporated by reference in its entirety. This invention does not give the right to consider it as previous to the earlier date of disclosure of this invention in the previous invention.

The term "immune response" refers to the overall systemic response to an antigen, and preferably refers to a cellular immune response, or both cellular and humoral immune response. The immune response may be protective/preventive/prophylactic and/or therapeutic.

"Providing an immune response" may mean that there was no immune response to a particular target antigen, target cell and/or tissue/target prior to providing an immune response, but it could also mean that there was some level of immune response to a particular target antigen, target cell and/or tissue/target until an immune response is provided, and after the formation of an immune response, said immune response is enhanced. Thus, "producing an immune response" includes "inducing an immune response" and "enhancing an immune response". Preferably, after providing an immune response in the subject, the specified subject is protected from the development of a disease, such as cancer, or the intensity of the disease state is reduced by providing an immune response. For example, an immune response to a tumor antigen can be generated in a cancer patient or in a subject at risk of developing cancer. Providing an immune response in this case may mean that the intensity of the disease state is reduced, that the subject does not develop metastases, or that the subject at risk of developing cancer does not develop cancer.

The terms "cell-mediated immunity" or "cellular immunity" or similar terms are intended to include a cellular response directed to cells expressing antigen, in particular those characterized by antigen presentation by class I or class II MHC molecules. The cellular response refers to cells called T cells or T lymphocytes, which act as either "helper" or "killer" cells. Helper T cells (also called CD4 ⁺ T cells) play a major role by regulating the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8 ⁺ T cells, or CTLs) kill diseased cells, for example, such as cancer cells, preventing the production of new diseased cells.

The term "antigen" refers to an agent containing an epitope against which an immune response is to be generated and/or directed. Preferably, an antigen in the context of the present invention means a molecule which, optionally after processing, induces an immune response which is preferably specific for the antigen or cells expressing the antigen, preferably on the cell surface. The term "antigen" includes in particular proteins and peptides. The antigen is preferably a product that matches a naturally occurring antigen or is derived using a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to a product of natural origin, such as a viral protein, or part thereof.

The term "pathogen" refers to pathogenic microorganisms and includes viruses, bacteria, fungi, unicellular organisms and parasites. Examples of pathogenic viruses are human immunodeficiency virus (HIV, HIV), cytomegalovirus (CMV), herpes virus (HSV), hepatitis A virus (HAV), HBV, HCV, papillomavirus and human T-lymphotropic virus (HTLV). Single-celled organisms include plasmodia, trypanosomes, amoeba, etc.

In a preferred embodiment, the antigen is a disease-specific or disease-associated (pathogenic) antigen. The term "disease-specific antigen" or "disease-associated antigen" refers to all antigens that have pathological significance. According to one particularly preferred embodiment, the antigen is present in diseased cells, tissues and/or organs, while it is absent or present in reduced amounts in healthy cells, tissues and/or organs, and therefore can be used to target diseased cells, tissues and/or organs, for example, using T cells carrying an antigen receptor targeted to the antigen. In one embodiment, an antigen specific to, or associated with, a particular disease is present on the surface of a diseased cell.

According to a preferred embodiment of the invention, the antigen is a tumor antigen or tumor-associated antigen, an element of a cancer cell that can be formed using the cytoplasm, the cell surface and the cell nucleus, in particular those antigens that are produced, preferably in large quantities, in the form of surface antigens on cancer cells.

As used herein, the term "tumor antigen" or "tumor-associated antigen" refers to proteins that are normally specifically expressed in a limited number of tissues and/or organs or at particular developmental stages, e.g., a tumor antigen may normally be specifically expressed in tissue. gastric mucosa, preferably in the gastric mucosa, in reproductive organs such as the testes, in trophoblastic tissue such as the placenta, or in germline cells, and are expressed or aberrantly expressed in one or more tumor or cancerous tissue. As used herein, the term "limited number" preferably means no more than 3, more preferably no more than 2. Tumor antigens in the context of the present invention include, for example, differentiation antigens, preferably cell type specific. differentiation antigens, i.e. proteins that under normal conditions are specifically expressed in cells of a certain type at a certain stage of differentiation, cancer-testicular antigens, i.e. proteins that under normal conditions are specifically expressed in the testes and sometimes in the placenta, and specific germline antigens. In the context of the present invention, the tumor antigen is preferably associated with the cell surface of the cancer cell and is preferably not expressed or very rarely expressed in normal tissues. Preferably, the tumor antigen or aberrant expression of the tumor antigen allows the tumor cells to be identified. In the context of the present invention, a tumor antigen that is expressed by a cancer cell in a subject, eg a cancer patient, is preferably the subject's own protein. According to preferred embodiments, the tumor antigen, in relation to the present invention, is expressed under normal conditions mainly in tissues or organs that are not essential, i. in tissues or organs, the damage of which, when damaged by the immune system, does not lead to the death of the subject, or in organs or structures of the body that are inaccessible or hardly accessible to the immune system. Preferably, the amino acid sequence in the tumor antigen which is expressed in normal tissues and in the tumor antigen which is expressed in cancer tissues is identical.

Examples of tumor antigens that can be used in the present invention are p53, ART-4, BAGE, beta-catenin/m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, cell surface proteins of the claudin family, such as, for example, CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan -A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1/RARa, PRAME , proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP- 2, TRP-2/INT2, TRTE and WT. Particularly preferred tumor antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).

The term "CLDN" or simply "Cl" in the context of this application means claudin and includes CLDN6 and CLDN18.2. Preferably, claudin means human claudin. Claudins are a family of proteins that are the most important components of tight junctions because they establish a paracellular barrier that controls the flow of molecules in the intracellular space between epithelial cells. Claudins are 4-fold membrane-spanning transmembrane proteins with both their N-terminal region and C-terminal region located in the cytoplasm. The first extracellular loop, labeled EC1 or ECL1, has an average of 53 amino acids, and the second extracellular loop, labeled EC2 or ECL2, has about 24 amino acids. Cell surface proteins of the claudin family are expressed in tumors of various origins and are particularly suitable as target structures for targeted cancer immunotherapy due to their selective expression (lack of expression in normal tissue of relevant toxicity) and plasma membrane localization.

CLDN6 and CLDN18.2 have been identified as differentially expressed in tumor tissues, with the only normal tissue expressing CLDN18.2 being the stomach (differentiated gastric mucosal epithelial cells) and the only normal tissue expressing CLDN6 being the placenta.

CLDN18.2 is expressed in malignant tumors of various origins such as pancreatic cancer, esophageal cancer, gastric carcinoma, bronchial carcinoma, breast carcinoma, and ENT tumors. CLDN18.2 is an important target for the prevention and/or treatment of primary tumors such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, cancer liver, head and neck cancer and gallbladder cancer, and their metastases, in particular gastric cancer metastases such as Krukenberg tumors, peritoneal metastases and lymph node metastases. Antigenic receptors targeting at least CLDN18.2 are useful in the treatment of these cancers.

CLDN6 has been found to be expressed, for example, in ovarian cancer, lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanomas, head and neck cancer, sarcomas, bile duct cancer, renal cell carcinoma and gallbladder cancer. CLDN6 is a particularly preferred target for the prevention and/or treatment of ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma , prevention and / or treatment of gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, in particular malignant pleomorphic adenoma, sarcoma, in particular , synovial sarcoma and carcinosarcoma, cancer of the bile duct, bladder cancer, in particular transitional cell carcinoma and papillary cancer, kidney cancer, in particular renal cell cancer, including clear cell renal cell carcinoma and papillary kidney cancer, colon cancer, small intestine cancer, including ileal cancer, in particular small intestine adenocarcinoma and ileal adenocarcinoma, testicular fetal cancer, choriocarcinoma of the placental bed, cervical cancer, ovarian cancer, in particular testicular seminoma, testicular teratoma, testicular teratoma and fetal testicular cancer, uterine cancer, embryonic cell tumors such as, for example, teratocarcinoma or embryonic carcinoma, in particular testicular embryonic cell tumors and their metastatic forms. Antigenic receptors that target at least CLDN6 are useful in the treatment of such cancers.

In the context of embodiments of the present invention, the antigen is preferably present on the surface of a cell, preferably an antigen-presenting cell or a diseased cell. According to the invention, an antigen, when bound by an antigen receptor, is preferably capable of inducing, optionally in the presence of appropriate costimulatory signals, stimulation, priming and/or expansion of a T cell carrying an antigen receptor that binds the antigen. Recognition of an antigen on the surface of a diseased cell can lead to an immune response against the antigen (or the cell expressing the antigen).

According to various aspects of the invention, the aim is preferably to provide an immune response against diseased cells expressing an antigen, such as a tumor antigen, in particular CLDN6 or CLDN18.2, and treatment of a disease, such as a cancer, including cells expressing an antigen, such as a tumor antigen. Preferably, the invention includes administering antigen receptor-modified effector cells of the immune system, such as T cells, targeting diseased antigen-expressing cells. Cells expressing an antigen on the surface can be targeted by effector cells of the immune system that carry an antigen receptor that targets the antigen.

The term "cell surface" is used in accordance with the meaning usual in the art, and therefore includes the outer surface of the cell, which is available for binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located on the surface of said cells and is available for binding by antigen-binding molecules, such as, for example, antigen receptors or antibodies specific for the antigen added to the cells. According to one embodiment of the present invention, the antigen expressed on the cell surface is an integral membrane protein having an extracellular region recognized by the antigen receptor. An antigenic receptor is expressed on the surface of cells if it is located on the surface of said cells and is available for binding, for example, by an antigen for which the antigen receptor added to the cells is specific. In one embodiment, the antigen receptor expressed on the cell surface is an integral membrane protein having an extracellular region that recognizes the antigen.

The term "extracellular region" or "ectodomain" in the context of the present invention refers to a part of a molecule, such as a protein, which is located opposite the extracellular space of the cell and, preferably, is accessible from the outside of said cell, for example, by binding molecules, such as antibodies, localized outside the cell . Preferably, the term refers to one (or one) or more extracellular loops or domains or fragments thereof.

The terms "section" or "part" are used in this application as synonyms and refer to a continuous or discontinuous element of the structure, such as, for example, an amino acid sequence. The term "fragment" refers to a continuous element of the structure, such as, for example, an amino acid sequence. The region or part of the protein sequence preferably contains at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive and/or non-consecutive amino acids of a protein sequence. A fragment of a protein sequence preferably contains at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids protein sequence. A site, part or fragment of a structure preferably contains one or more functional properties, for example, antigenic, immunological and/or binding properties of said structure. For example, the variable region portion of the T cell receptor chain is preferably capable of forming an antigen recognition site and binding the antigen. Thus, if the variable region of the T cell receptor chain is V alpha, its portion is preferably still capable of interacting with the corresponding V beta region or portion thereof to form a functional antigen recognition site. If the T cell receptor chain variable region is V beta, its region is preferably still capable of interacting with the corresponding V alpha region or region thereof to form a functional antigen recognition site. Similarly, the constant region portion of the T cell receptor chain is preferably capable of performing its signal transduction function.

According to the invention, an antigen is (substantially) not expressed in a cell if the expression level is below the detection limit and/or if the expression level is too low to allow binding by antigen-specific antibodies added to the cell. According to the present invention, an antigen is expressed in a cell if the expression level is above the detection limit and/or if the expression level is high enough to allow binding by antigen-specific antibodies added to the cell. Preferably, the antigen expressed in the cell is expressed or displayed, i. e. is present on the surface of said cell and is therefore available for binding by antigen-specific molecules such as antibodies or antigen receptors added to the cell.

"Target cell" shall mean a cell that is the target of an immune response, such as a cellular immune response. Target cells include any unwanted cell such as a cancer cell. In preferred embodiments, a target cell means a cell expressing a target antigen, in particular an antigen specific for a particular disease, which is preferably present on the surface of the cell.

The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, i. to the part or fragment of the molecule that is (th) recognized, i.e. binds, the immune system, for example, is recognized by an antibody or antigen receptor. For example, epitopes are discrete three-dimensional sites on an antigen that are recognized by the immune system. Epitopes usually consist of reactive surface groupings of molecules, such as amino acid or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not to the latter, is lost in the presence of denaturing solvents. Preferably, the epitope is capable of eliciting an immune response to an antigen or antigen-expressing cell. Preferably, the term refers to the immunogenic region of an antigen. An epitope of a protein, such as a tumor antigen, preferably contains a continuous or discontinuous section of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length, for example, an epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

"Antigen processing" refers to the cleavage of an antigen into processing products that are fragments of said antigen (e.g., cleavage of a protein into peptides) and the association of one or more such fragments (e.g., via binding) to MHC molecules for presentation by cells, preferably antigen-presenting cells, specific T cells.

An antigen presenting cell (APC) means a cell that presents an antigen in the context of a major histocompatibility complex (MHC) on its surface. T cells can recognize this complex using their T cell receptor (TCR). Antigen-presenting cells process antigens and present them to T cells. According to the invention, the term "antigen-presenting cell" includes professional antigen-presenting cells and non-professional antigen-presenting cells.

Professional antigen presenting cells are very efficient at internalizing an antigen, either through phagocytosis or receptor-mediated endocytosis, and then displaying an antigen fragment bound to an MHC class II molecule on its membrane. The T cell recognizes and interacts with the MHC class II antigen-molecule complex on the membrane of the antigen presenting cell. The antigen-presenting cell then produces an additional costimulatory signal, leading to the activation of the T cell. Expression of co-stimulatory molecules is a defining feature of professional antigen-presenting cells. The main types of professional antigen presenting cells are dendritic cells, which have the widest range of antigen presentation and are perhaps the most important antigen presenting cells, macrophages, B cells, and some activated epithelial cells.

Nonprofessional antigen-presenting cells do not consistently express MHC class II proteins required for interaction with naive T cells; these proteins are only expressed when non-professional antigen-presenting cells are stimulated by certain cytokines, such as IFNγ, for example.

Dendritic cells (DCs) are populations of leukocytes that present antigens captured in peripheral tissues to T cells by presenting both class II and class I MHC antigens. cells is a key step for the induction of antitumor immunity. Dendritic cells and progenitors can be obtained from peripheral blood, bone marrow, tumor infiltrating cells, tumor infiltrating cells surrounding the tumor, lymph nodes, spleen, skin, cord blood, or any other suitable tissue or fluid. For example, dendritic cells can be differentiated ex vivo by adding a combination of cytokines, such as GM-CSF, IL-4, IL-13 and/or TNFa, to cultures of monocytes selected from peripheral blood. Alternatively, CD34 positive cells selected from peripheral blood, cord blood, or bone marrow can be differentiated into dendritic cells by adding combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and/or other to the culture medium. (s) compound(s) that induce differentiation, maturation and proliferation of dendritic cells. Dendritic cells are conventionally categorized as "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-studied phenotypes. However, these terms should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with a high ability to capture and process antigen, which correlates with high levels of Fcγ receptor and mannose receptor expression. The mature phenotype is typically characterized by reduced expression of these markers but high expression of cell surface molecules responsible for T cell activation, such as MHC class I and class II molecules, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., , CD40, CD80, CD86 and 4-1BB). Dendritic cell maturation is referred to as a dendritic cell activation status in which such antigen-presenting dendritic cells induce T cell priming while presentation by immature dendritic cells induces tolerance. The maturation of dendritic cells is mainly driven by biomolecules with microbial hallmarks, detected by the body's natural receptors (bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines (TNF, IL-1, IFNs), CD40 ligation on the surface of dendritic cells via CD40L, and substances released from cells undergoing stress-related cell death. Dendritic cells can be obtained in vitro by culturing bone marrow cells with cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor alpha.

The term "immunogenicity" refers to the relative effectiveness of an antigen in stimulating an immune response.

The term "immune effector functions" as used herein includes any functions mediated by components of the immune system that cause, for example, the killing of diseased cells, such as tumor cells, or inhibition of tumor growth and/or inhibition of tumor development, including inhibition of seeding and tumor metastasis. . Preferably, the immune effector functions for the purposes of the present invention are T-cell mediated effector functions. Such functions include, in the case of a helper T cell, (CD4 ⁺ T cells), release of cytokines such as Interleukin-2 and/or activation of CD8 ⁺ lymphocytes (CTLs) and/or B cells, and, in the case of CTL, elimination of cells, i.e. cells characterized by antigen expression, for example, through apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of antigen-expressing target cells.

The term "immunoreactive cell" or "immune effector cell", as used in the present invention, refers to a cell that exhibits effector functions in an immune response. An "immunoreactive cell" is preferably capable of binding an antigen, such as, for example, an antigen expressed on the cell surface, and mediating an immune response. For example, such cells secrete cytokines and/or chemokines, kill microbes, secrete antibodies, recognize infected or cancerous cells, and optionally eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in relation to the present invention, "immunoreactive cells" means T cells, preferably CD4 ⁺ and/or CD8 ⁺ T cells. According to the present invention, the term "immunoreactive cell" also includes a cell that can mature into an immune cell (such as a T cell, in particular a helper T cell or a cytolytic T cell) with appropriate stimulation. Immunoreactive cells include CD34 ⁺ hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. Differentiation of T cell precursors into a cytolytic T cell, when exposed to an antigen, is similar to clonal selection using the mechanisms of the immune system.

Preferably, the "immunoreactive cell" or "immune effector cell" recognizes the antigen with a certain degree of specificity, in particular if it is on the surface of an antigen presenting cell or a diseased cell, such as a cancer cell. Preferably, said recognition enables the cell that recognizes the antigen to be responsive or reactive. If the cell is a helper T cell (CD4 ⁺ T cell), such responsiveness or reactivity may include cytokine release and/or activation of CD8 ⁺ lymphocytes (CTLs) and/or B cells. If the cell is a CTL, such responsiveness or reactivity may include cell elimination, ie. cells characterized by antigen expression, for example, through apoptosis or perforin-mediated cell lysis. According to the invention, CTL responsiveness may include sustained calcium transfer, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen-expressing target cells. CTL responses can also be determined using an artificial reporter that accurately indicates CTL responses. Such CTLs that recognize an antigen and are responsive or reactive are also referred to herein as "antigen-responsive CTLs".

"Lymphoid cell" means a cell which, optionally after appropriate modification, for example, after the transfer of a T-cell receptor or an antigen receptor, is capable of eliciting an immune response, such as a cellular immune response, or a progenitor cell of such a cell, and includes lymphocytes, preferably T-lymphocytes, lymphoblasts and plasma cells. The lymphoid cell may be an immunoreactive cell or an immune effector cell as described herein. A preferred lymphoid cell is a T cell that can be modified to express a T cell receptor or an antigen receptor on the cell surface. According to one embodiment of the present invention, the lymphoid cell lacks endogenous expression of the T cell receptor.

The terms "T cell" and "T lymphocyte" are used herein as synonyms and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), which include cytolytic T- cells.

The T cell belongs to a group of white blood cells known as lymphocytes and plays a central role in cell-mediated immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence on their surface of special receptors called T cell receptors (TCRs). The thymus is the main organ responsible for the maturation of T cells. Several different subpopulations of T cells have been found, each with an individual function.

Helper T cells assist other white blood cells in immunological processes, including but not limited to the maturation of B cells to plasma cells and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells become activated when they are presented with peptide antigens by class II MHC molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or promote an active immune response.

Cytotoxic T cells destroy virus-infected and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to an antigen associated with MHC class I, which is present on the surface of almost every cell in the body.

Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor consists of two separate peptide chains that are derived using independent T cell receptor alpha and beta genes (TCRα and TCRβ) and are called α- and β-TCR chains, γδ T cells (gamma delta T cells) represent a small subpopulation of T cells that have a distinct T cell receptor (TCR) on their surface. At the same time, in γδ T-cells, the TCR consists of one γ-chain and one δ-chain. This group of T cells is significantly less abundant (2% of total T cells) than αβ T cells.

Each chain of the T-cell receptor consists of two extracellular (extracellular) domains: the variable (V) region and the constant (C) region. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide/MHC complex. In the context of the present invention, the term "constant region of the T-cell receptor chain or part thereof" also includes variants in which the constant region of the T-cell receptor chain (from N-terminus to C-terminus) is followed by a transmembrane region and a cytoplasmic tail, for example, the transmembrane region and the cytoplasmic tail, which are naturally associated with the constant region of the T-cell receptor chain.

All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors derived from hematopoietic stem cells colonize the thymus and proliferate by cell division, creating a large population of immature thymocytes. 'Early' thymocytes express neither CD4 nor CD8 and are therefore classified as double negative (CD4-CD8-) cells. As they progress during development, they become doubly positive (CD4+CD8+) thymocytes, and finally mature into monopositive (CD4+CD8- or CD4-CD8+) thymocytes, which are then released from the thymus into peripheral tissues.

Typically, T cells can be obtained in vitro or ex vivo using standard methods. For example, T cells can be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood from a mammal, such as a patient, using a commercially available cell separation system. Alternatively, T cells can be obtained from related or unrelated humans, animals, non-human animals using cell lines or cultures. The sample containing T cells may, for example, be peripheral blood mononuclear cells (PBMCs).

T cells for use according to the invention may express the endogenous T cell receptor, or may not express the endogenous T cell receptor.

Nucleic acids, such as RNA encoding an antigen receptor, can be introduced into T cells with lytic potential, in particular lymphoid cells.

The term "antigen receptor targeting an antigen" or similar terms refers to an antigen receptor which, when present on an immune effector cell such as a T cell, recognizes an antigen that is on the surface of antigen presenting cells or diseased cells such as cancer cells. cells, whereby the immune effector cell is stimulated, primed and/or propagated or affects the effector functions of the immune effector cells described above.

The term "antigen-specific T cell" or similar terms refers to a T cell that, in particular, when presented with an antigen receptor, recognizes the antigen targeted by the antigen receptor, for example, on the surface of antigen presenting cells or diseased cells such as cancer cells. , and preferably affects the T cell effector functions described above.

T cells and other lymphoid cells are considered specific for an antigen if these cells kill target cells expressing the antigen. T cell specificity can be determined using any of a number of standard methods, such as the radioactive chromium test or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-γ) can be quantified.

The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I molecules and MHC class II molecules and refers to a gene complex that occurs in all vertebrates. MHC proteins or molecules are essential for signaling between lymphocytes and antigen presenting cells or diseased cells in immune responses, where MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. MHC-encoded proteins are expressed on the cell surface and are exposed to the T cell as both self-antigens (peptide fragments from the cell itself) and non-self-antigens (eg, fragments of invasive microorganisms).

According to the present invention, the term "antigenic receptor" includes engineered receptors that confer arbitrary specificity, such as that of a monoclonal antibody, for example, to an immune effector cell, such as a T cell. As a result, a large number of antigen-specific T cells can be obtained, which can be created for adoptive cell transfer. As a consequence, the antigen receptor of the invention may be present on T cells, for example, instead of or in addition to the T cell's own T cell receptor. Such T cells do not necessarily require antigen processing and presentation to recognize the target cell, but rather can recognize, preferably using specificity, any antigen present on the target cell. Preferably, said antigen receptor is expressed on the cell surface. In relation to the present invention, T cells containing an antigen receptor are encompassed by the term "T cell" in the context of this application. Namely, according to the present invention, the term "antigen receptor" includes artificial receptors containing a single molecule or complex of molecules that recognize, i. bind to a target structure (eg, an antigen) on a target cell, such as a cancer cell (eg, by binding an antigen-binding site or antigen-binding domain to an antigen expressed on the surface of the target cell), and can confer specificity on an immune effector cell, such as T -cell expressing the specified antigen receptor on the surface of the cell. Preferably, recognition of a target structure by an antigen receptor results in activation of an immune effector cell expressing said antigen receptor. An antigenic receptor may contain one or more protein elements, said protein elements containing one or more domains as described herein. The term "antigen receptor" preferably does not include T-cell receptors. According to the invention, the term "antigen receptor" is preferably synonymous with the terms "chimeric antigen receptor (CAR)", "chimeric T-cell receptor" and "artificial T-cell receptor".

According to the present invention, an antigen can be recognized by an antigen receptor through any of the antigen recognition domains (also referred to herein simply as "domains") capable of forming an antigen-binding site, for example, using antigen-binding regions of antibodies and T-cell receptors, which can be on different peptide chains. In one embodiment, the two domains that form the antigen-binding site are obtained using an immunoglobulin. In another embodiment, the two domains that form the antigen-binding site are derived from a T-cell receptor. Particularly preferred are antibody variable domains, such as, for example, single chain variable fragments (scFv) derived from the variable domains of monoclonal antibodies and T cell receptors, in particular TCR alpha and beta single chains. In fact, almost anything that binds a given target with high affinity can be used as an antigen recognition domain.

According to one embodiment of the invention, the antigen receptor of the invention comprises at least four immunoglobulin variable domains forming at least two binding sites, where the two binding sites may bind to the same epitope or to different epitopes, each epitope may be on the same the same antigen or different antigens. In one embodiment, the antigen receptor comprises an immunoglobulin heavy chain (VH) variable domain (or region) with a first epitope specificity (VH(1)), an immunoglobulin light chain (VL) variable domain (or region) with a first epitope specificity (VL( 1)), an immunoglobulin heavy chain (VH) variable domain (or region) with a second epitope specificity (VH(2)), and an immunoglobulin light chain (VL) variable domain (or region) with a second epitope specificity (VL(2 )), wherein the first and second epitopes may be the same or different and may be on the same or on different antigens. According to one variant, VH(1) is able to interact and form an antigen-binding site with VL(1), and VH(2) is able to interact and form an antigen-binding site with VL(2), while the VH(1) domain is not able to interact and form an antigen-binding site with VL(2), and the VH(2) domain is unable to interact and form an antigen-binding site with VL(1). However, according to another variant, the VH(1) domain is able to interact and form an antigen-binding site with VL(1), as well as VL(2), and VH(2) is able to interact and form an antigen-binding site with VL(2), as well as VL(1). In the latter embodiment, VH(1) and VH(2) may be identical or at least derived from the same immunoglobulin, and VL(1) and VL(2) may be identical or at least derived from the same immunoglobulin.

In one aspect, this invention relates to an antigen receptor, also referred to herein as a combined antigen receptor, wherein such a receptor comprises a first peptide chain and a second peptide chain, the first peptide chain comprising a first domain, a second domain, a T-cell receptor chain variable region or its site and the domain of signal transmission from the immunoreceptor; the second peptide chain comprises a first domain, a second domain, a T cell receptor variable region or portion thereof, and an immunoreceptor signal transduction domain; wherein the first domain from the first peptide chain, together with one of the domains from the second peptide chain, forms the first antigen-binding site, while the second domain from the first peptide chain, together with another domain from the second peptide chain, forms the second antigen-binding site.

According to another embodiment, the combined antigen receptor of the invention comprises a heavy chain variable domain associated with a light chain variable domain on each of both peptide chains, wherein two antigen binding sites are formed by interaction between the heavy chain variable domain and the light chain variable domain on different peptide chains. . In one embodiment, the combined antigen receptor of the invention comprises two peptide chains, with one peptide chain containing VL(1) and VH(2) and the second polypeptide chain containing VH(1) and VL(2). In another embodiment, the combined antigen receptor of the invention comprises a heavy chain variable domain linked to a heavy chain variable domain on one peptide chain and a light chain variable domain linked to a light chain variable domain on the other peptide chain, wherein two antigen-binding sites are formed. occurs through interaction between the heavy chain variable domain and the light chain variable domain on different peptide chains. In one embodiment, the combined antigen receptor of the invention comprises two peptide chains, with one peptide chain containing VH(1) and VH(2) and the other peptide chain containing VL(1) and VL(2).

According to one embodiment of the present invention, the combined antigen receptor of the invention comprises a first peptide chain wherein the heavy chain variable region (VH) and the light chain variable region (VL) are preferably N-terminal to C-terminal in the following order: VH (1)-VL(2), and a second peptide chain, wherein the heavy chain variable region (VH) and the light chain variable region (VL) are preferably located, from the N-terminus to the C-terminus, in the following order: VL( 1)-VH(2). The variable region of the T-cell receptor chain or portion thereof and the immunoreceptor signaling domain are preferably located C-terminally to the location of the variable regions. The T cell receptor variable chain region or portion thereof and the immunoreceptor signal transduction domain preferably comprise a T cell receptor alpha chain variable region or portion thereof and a T cell receptor alpha chain constant region or portion thereof located on one of the peptide chains, and a T-cell receptor beta chain variable region or portion thereof, and a T-cell receptor beta chain constant region or portion thereof, located on another peptide chain. In one embodiment, the T cell receptor alpha chain variable region or portion thereof and the T cell receptor alpha chain constant region or portion thereof comprise the T cell receptor alpha chain. In one embodiment, the T cell receptor beta chain variable region or portion thereof and the T cell receptor beta chain constant region or portion thereof comprise a T cell receptor beta chain. The T cell receptor alpha chain and the T cell receptor beta chain are preferably derived from the same T cell receptor.

In another embodiment, the combined antigen receptor of the invention comprises a first peptide chain wherein the heavy chain variable region (VH) and the light chain variable region (VL) are preferably N-terminal to C-terminal in the following order: VH(1 )-VH(2), and a second peptide chain wherein the heavy chain variable region (VH) and the light chain variable region (VL) are preferably N-terminal to C-terminal in the following order: VL(1) -VL(2). The variable region of the T-cell receptor chain, or portion thereof, and the immunoreceptor signal transduction domain are located C-terminally to the location of the variable regions. The T cell receptor variable chain region, or portion thereof, and the immunoreceptor signal transduction domain preferably comprise a T cell receptor alpha chain variable region or portion thereof, and a T cell receptor alpha chain constant region or portion thereof, located on the same of peptide chains, and a T-cell receptor beta chain variable region or portion thereof, and a T-cell receptor beta chain constant region or portion thereof, located on another peptide chain. In one embodiment, the T cell receptor alpha chain variable region or portion thereof and the T cell receptor alpha chain constant region or portion thereof comprise the T cell receptor alpha chain. In one embodiment, the T cell receptor beta chain variable region or portion thereof and the T cell receptor beta chain constant region or portion thereof comprise a T cell receptor beta chain. The T cell receptor alpha chain and the T cell receptor beta chain are preferably derived from the same T cell receptor.

The antigen receptors of the invention have at least two antigen binding sites and are therefore at least divalent. As noted above, the antigen receptor binding sites of the invention may bind to the same epitope or to different epitopes, which epitopes may be on the same antigen or on different antigens. If the binding sites bind to the same epitopes, in particular on the same antigen, the two binding sites may be identical or substantially identical and/or may be formed by identical or substantially identical domains, such identical or substantially identical domains may be derived from the same immunoglobulin. If the binding sites bind to different epitopes, either on the same or different antigens, the two binding sites are different and are formed by different domains, and these different domains may come from different immunoglobulins. In the case of such different domains, it is preferable that domains having different epitope specificities do not interact or do not substantially interact with each other, i. e. VH(1) is unable to interact and form an antigen binding site with VL(2), and VH(2) is unable to interact and form an antigen binding site with VL(1). Therefore, VH(1) interacts and forms an antigen-binding site with VL(1), and VH(2) interacts and forms an antigen-binding site with VL(2). If the combined antigen receptor of the present invention contains two peptide chains, with one peptide chain containing VH(1) and VL(2) and the other peptide chain containing VH(2) and VL(1), the result is peptide chains that do not capable of forming antigen-binding sites through intramolecular domain interaction.

The two antigen receptor domains of the invention forming the antigen-binding site may also originate from the T-cell receptor and may be fragments or regions thereof which retain antigen-specific binding, in particular binding to the peptide-MHC complex, such as the T-variable regions. cell receptor.

According to the invention, the term "T cell receptor variable region" refers to the variable domains of the TCR chains. The variable region of both the α-chain and the β-chain of the TCR has three hypervariable regions, or complementarity determining regions (CDRs), while the β-chain variable region has an additional hypervariability region (HV4) that does not normally contact antigen and, therefore not considered a CDR. CDR3 is the main CDR responsible for recognition of the processed antigen, although the α-chain CDR1 has also been shown to interact with the N-terminal portion of the antigenic peptide, while the β-chain CDR1 interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize the MHC. The β-chain CDR4 is not thought to be involved in antigen recognition, but this region has been shown to interact with superantigens.

The above information relating to immunoglobulin variable domains applies in a certain way to T-cell receptor variable domains. The antigenic receptor of the invention, instead of an immunoglobulin heavy chain (VH) variable domain with a specificity for the first epitope (VH(1)) and an immunoglobulin light chain variable domain (VL) with a specificity for the first epitope (VL(1)) may contain an α variable domain TCR α-chains of the TCR receptor with specificity for the first epitope; and the TCR β-chain variable domain of the TCR receptor with specificity for the second epitope. Alternatively or in addition, the antigen receptor of the invention, instead of an immunoglobulin heavy chain (VH) variable domain with a second epitope specificity (VH(2)), and an immunoglobulin light chain variable domain (VL) with a second epitope specificity (VL(2) ) may contain a TCR α-chain variable domain of the TCR receptor with a specificity for a second epitope and a TCR β-chain variable domain of a TCR receptor with a specificity for a second epitope.

Since each antigen-binding site is formed using two domains, each domain may contain a region or fragment of an immunoglobulin or a T-cell receptor, respectively. A single site or fragment, on its own, will not be able to bind an antigen, but when two individual sites or fragments bind, they together form or recreate the antigen-binding structure of the original immunoglobulin or T-cell receptor and, therefore, are able to bind the same antigen, preferably, with the same affinity.

After antigen recognition, the receptors are preferably clustered and the signal is transmitted to the cell. In this regard, "immunoreceptor signal transduction domain" or "T cell signal transduction domain" means a domain that is involved in transduction of an activation signal to a T cell after antigen binding. The possibility of such signal transmission is provided by antigen receptors according to the invention, containing a constant or conserved region of the T-cell receptor chain or a constant or conserved region of the Fc chain of an immune receptor or a portion of a constant or conserved region, such as a constant region of the T-cell receptor alpha chain or a portion thereof, on the same peptide chain, or containing a constant or conserved region of a T-cell receptor chain or a constant or conserved region of the Fc chain of an immune receptor, or a portion of a constant or conserved region, such as a constant region of the T-cell receptor beta chain or a portion thereof, on another peptide chain . In this case, the CD3 complex designates an antigen that is expressed on mature human T cells, thymocytes, and a subpopulation of natural killer cells as part of a multimolecular T cell receptor (TCR) complex. The T cell coreceptor is a protein complex and consists of four separate chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and with the ζ chain, generating an activation signal in T lymphocytes. TCR molecules, ζ-chains and CD3, together, make up the TCR complex. CD3 is responsible for TCR signal transduction. As described in Lin and Weiss, Journal of Cell Science 114, 243-244 (2001), activation of the TCR complex via binding of MHC-presented specific antigenic epitopes results in phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) by Src family kinases, triggering the recruitment of other kinases, entails the activation of T cells, including the release of Ca ²⁺ . Clustering of CD3 on T cells, for example by immobilized anti-CD3 antibodies, leads to T cell activation similar to T cell receptor involvement, but independent of their usual clone specificity.

The antigen receptor signal transduction domain, at least preferably, serves to interact with the cell's native signal transduction complex, e.g., the CD3 complex, which is responsible for signaling antigen binding to the antigen receptor into the cell, resulting in immune cell activation. The identity of the signal transduction domain is only limited by the fact that it has the ability to interact with the native signal transduction complex to induce immune cell activation upon antigen binding to the antigen receptor.

Preferably, the signal transduction domain on one peptide chain forms a dimer with the signal transduction domain on the second strand, for example through disulfide bridges. Preferred signal transduction domains may comprise a T cell receptor chain constant or conserved region, or an immune cell Fc receptor chain constant or conserved region, or a portion of a constant or conserved region. Preferred signal transduction domains may comprise the alpha, beta, gamma or delta chains of the T cell receptor or a portion thereof, as well as the D2 or D3 conserved regions of the immune cell Fc receptor constant domain or portion thereof. In a preferred embodiment, the first peptide chain comprises a T cell receptor alpha chain constant region or portion thereof, and the second peptide chain comprises a T cell receptor beta chain constant region or portion thereof. In this embodiment, the first peptide chain preferably comprises a T cell receptor alpha chain variable region or portion thereof, and the second peptide chain preferably comprises a T cell receptor beta chain variable region or portion thereof, with the variable regions located at the N-terminus of the constant regions. Alternatively, the first peptide chain preferably comprises a T cell receptor beta chain constant region or portion thereof, and the second peptide chain preferably comprises a T cell receptor alpha chain constant region or portion thereof. In this embodiment, the first peptide chain preferably comprises a T cell receptor beta chain variable region or portion thereof, and the second peptide chain preferably comprises a T cell receptor alpha chain variable region or portion thereof, with the variable regions located at the N-terminus of the constant regions. In another embodiment, the first peptide chain comprises a T cell receptor gamma chain constant region or portion thereof, and the second peptide chain comprises a T cell receptor delta chain constant region or portion thereof. In this embodiment, the first peptide chain preferably comprises a T cell receptor gamma chain variable region or portion thereof, and the second peptide chain comprises a T cell receptor delta chain variable region or portion thereof, with the variable regions located at the N-terminus of the constant regions. Alternatively, the first peptide chain comprises the T cell receptor delta chain constant region or portion thereof, and the second peptide chain comprises the T cell receptor gamma chain constant region or portion thereof. In this embodiment, the first peptide chain preferably comprises a T cell receptor delta chain variable region or portion thereof, and the second peptide chain comprises a T cell receptor gamma chain variable region or portion thereof, with the variable regions located at the N-terminus of the constant regions. Optionally, the signal transduction domains or variable region of the T cell receptor chain or portion thereof can be modified such that additional disulfide bonds are created between the chains, resulting in more efficient formation and increased stability of the dimer.

Without wishing to be limited to a specific mechanism of action, it is believed that the two antigen receptor peptide chains of the invention, when expressed on the surface of an immune cell, form a dimer due to an interaction (e.g., disulfide bonding) between at least separate immunoreceptor signaling domains on the two chains, and also form a complex with the endogenous CD3 complex involved in the physiological process of signal transduction from the T-cell receptor. However, the invention may also include direct fusion to CD3ζ or any other immune cell signaling domain (CD3, CD3 subunit of FcγR) instead of the TCR Cα and Cβ domains. It is believed that when the antigen binds, the signal is transmitted into the cell, leading to the activation of the immune cell and the formation of an antigen-specific immune response. Interchain binding is also believed to result in a more stable antigen-antigen receptor-endogenous CD3 signal transduction module, the increased stability of which, in turn, provides more effective stimulation of an antigen-specific immune response, compared with monovalent receptors and bivalent receptors capable of only intrachain binding. with antigen. This increased stability is believed to allow for the use of human-only immunoreceptor signal transduction domains (eg, with minimal or no substitutions in the human amino acid sequence to an amino acid sequence from an organism of another species, such as a mouse). In this way, any possible unwanted immune response to the antigen receptor itself can be avoided.

The antigen receptors of the invention, or their peptide chains, in addition to domains forming antigen-binding sites, variable regions of the T-cell receptor chain or portion thereof, immunoreceptor signaling domains including CD3ζ, or any other immune cell signaling domain, may also contain one or more costimulatory domains. Costimulatory domains serve to enhance the proliferation and survival of T cells, such as cytotoxic T cells, upon binding of an antigen receptor to a target component. The identity of co-stimulatory domains is limited in that they have the ability to enhance cell proliferation and increase survival upon binding of the target component to the antigen receptor. Relevant costimulatory domains include CD28, CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNF) receptor family, CD134 (OX40), a member of the TNFR superfamily of receptors, and CD278 (ICOS), a costimulatory molecule of the CD28 superfamily expressed on activated T -cells. One skilled in the art will appreciate that sequence variants of these co-stimulatory domains above can be used without adversely affecting the invention, as long as the variants have the same or similar activity as the domain they are modeled on. Such variants have at least about 80% sequence identity with the amino acid sequence of the domain from which they are formed. In some embodiments, antigen receptor constructs or peptide chains thereof contain two co-stimulatory domains. While individual combinations include all possible variants of the four labeled domains, specific examples include CD28+CD137 (4-1BB) and CD28+CD134 (OX40).

The antigen receptors of the present invention, or peptide chains thereof, may contain one or more co-stimulatory domains and immunoreceptor signal transduction domains linked in an N-terminal to C-terminal direction. However, the antigen receptors of the present invention or their peptide chains are not limited to this order, and other orders are acceptable and include immunoreceptor signal transduction domains and one or more co-stimulatory domains.

It is understood that since the domains that form the antigen binding sites must be free in order to bind the antigen, the location of these domains in the fusion protein is usually such as to achieve exposure of the region on the outer surface of the cell. Similarly, since co-stimulatory and immunoreceptor signaling domains help stimulate T cell activity and proliferation, the fusion protein will typically expose these domains to the interior of the cell. Antigenic receptors may include additional elements such as a signal peptide to ensure that the fusion protein is correctly exported to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain (or spacer region) that confers flexibility to the domains. , forming antigen-binding sites and provides a strong bond with the antigen.

Optionally, the antigen receptors of the invention may also contain a linker, which linker may be an arbitrary amino acid sequence or other chemical compound useful as a spacer between amino acid sequences. The linker is generally designed to provide flexibility and resistance to proteases. For example, the linker may be between the first and second domains on the first peptide chain and/or between the first and second domains on the second peptide chain of the combined antigen receptor of the invention. Optionally, the linker may be between the domains that form the antigen-binding sites and the T-cell receptor chain variable region or portion thereof. Any type of linker known in the art that allows the domains to form an antigen-binding site or does not interfere with antigen binding is within the scope of the invention. In specific embodiments, the linker may be an arbitrary amino acid sequence and may be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 in length. , 90, 95 or at least 100 amino acid residues. The amino acid linker is typically enriched in glycine for flexibility, and serine and threonine for solubility. In one embodiment, the linker is one or more (1, 2, 3, 4, 5, 6, 7, 8, or 9) repeats of four glycine residues followed by a serine residue (Gly4Ser). In some embodiments, the linker is an antibody hinge region or a fragment thereof.

The antigen receptor of the invention may also contain another domain that anchors the antigen receptor to the membrane, such as, for example, the classical transmembrane domain. Preferably, the transmembrane domain is included in or part of the signal transduction domain.

In other embodiments, the antigen receptors or antigen receptor peptide chains of the invention may also contain other domains, such as additional domains involved in antigen binding or stimulating antigen binding, signal sequences for membrane-bound expression or for secretion, domains that allow for increased dimerization, and the transmembrane domain when it is no longer part of the immunoreceptor signal transduction domain. In some embodiments, the transmembrane domain may be a hydrophobic alpha helix that spans the membrane.

Preferably, the signal sequence or signal peptide is a sequence or peptide that allows adequate passage through the secretory pathway and expression on the cell surface so that the antigen receptor, for example, can bind an antigen found in the extracellular environment. Preferably, the signal sequence or signal peptide is (or is) capable of being cleaved and removed from mature peptide chains. The signal sequence or signal peptide is preferably (or selected) based on the cell or organism in which the peptide chains are produced.

According to a specific embodiment of the present invention, the peptide chain of the combined antigen receptor of the invention may be the structure: NH2 signal peptide - first domain involved in antigen binding - optional linker - second domain involved in antigen binding - optional linker - variable region of the T-cell chain receptor or its site-the domain of signal transmission from the immunoreceptor-COOH.

Examples of antigen receptors of the present invention include, but are not limited to, receptors formed by first and second peptide chains having the structure shown in Table I below (VH denotes an immunoglobulin heavy chain variable region or portion thereof; VL denotes an immunoglobulin light chain variable region or a portion thereof). region; V1 and V2 are T-cell receptor chain variable regions or regions that form a dimer with each other, C1 and C2 are immunoreceptor signal transduction domains that form a dimer with each other, e.g., the constant or conserved region of the immune receptor Fc chain or a constant or conserved region of a T-cell receptor chain or a portion of a constant or conserved region). If C1 and C2 are each T-cell receptor chain constant regions, V1 and C1 are from the same T-cell receptor chain, and V2 and C2 are preferably from the same T-cell receptor chain, with the T- cell receptor is preferably derived from the same T-cell receptor. In particular, according to one of the options, V1-C1 essentially corresponds to the sequence of the T-cell receptor chain (TCR alpha or TCR beta) and V2-C2 essentially corresponds to the sequence of the complementary chain of the T-cell receptor (TCR beta, if V1-C1 is TCR alpha, or TCR alpha if V1-C1 is TCR beta).

As stated above, the antigen receptor comprises an immunoglobulin heavy chain variable domain (VH) with a first epitope specificity (VH(1)), an immunoglobulin light chain variable domain (VL) with a first epitope specificity (VL(1)), a heavy chain variable domain an immunoglobulin chain (VH) with a specificity for a second epitope (VH(2)) and an immunoglobulin light chain (VL) variable domain with a specificity for a second epitope (VL(2)), wherein the first and second epitopes may be the same or different and may be located on the same or different antigens. According to one variant, VH(1) is able to interact and form an antigen binding site with VL(1) and VH(2) is able to interact and form an antigen binding site with VL(2), while VH(1) is not able to interact and form an antigen binding site with VL(2) and VH(2) is not able to interact and form an antigen-binding site with VL(1). However, according to another embodiment, VH(1) is able to interact and form an antigen-binding site with VL(1) as well as VL(2), and VH(2) is able to interact and form an antigen-binding site with VL(2) as well as VL(1 ). In the latter embodiment, VH(1) and VH(2) may be identical or at least derived from the same immunoglobulin, and VL(1) and VL(2) may be identical or at least derived from the same immunoglobulin.

In specific embodiments, the V1 and V2 domains of the first and second peptide chains shown in Table 1 are T-cell receptor alpha and beta chain variable regions, respectively, or portions thereof. In specific embodiments, the C1 and C2 domains of the first and second peptide chains listed in Table 1 are T-cell receptor alpha and beta chain constant regions, respectively, or portions thereof.

In one embodiment, the V1 and C1 domains of the first peptide chain listed in Table 1 are the variable and constant regions of the T-cell receptor alpha chain or portions thereof. In this embodiment, the V2 and C2 domains of the second peptide chain listed in Table 1 are preferably the variable and constant regions of the T-cell receptor beta chain or regions thereof.

In one embodiment, the V1 and C1 domains of the first peptide chain listed in Table 1 are the variable and constant regions of the T-cell receptor beta chain or portions thereof. In this embodiment, the V2 and C2 domains of the second peptide chain listed in Table 1 are preferably the variable and constant regions of the T-cell receptor alpha chain or portions thereof.

In a preferred embodiment, when two domains of one peptide chain are both an immunoglobulin heavy chain variable region or a portion thereof and two domains of the other chain are both an immunoglobulin light chain variable region or a portion thereof, there is a linker between the first and second domain of both peptide chains. The linker can be an arbitrary amino acid sequence between 10 and 25 amino acids in length, more preferably 15 amino acids in length. In a specific embodiment, the linker contains 3 repeats of a 5-mer amino acid sequence (Gly4Ser).

In some embodiments of the present invention, the amino acid sequences of the first and second peptide chains, such as those containing one or more domains that form antigen-binding sites, a T-cell receptor chain variable region or portion thereof, or an immunoreceptor signal transduction domain, are mammalian, preferably murine, sequences, and more preferably are of human origin. In one embodiment, the amino acid sequences are of human origin, but murine sequences have been introduced into them by replacing one or more amino acids in the human sequence with an amino acid at the corresponding position in the murine sequence. Such a substitution may contribute to a greater degree of dimerization or stability, or the ability to transmit a signal to the cell after antigen binding. In yet another embodiment, the amino acid sequences are of murine origin and have been humanized.

According to the present invention, an antigen receptor can replace the function of a T cell receptor as described above, and in particular can confer reactivity, eg, cytolytic activity, on a cell, such as a T cell, as described above. However, in contrast to the binding of the T cell receptor to the antigenic peptide-MHC complex as described above, the antigenic receptor in some embodiments can bind to an antigen, especially when expressed on a cell surface.

The amino acid sequences of the peptide chains, including any of the domains or linkers, may be modified. For example, as will be appreciated by those skilled in the art, antibody and T cell receptor variable region sequences can be modified without losing the ability to bind to a target, and therefore the amino acid sequence of antigen binding sites can be similarly modified without losing the ability to bind to a target. For example, the amino acid sequence of the domain forming the antigen binding site may be identical or highly homologous to the variable region of the antibody from which it is derived. By "highly homologous" is meant a sequence in which 1 to 5, preferably 1 to 4, eg 1 to 3 substitutions or 1 or 2 substitutions can be made. In one embodiment, the peptide chain may include natural amino acids and non-natural amino acids. In another embodiment, the peptide chain simply includes naturally occurring amino acids. The term "unnatural amino acid" refers to an amino acid having a structure different from the structures of the 20 natural amino acids. Since non-natural amino acids have a structure similar to that of naturally occurring amino acids, non-natural amino acids can be classified as derivatives or analogues of the naturally occurring amino acids in question.

In one embodiment, the amino acid sequence of one or more T-cell receptor variable regions or portions thereof, especially one that does not form antigen-binding sites, can be modified to remove (residual) binding to its antigen. In particular, such "silencing" modification can be carried out by introducing one or more mutations in the CDR3 variable region of TCR alpha and/or TCR beta.

The present invention also covers derivatives of the antigen receptors and peptide chains described in this application. According to the invention, "derivatives" are modified forms of proteins and peptides. Such modifications include any chemical modification and include single or multiple substitutions, deletions and/or additions of any antigen receptor or peptide chain associated molecules such as carbohydrates, lipids and/or proteins or peptides. In one embodiment, "derivatives" of proteins or peptides include modified analogs obtained by glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, protection/blocking groups, proteolytic cleavage, or antigen binding. The term "derivative" also covers all functional chemical equivalents of said antigen receptors and peptide chains. Preferably, the modified antigen receptor or its peptide chain has stronger binding or dimerization and/or increased activation of the immune system.

The cells used in connection with the antigen receptor system of this invention are preferably T cells, in particular cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer cells (NK cells) and lymphokine-activated killer cells (LAK). Upon activation, each of these cytotoxic lymphocytes triggers the destruction of target cells. For example, cytotoxic T cells trigger the destruction of target cells in one or both of the following ways. First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulosin. Perforin and granulosin create pores in the target cell, and granzymes enter the cell and trigger a caspase cascade in the cytoplasm that induces apoptosis (programmed cell death). Second, apoptosis can be induced through the interaction of the Fas-Fas ligand system between T cells and target cells. The cytotoxic lymphocytes are preferably autologous cells, although heterologous or allogeneic cells may be used.

The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably antigen receptors such as antibodies or the B cell receptor (BCR). Immunoglobulins are characterized by a structural domain, that is, an immunoglobulin domain having a characteristic fold of immunoglobulin (Ig) chains. The term includes membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are usually part of the BCR. Soluble immunoglobulins are commonly referred to as antibodies. Immunoglobulins usually contain several chains, usually two identical heavy chains and two identical light chains, which are linked by disulfide bonds. These chains are mainly composed of immunoglobulin domains such as the V _L (light chain variable domain), C _L (light chain constant domain) and C _H (heavy chain constant region) of the C _H 1, C _H 2, C _H 3 domains. and _CH 4. There are five types of immunoglobulin heavy chains in a mammal, namely α, δ, ε, γ and μ, which account for the presence of different classes of antibodies, namely IgA, IgD, IgE, IgG and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy terminus. Mammals have two types of light chains, namely lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved across immunoglobulin isotypes, while the variable region is highly diverse and is responsible for antigen recognition.

The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain contains a heavy chain variable region (referred to herein as VH) and a heavy chain constant region. Each light chain contains a light chain variable region (referred to herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), between which are regions that are more conserved and called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the amino to carboxy direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The term "monoclonal body" as used in this application refers to a population of antibody molecules of the same molecular composition. A monoclonal antibody exhibits one binding specificity and affinity. In one embodiment, the monoclonal antibodies are produced by a hybridoma that includes a B cell derived from a non-human animal, such as a mouse, fused to an immortalized cell.

The term "recombinant antibody" as used in this application includes all antibodies that are obtained, expressed, created or isolated by a recombinant method, for example (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transchromosomal with respect to genes immunoglobulin or hybridoma derived therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g. from a transfectoma, (c) antibodies isolated from a library of recombinant, combinatorial antibodies, and (d) antibodies obtained, expressed, created or isolated by any other methods that involve the splicing of immunoglobulin gene sequences into other DNA sequences,

The term "human antibody" as used herein includes antibodies having variable and constant regions derived from human immunoglobulin germline sequences. Human antibodies may include amino acid residues not encoded by human immunoglobulin germline sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation).

The term "humanized antibody" refers to a molecule having an antigen binding site that is essentially derived from an immunoglobulin of a non-human species, where the rest of the structure of the immunoglobulin molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may include either complete variable domains fused to constant domains or only complementarity determining regions (CDRs) grafted onto the appropriate framework regions in the variable domains. Antigen binding sites may be wild-type or modified by substitution of one or more amino acids, for example, modified to more closely resemble human immunoglobulin. Some forms of humanized antibodies retain all CDR sequences (eg, a humanized mouse antibody that contains all six CDRs from a mouse antibody). Other forms contain one or more CDRs that are altered from the original antibody.

The term "chimeric antibody" refers to those antibodies in which one portion of each amino acid sequence of the heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining chain segment is homologous to corresponding sequences from other types. Typically, the variable region of both the light and heavy chains mimics the variable regions of antibodies derived from one mammalian species, while the constant regions are homologous to antibody sequences derived from another species. One distinct advantage of chimeric antibodies is that the variable region can be readily obtained from sources known to date, using readily available B cells or hybridomas from non-human hosts, in combination with constant regions derived from, for example, populations of human cells. While the variable region has the advantage of being easy to obtain and the source does not affect specificity, a human-derived constant region is less likely to elicit an immune response in humans when injected with antibodies compared to a constant region from a non-human source. However, the definition is not limited to this particular example.

Antibodies of the present invention can be obtained from various species, including, but not limited to, mouse, rat, rabbit, hamster, and human.

The antibodies described in this application include IgA, such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, especially IgG1, kappa or IgG1 isotypes, lambda (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), IgG3 antibody (eg IgG3, κ, λ) or IgG4 antibody (eg IgG4, κ, λ).

The antigen receptors described in this application may include antigen-binding sites of one or more antibodies. The terms "antigen-binding site" of an antibody (or simply "binding site") or "antigen-binding fragment" of an antibody (or simply "binding fragment") or similar terms refer to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the function of an antibody to bind an antigen can be performed by fragments of a full length antibody. Examples of binding fragments encompassed by the term "antigen binding site" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab') ₂ fragments, bivalent fragments, comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of one antibody arm, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546) which consist of a VH domain; (vi) isolated complementarity determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, which may optionally be joined by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected using recombinant techniques with a synthetic linker that allows them to form a single protein chain in which the VL and VH regions pair to form monovalent molecules ( known as single chain Fv (scFv) see for example Bird et al (1988) Science 242: 423-426 and Huston et al (1988) Proc Natl Acad Sci USA 85: 5879-5883) . Such single chain antibodies are also encompassed by the term "antigen binding fragment" of an antibody. Another example are immunoglobulin-binding domain fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge polypeptide (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge, and (iii) a heavy chain CH3 constant region. an immunoglobulin chain fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Fusion proteins "binding domain-immunoglobulin" are also described in US patent applications No. 2003/0118592 and 2003/0133939. These antibody fragments were obtained using conventional methods known to those skilled in the art, and such fragments were screened for applicability in the same manner as for intact antibodies.

A single chain variable fragment (scFv) is a fusion protein between the heavy (VH) and light (VL) immunoglobulin variable regions connected by a peptide linker. This linker can connect either the N-terminus of the VH to the C-terminus of the VL, or vice versa. Bivalent (or bivalent) single chain variable fragments (di-scFvs, bi-scFvs) can be generated by linking two scFvs. This can be done by making a single stranded peptide chain with two VH and two VL regions to form tandem scFvs.

The term "binding domain" or simply "domain" in connection with the present invention is characterized by a structure, for example, an antibody, which binds/interacts with a given target structure/antigen/epitope, optionally by interacting with another domain. These domains are referred to as "antigen binding sites" according to the invention.

Antibodies and antibody derivatives are useful for generating binding domains such as antibody fragments, in particular for generating VL and VH regions.

The binding domains for an antigen, which may be located in the antigen receptor, have the ability to bind to the antigen (antigen targeting), that is, the ability to bind to an epitope (epitope targeting) located in the antigen, preferably an epitope located in the extracellular domain antigen. Preferably, the binding domains for the antigen are specific for the antigen. Preferably, the binding domains for the antigen bind to the antigen expressed on the cell surface. In particular preferred embodiments, the binding domains for an antigen bind to native antigen epitopes found on the surface of living cells.

All antibodies and antibody derivatives, such as antibody fragments described in this application, for the purposes of this invention are covered by the term "antibody".

Antibodies can be obtained by various methods, including the usual procedure using monoclonal antibodies, for example, using the standard method of hybridization of somatic cells, described in the publication of Koehler and Milstein, see Nature 256: 495 (1975). Although somatic cell hybridization methods are preferred, other methods for producing monoclonal antibodies can in principle be used, such as viral or oncogenic transformation of B lymphocytes or phage display using antibody gene libraries.

The preferred system for producing hybridomas that secrete monoclonal antibodies is the murine system. The production of a hybridoma using a mouse is a well established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg mouse myeloma cells) and fusion technique are also known.

Other preferred animal systems for producing hybridomas that secrete monoclonal antibodies are the rat and rabbit systems (for example, those described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92: 9348 (1995), see also Rossi et al., Am J Clin Pathol 124: 295 (2005)).

To generate antibodies, mice can be immunized with peptides conjugated to carriers derived from the sequence of the antigen, i.e., the sequence against which the antibodies are to be directed, enriched in a population of recombinantly expressed antigen or fragments thereof, and/or cells expressing the antigen, as described in this application. Alternatively, mice can be immunized with DNA encoding the antigen or fragments thereof. In the event that immunization using a purified or enriched antigen population does not result in antibody production, mice can be immunized with antigen expressing cells, such as a cell line, to promote an immune response.

The immune response can be monitored during the immunization protocol, with plasma and serum samples obtained using plasma and serum samples obtained from the tail vein or by blood sampling by retroorbital phlebotomy. Mice with sufficient immunoglobulin titers can be used for fusion. It is possible to perform intraperitoneal or intravenous reimmunization of mice with cell-expressing antigen 3 days before sacrifice and removal of the spleen to increase the rate of specific antibody secretion by hybridomas.

To generate monoclonal antibody-producing hybridomas, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Using the immunofluorescent method and FACS analysis using cells expressing antigens, antibodies with specificity for the antigen can be identified. The antibody-secreting hybridomas can be replated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned using limiting dilution. Stable subclones can then be cultured in vitro to generate antibody for characterization in tissue culture medium.

The ability of antibodies and other binding agents to bind to antigens can be determined using standard binding assays (eg, ELISA, Western blotting, immunofluorescence, and flow cytometric analysis).

According to the present invention, the term "binding" preferably refers to specific binding.

According to the present invention, an agent, such as an antigen receptor, is capable of binding to a given target (targeting a given target) if it has significant affinity for said given target and binds to said given target in a standard assay. "Affinity" or "binding affinity" is often measured by the dissociation equilibrium constant (K _D ). Preferably, when the term "high affinity" refers to the binding of a given target with a dissociation constant (K _D ) value of 10 ^-5 M or less, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, or 10 ^-12 M or less.

An agent is (substantially) unable to bind (target) a target unless it has a high affinity for said target and does not bind significantly, in particular binds detectably, to said target in a standard assay. Preferably, such an agent does not bind definitively to said target if present at a concentration of up to 2, preferably up to 10, more preferably up to 20, in particular up to 50 or 100 µg/ml or more. Preferably, when an agent does not have high affinity for a target, if it binds to said target with a K _D value that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times greater than K _D when binding a given target with which this agent is able to bind. For example, if the K _D value for binding an agent to a target for which the agent is able to bind is 10 ^-7 M, the K _D for binding to a target for which the agent has no significant affinity will be at least 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

An agent is specific for a given target if it is able to bind to said given target, that is, it does not have high affinity for other targets and does not bind to other targets to a significant extent in standard assays. Preferably, the agent is specific for a given target if the affinity and binding to such other targets does not significantly exceed the affinity for or binding to proteins that are not related to the given target, such as bovine serum albumin (BSA), casein or albumin human serum (HSA). Preferably, an agent is specific for a given target if it binds to said target with a K _D value that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times less than than the K _D value when binding to a target for which it is not specific. For example, if the K _D value for binding an agent to a target for which it is specific is 10 ^-7 M, the K _D value for binding to a target for which it is not specific will be at least 10 ^{- 6} M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

The binding of an agent to a target can be determined using a suitable method; see, for example, see Berzofsky et al., "Antibody-Antigen Interactions". In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described in this application. Affinity can be easily determined using standard methods such as equilibrium dialysis; using the biosensor BIAcore 2000, using conventional methods developed by the manufacturer; radioimmunological method of analysis using radiolabeled target antigen; or other method known to the person skilled in the art. Affinity results can be analyzed, for example, by the method described by Scatchard et al. at Ann NY Acad. Sci, 51:660 (1949). The measured value of affinity during the interaction of specific antibodies-antigen can vary if it is measured under different conditions, for example, it can be a different salt concentration, a different pH value. Thus, measurement of affinity and other antigen binding parameters, eg K _D , IC ₅₀ , is preferably performed using standardized antibody and antigen solutions and a standardized buffer.

The present invention may involve the introduction, ie, transfection, of nucleic acids encoding antigen receptors into cells, such as T cells, in vitro or in vivo.

For the purposes of this invention, the term "transfection" includes the introduction of a nucleic acid into a cell or the absorption of a nucleic acid by a cell, while such a cell may be in the body of a subject, for example, a patient. Thus, according to the present invention, the nucleic acid transfection cell described in this application may be in vitro or in vivo, for example, the cell may form part of an organ, tissue and/or body of a patient. In accordance with this invention, transfection may be transient or stable. For some use cases, transfection is sufficient if the transfected genetic material is only transiently expressed. Since the nucleic acid introduced during transfection usually does not integrate into the nuclear genome, the foreign nucleic acid will be diluted during mitosis or degraded. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the cell's genome and its daughter cells, stable transfection must occur. The RNA can be transfected into cells for transient expression of its encoded protein.

According to the present invention, any method can be used to introduce, ie to transfer or transfect, nucleic acids into a cell. Preferably, the nucleic acid, such as RNA, is transfected into cells by standard methods. Such methods include electroporation, lipofection and microinjection. In accordance with one particularly preferred embodiment of the present invention, RNA is introduced into cells by electroporation. Electroporation or electropermeabilization refers to a significant increase in the electrical conductivity and permeability of the cytoplasmic membrane caused by the application of an external electric field. It is commonly used in molecular biology as a method of introducing a substance into a cell. According to the present invention, it is preferable that the introduction of a nucleic acid encoding a protein or peptide into cells results in the expression of said protein or peptide.

Various methods can be used to introduce antigen receptor constructs into T cells, including transfection using non-viral DNA, transposon-based systems, and viral-based systems. Transfection using non-viral DNA is characterized by a low risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain an integrating element. Virus-based systems include the use of γ-retroviruses and lentiviral vectors. γ-Retroviruses are relatively easy to produce, efficiently and consistently transduce T cells and have been shown to be tentatively safe in terms of integration into primary human T cells. Lentiviral vectors also efficiently and consistently transduce T cells, but are more expensive to produce. They are also potentially more secure than retrovirus-based systems.

For transfection of cells in vivo, a pharmaceutical composition comprising a nucleic acid encoding an antigen receptor can be used. A delivery vehicle that targets the nucleic acid to a specific cell, such as a T cell, can be administered to a patient resulting in transfection that occurs in vivo.

According to the present invention, it is preferable to administer the nucleic acid encoding the antigen receptor in "naked form" or on a carrier. Carriers, such as lipid carriers, contemplated for use in accordance with the present invention include any substances or excipients with which a nucleic acid, such as RNA, can be associated, for example, by complexing with the nucleic acid or by forming vesicles in which the nucleic acid is enclosed or encapsulated. nucleic acid. This can lead to increased stability of the nucleic acid compared to the naked nucleic acid. In particular, the stability of the nucleic acid in the blood can be improved. For example, RNA formulations in the form of nanoparticles of defined size can be used, such as lipoplexes of RNA and liposomes, for example, lipoplexes including DOTMA and DOPE or DOTMA and cholesterol.

As used herein, the term "nanoparticle" refers to any particle, in particular a nucleic acid particle, having a diameter making the particle suitable for systemic, in particular parenteral, administration, typically less than 1000 nanometers (nm) in diameter. In some embodiments, the nanoparticle has a diameter of less than 600 nm. In some embodiments, the nanoparticle has a diameter of less than 400 nm.

Used in this application, the term "composition of nanoparticles" or similar terms refers to any substance that contains at least one nanoparticle. In some embodiments, the nanoparticle composition is a homogeneous mass of nanoparticles. In some embodiments, the nanoparticle compositions are a dispersion or an emulsion. In general, a dispersion or emulsion is formed when at least two immiscible materials are combined.

The term "lipoplex" or "nucleic acid lipoplex", in particular "RNA lipoplex", refers to a complex of lipids and nucleic acids, in particular RNA. Lipoplexes form spontaneously when cationic liposomes, which often also include a neutral "helper" fluid, are mixed with nucleic acids.

Cationic lipids, cationic polymers and other substances with positive charges can form complexes with negatively charged nucleic acids. These cationic molecules can be used to complex with nucleic acids to form, for example, so-called lipoplexes or polyplexes, respectively, and these complexes have been shown to deliver nucleic acids to cells.

Nucleic acid preparations in the form of nanoparticles for use according to the present invention can be obtained according to various protocols and from various compounds that form complexes with nucleic acids. Typical complexing agents are lipids, polymers, oligomers or amphiphiles. In one embodiment, the complexing compound comprises at least one agent selected from the group consisting of protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine, or histone.

In accordance with this invention, protamine is suitable as a cationic carrier. The term "protamine" refers to any of various relatively low molecular weight strongly basic proteins that are rich in arginine and are especially associated with DNA in place of somatic histones in sperm cells of various animals (such as fish). In particular, the term "protamine" refers to proteins found in fish semen that are strongly basic, water-soluble, do not coagulate when heated, and upon hydrolysis form mainly arginine. In their pure form, they are used in a formulation based on long-acting insulin and to neutralize the anticoagulant effect of heparin.

As used herein, the term "protamine" is intended to include any protamine amino acid sequence derived or derived from native or biological sources, including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof. In addition, this term covers (synthesized) polypeptides that are artificial and specially designed for specific purposes and cannot be isolated from native or biological sources.

The protamine used according to the present invention may be sulfonated protamine or protamine hydrochloride. In a preferred embodiment, the source of protamine used to prepare the nanoparticles described herein is Protamine 5000, which contains protamine in excess of 10 mg/ml (5000 heparin neutralizing units per ml) in isotonic saline.

Liposomes are microscopic lipid vesicles, often having one or more bilayers of a vesicle-forming lipid, such as a phospholipid, and are capable of encapsulating a drug. Various types of liposomes can be used in the context of the present invention, including, without limitation, multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), sterically stabilized liposomes (SSLs), multivesicular vesicles (MVs), and large multivesicles. vesicles (LMV), as well as other two-layer forms known in this field. The size and lamellarity of the liposome will depend on the method of preparation, and the choice of the type of vesicles to be used will depend on the preferred route of administration. There are several other forms of supramolecular organization in which lipids can be found in an aqueous medium, including lamellar phases, hexagonal and inverse hexagonal phases, cubic phases, micelles, reverse micelles consisting of monolayers. These phases can also be obtained in combination with DNA or RNA, and interaction with RNA and DNA can significantly affect the phase state. The phases described may be present in the nucleic acid nanoparticle formulations of the invention.

Any suitable method can be used to form nucleic acid lipoplexes from nucleic acid and liposomes as long as it provides the formation of the intended nucleic acid lipoplexes. Liposomes can be formed using standard methods such as the vapor reversal (REV) method, the ethanol injection method, the dehydration-rehydration (DRV) method, the use of ultrasound, or other suitable methods.

Once the liposomes are formed, the liposomes can be sorted by size to obtain a composition of liposomes having a substantially uniform size range.

Lipids forming bilayers usually have two hydrocarbon chains, especially acyl chains, and a head group, either polar or non-polar. Bilayered lipids are composed of either naturally occurring lipids or synthetically derived lipids, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbon chains typically contain about 14-22 carbon atoms, and have various degrees of unsaturation. Other suitable lipids for use in the composition of this invention include glycolipids and sterols such as cholesterol and its various analogs, which can also be used in liposomes.

Cationic lipids typically contain a lipophilic moiety such as a sterol, acyl, and diacyl chain and carry a net positive charge. The head group of a lipid usually carries a positive charge. The cationic lipid preferably contains a positive charge of 1 to 10, more preferably a positive charge of 1 to 3, and even more preferably a positive charge of 1. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane ( DOTMA); dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamine trifluoroacetate (DOSPA). Preferred are DOTMA, DOTAP, DODAC and DOSPA. Most preferred is DOTMA.

In addition, the nanoparticles described in this application preferably also include a neutral lipid due to structural stability and the like. The neutral lipid may be appropriately selected based on the delivery efficiency of the nucleic acid-lipid complex. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine , diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, sterol and cerebroside. DOPE and/or DOPC are preferred. DOPE is the most preferred. In the case where the cationic liposome includes both a cationic lipid and a neutral lipid, the mole ratio of the cationic lipid to the neutral lipid can be appropriately determined in consideration of the stability of the liposome and the like.

According to one of the options, the nanoparticles described in this application may include phospholipids. The phospholipids may be glycerophospholipids. Examples of glycerophospholipids include, but are not limited to, three types of lipids: (i) zwitterionic phospholipids, which include, for example, phosphatidylcholine (PC), egg yolk phosphatidylcholine, PC from natural soybeans, in partially hydrogenated or fully hydrogenated form, dimyristoylphosphatidylcholine (DMPC) , sphingomyelin (SM); (ii) negatively charged phospholipids which include, for example, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), dipalmitoyl-PG, dimyristoylphosphatidylglycerol (DMPG); synthetic derivatives in which the conjugate is a zwitterionic phospholipid, negatively charged, such as in the case of methoxy-polyethylene glycol-distearoyl-phosphatidylethanolamine (mPEG-DSPE); and (iii) cationic phospholipids which include, for example, phosphatidylcholine or sphingomyelin whose phosphomonoesters have been O-methylated to form cationic lipids.

The association of a nucleic acid with a lipid carrier can occur, for example, by filling the intercellular space of the carrier with the nucleic acid so that the carrier physically captures the nucleic acid, or by forming a covalent, ionic or hydrogen bond, or by adsorption upon non-specific bonding. Whatever the mechanism of association, the nucleic acid must retain its therapeutic, ie coding, properties.

According to the present invention, the nucleic acid encoding the antigen receptor, in accordance with one embodiment, is an RNA, preferably mRNA. RNA is preferably obtained by in-vitro transcription.

The term "nucleic acid" as used in this application includes DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized. The nucleic acid can be single stranded or double stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, the nucleic acid is preferably an isolated nucleic acid.

Nucleic acids may be enclosed in a vector. The term "vector" as used in this application includes any vectors known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus, or artificial chromosomal vectors such as as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). These vectors include expression as well as cloning vectors. Expression vectors include plasmids as well as viral vectors, and typically contain the desired coding sequence and the corresponding DNA sequences necessary to express the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in vitro. expression systems. Cloning vectors are typically used to construct and amplify some desired DNA fragment and may lack the functional sequences required to express the desired DNA fragments.

In the context of the present invention, the term "RNA" refers to a molecule that includes ribonucleotide residues and preferably consists entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. The term encompasses double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant-derived RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such changes may include the addition of non-nucleotide material, eg, at the end(s) of the RNA or intrastrand, eg, within one or more nucleotides of the RNA. Nucleotides in RNA molecules may also include non-standard nucleotides such as non-natural or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be considered analogs or analogs of naturally occurring RNAs.

According to the present invention, the term "RNA" includes and preferably refers to "mRNA", which means "messenger RNA", and refers to a "transcript", which can be obtained using DNA as a template and encodes a peptide or protein. An mRNA typically includes a 5' untranslated region (5'UTR) encoding a peptide or protein and a 3' untranslated region (3'UTR). mRNA has a limited half-life in cells and in vitro. Preferably, the mRNA is obtained by in vitro transcription using template DNA. According to one embodiment of the present invention, RNA is obtained by in vitro transcription or by chemical synthesis. The technique of in vitro transcription is known to the person skilled in the art. For example, there are a variety of commercially available in vitro transcription kits.

According to one embodiment of the present invention, the RNA is a self-replicating RNA, such as a single-stranded self-replicating RNA. In one embodiment, the self-replicating RNA is a single stranded RNA with positive polarity. In one embodiment, the self-replicating RNA is viral RNA or RNA derived from viral RNA. In one embodiment, the self-replicating RNA is alpha viral genomic RNA or derived from alpha viral genomic RNA. In one embodiment, the self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is a Semliki forest virus. According to one variant, self-replicating RNA contains one or more transgenes, at least one of these transgenes, encoding agents described in this application. In one embodiment, if the RNA is viral RNA or derived from viral RNA, the transgenes may partially or completely replace viral sequences, such as viral sequences encoding structural proteins. In one embodiment, the self-replicating RNA is RNA transcribed in vitro.

In order to enhance the expression and/or increase the stability of the RNA used according to the present invention, it can be modified, preferably without changing the sequence of the expressed peptide or protein.

The term "modification" in the context of RNA, used according to this invention, includes any modification of the RNA, which itself is present in the specified RNA.

According to one embodiment of the present invention, the RNA used according to the invention does not contain uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with phosphatase.

According to the present invention, RNA may contain modified natural or synthetic ribonucleotides in order to increase its stability and/or reduce cytotoxicity. For example, according to one variant in the RNA used according to this invention, 5-methylcytidine is replaced partially or completely, preferably completely, by cytidine. Alternatively or additionally, according to one embodiment, in the RNA used according to this invention, pseudouridine may be replaced partially or completely, preferably completely, by uridine.

In one embodiment, the term "modification" refers to providing the RNA with a 5' cap or 5' cap analog. The term "5' cap" refers to a cap structure found at the 5' end of an mRNA molecule and this structure typically consists of a guanosine nucleotide linked to the mRNA via an atypical 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at the 7-position. The term "normal 5'-cap" refers to the 5'-cap of naturally occurring RNA, preferably the 7-methylguanosine (m7G) cap. In the context of this invention, the term "5'-cap" embraces a 5'-cap analog that resembles the structure of an RNA cap and is modified to confer RNA stabilization when attached to it, preferably in vivo and/or in a cell.

Providing RNA with a 5'-cap or 5'-cap analog can be achieved by in vitro transcription of template DNA in the presence of said 5'-cap or 5'-cap analog, said 5'-cap being introduced into the generated RNA strand by co-transcription or the RNA can be obtained, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-translationally using capping enzymes, for example, vaccinia virus capping enzymes.

The RNA may include other modifications. For example, a modification of the RNA used according to the invention may be the insertion or truncation of a naturally occurring poly(A) tail, or alteration of the 5' or 3' untranslated regions (UTRs), such as introducing a UTR that is not related to the coding region of said RNA. eg insertion of one or more, preferably two copies of the 3'UTR derived from a globin gene such as alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

Therefore, in order to increase the stability and/or enhance the expression of the RNA used according to the present invention, it can be modified so as to be in conjunction with a poly-A sequence, preferably containing a chain length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues. In a particularly preferred embodiment, the poly A sequence is approximately 120 adenosine residues in length. In addition, the introduction of two or more 3'-untranslated regions (UTRs) into the 3'-untranslated region of the RNA molecule can lead to an increase in the efficiency of translation. In one specific embodiment, the 3'UTR is derived from the human β-globin gene.

The term "stability" in relation to RNA refers to the "half-life" of RNA. The term "half-life (half-life)" refers to the period of time required for the disappearance of half of the activity, number or number of molecules. In the context of the present invention, the half-life of an RNA is indicative of the stability of said RNA. The half-life of RNA can influence the "length of expression" of the RNA. An RNA with a long half-life can be expected to be expressed over an extended period of time.

In the context of the present invention, the term "transcription" refers to the process whereby the genetic code in a DNA sequence is transcribed into RNA. Subsequently, RNA can be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", where the term "in vitro transcription" refers to the process by which RNA, in particular mRNA, is synthesized in vitro in a cell-free system, preferably using appropriate cell extracts. . To obtain transcripts, it is preferable to use cloning vectors. These cloning vectors are commonly referred to as transcription vectors and are encompassed by the term "vector" according to this invention.

According to the present invention, the term "translation" refers to a process in the ribosomes of a cell in which a strand of messenger RNA directs the assembly of amino acid sequences to produce a peptide or protein.

Nucleic acids according to this invention may be alone or in combination with other nucleic acids, which may be homologous or heterologous. In preferred embodiments, the nucleic acid is operably linked to expression control sequences, which may be homologous or heterologous to said nucleic acid. The term "homologous" means that the nucleic acids are also operably linked in a natural way, and the term "heterologous" means that the nucleic acids are not operably linked in a natural way.

A nucleic acid and an expression control sequence are "operably" linked to each other if they are covalently linked to each other in such a way that expression or transcription of said acid is controlled or influenced by said expression control sequence. If the nucleic acid is to be translated into a functional protein, then, with an expression control sequence operably linked to a coding sequence, induction of said expression control sequence results in transcription of said nucleic acid without causing a frameshift in the coding sequence, or said coding the sequence is not capable of being translated into the desired protein or peptide.

The term "expression control sequence" or "expression control element" according to the invention includes promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. According to specific embodiments of the invention, expression control sequences can be regulated. The exact structure of expression control sequences may vary as a function of cell species or cell types, but typically contains 5'-untranscribed and 5'- and 3'-untranslated sequences that are involved in transcription and translation initiation, respectively, such as the TATA box, a capping sequence, a CAA sequence, and the like. More specifically, the 5' non-transcribed expression control sequences include a promoter region that includes a promoter sequence to control transcription of the operably linked nucleic acid. Expression control sequences may also include enhancer or activating sequences.

The term "expression" according to this invention is used in its most general sense and includes the production of RNA and/or peptides or proteins, for example, by transcription and/or translation. In relation to RNA, the term "expression" or "translation" refers in particular to the production of peptides or proteins. It also covers the partial expression of nucleic acids. Moreover, expression can be transient or stable. According to the invention, the term "expression" also includes "aberrant expression" or "abnormal expression".

"Aberrant expression" or "abnormal expression" according to the present invention means that the expression is altered, preferably increased, compared to a control variant, for example, the condition of a subject not having a disease associated with aberrant or abnormal expression of some protein, for example, a tumor antigen. An increase in expression refers to an increase of at least 10%, in particular at least 20%, at least 50% or at least 100% or more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is repressed.

The term "specifically expressed" means that the protein is essentially expressed only in a particular tissue or organ. For example, a tumor antigen specifically expressed in the gastric mucosa means that said protein is predominantly expressed in the gastric mucosa and is not expressed in other tissues or is not significantly expressed in other types of tissue or organs. Thus, a protein that is exclusively expressed in gastric mucosal cells and to a much lesser extent in any other tissue, such as testicular tissue, is specifically expressed in gastric mucosal cells. In some embodiments, the tumor antigen may also be specifically expressed under normal conditions in more than one tissue or organ type, such as 2 or 3 different tissue or organ types, but preferably no more than 3 different tissue or organ types. In this case, the tumor antigen is specifically expressed in these organs. For example, if a tumor antigen is expressed under normal conditions, preferably approximately equally in the lung and stomach, said tumor antigen is specifically expressed in the lung and stomach.

According to the present invention, the term "encoding nucleic acid" means that the nucleic acid, if it is in an appropriate environment, preferably in a cell, can be expressed to produce the protein or peptide that it encodes.

The term "peptide" according to this invention includes oligo - and polypeptides and refers to a substance that contains two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more and preferably up to 8, 10, 20, 30, 40 or 50, in particular 100 amino acids linked covalently by peptide bonds. The term "protein" refers to large peptides, preferably peptides with more than 100 amino acid residues, but in general, the terms "peptide", "peptide chain" and "protein" are synonymous and are used interchangeably in this application.

As mentioned above, the amino acid sequences of the peptide chains and antigen receptors described in this application can be modified in such a way as to obtain variants of these amino acid sequences. Accordingly, the present invention includes variants of the peptide and protein sequences described in this application, and includes variants of amino acid sequences of natural origin, resulting in sequences that are functionally equivalent to said sequences, for example, amino acid sequences that exhibit properties identical or similar to those of said sequences. Important properties are the ability to retain the ability to bind an antigen receptor to its target, or to transduce an antigen-binding fragment into a cell, such as a T cell. In one embodiment, the variant molecule or sequence is immunologically equivalent to the parent molecule or sequence.

The term "variant" according to the invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, in particular those found in nature. An allelic variant refers to a change in the normal sequence of a gene, the meaning of which is often unclear. Whole gene sequencing often makes it possible to identify multiple allelic variants of a given gene. A species homologue is a nucleic acid or amino acid sequence of origin different from the origin of the given nucleic acid or amino acid sequence. The term "variant" embraces any post-translationally modified variants and conformational variants.

The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or substantially the same immunological properties and/or provides the same or substantially the same immunological effects, for example, in terms of the type of immunological effect. In the context of the present invention, the term "immunologically equivalent" is preferably used in relation to the immunological effects or properties of antigen receptors used for therapy.

It is known to those skilled in the art that, in particular, the hypervariable and variable regions of CDR sequences can be modified without losing the ability to bind to a target. For example, the CDR regions may be either identical or highly homologous to the regions of the parent antibodies. By "highly homologous" is meant that 1 to 5, preferably 1 to 4, eg 1 to 3 substitutions or 1 or 2 substitutions can be made in CDRs.

For the purposes of this invention, amino acid sequence "variants" include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. Amino acid deletion variants that include deletion at the N-terminus and/or C-terminus of the protein are also referred to as N-terminal and/or C-terminal truncation variants.

Variants of amino acid insertions include insertions of one or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

Variants of amino acid additions include amino-terminal and/or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

Amino acid deletion variants are characterized by the removal of one or more amino acids from a sequence, for example by removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can be in any position of the protein.

Variants of amino acid substitutions are characterized in that at least one residue in the sequence is deleted and another residue is inserted in its place. Preference is given to modifications at positions in the amino acid sequence that are not conservative between homologous proteins or peptides, and/or replacement of amino acids with other amino acids having similar properties. Preferably, the amino acid changes in protein variants are conservative amino acid changes, ie similarly charged or uncharged amino acid changes. Changing a conservative amino acid involves changing one acid from a family of amino acids that are found in the side chains. Amino acids of natural origin are usually divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

Preferably, the degree (percent) of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably indicated for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the total length of the reference amino acid sequence. For example, if the reference amino acid sequence is 200 amino acids, the degree of similarity or identity is preferably indicated for about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous chain amino acids. In preferred embodiments, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be done by methods known in the art, preferably using an optimal alignment method, for example using the Align program, with a package of standard parameters, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

"Sequence similarity" indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions. "Sequence identity" when considering the sequences of two amino acids indicates the percentage of amino acids that are identical in those sequences.

The term "percent identity" refers to the number of amino acid residues that are identical in compared sequences obtained after optimal alignment, while this percentage is purely statistical and the differences between the two sequences are distributed randomly and along their entire length. Comparison of two amino acid sequences is usually carried out by comparing these sequences after their optimal alignment, and this comparison is carried out segmentally or using a "comparison window" in order to identify and compare local areas of similar sequences. For comparison, comparison sequences can be aligned other than manual alignment using the local analogy algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, via the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443 using the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percent identity is calculated by determining the number of identical positions in the two compared sequences, dividing this number of identical positions by the total number of positions in the alignment window, and multiplying the result by 100 to obtain the percent identity of the two sequences.

The homologous amino acid sequences according to the invention are characterized by at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95 %, at least 98 or at least 99% identity of amino acid residues.

According to the invention, a variant, fragment, part or derivative of an amino acid, peptide or protein sequence preferably has a functional property of the amino acid, peptide or protein sequence, respectively, from which it is derived, i.e. it is a functional equivalent. In one embodiment, the variant, fragment, portion, region, or derivative of an amino acid sequence, peptide, or protein is functionally equivalent, eg, immunologically equivalent, to the amino acid sequence, peptide, or protein, respectively, from which it is derived. In one embodiment, the functional property is the ability to bind to an antigen or to transduce a binding signal into a cell.

The term "derivative" according to this invention means that a specific fragment, in particular a specific sequence, is contained in the object from which it is derived (derived), in particular in an organism or in a molecule. In the case of amino acid sequences, especially specific regions of the sequences, "derived" in particular means that the relevant amino acid sequence is derived (derived) from the amino acid sequence in which it is found.

The term "cell" or "host cell" preferably refers to an intact cell, that is, a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. The intact cell is preferably a viable cell, that is, a living cell capable of performing its normal metabolic functions. Preferably, according to this invention, the term refers to any cell that can be transfected with exogenous nucleic acid. Preferably, the cell, when transfected with an exogenous nucleic acid and brought into the cell, can express the nucleic acid in the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fungal cells or mammalian cells. Suitable bacterial cells include cells from Gram-negative bacterial strains such as Escherichia coli, Proteus and Pseudomonas strains and cells from Gram-positive bacterial strains such as Bacillus, Streptomyces, Staphylococcus and Lactococcus strains. Suitable fungal cells include Trichoderma, Neurospora and Aspergillus species. Suitable yeast cells include Saccharomyces species (eg Saccharomyces cerevisiae), Schizosaccharomyces (eg Schizosaccharomyces pombe), Pichia (eg Pichia pastoris and Pichia methanolicd), and Hansenula. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, HEK 293 cells, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art to express heterologous proteins can also be used. Mammalian cells of choice for adoptive transfer are human, mouse, hamster, pig, goat, and primate cells. These cells can be obtained from a wide variety of tissue types and include primary cells and cell lines such as cells of the immune system, in particular antigen presenting cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells. and other types of cells. Particularly preferred cells for use according to the invention are immunoreactive or immune effector cells, in particular T cells.

A cell that contains a nucleic acid molecule preferably expresses the peptide or protein encoded by the nucleic acid.

The term "clonal expansion" or "expansion" refers to the process where a particular substance is multiplied. In the context of the present invention, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, a proliferate, and a particular lymphocyte recognizing said antigen is amplified. Preferably, clonal expansion results in lymphocyte differentiation. The term "priming" refers to the process where a T cell first encounters a particular antigen and causes differentiation into effector T cells.

Molecules such as nucleic acids, peptide chains and antigen receptors or cells described in this application may be recombinant and/or isolated.

The term "isolated", as used in this application, refers to a substance that is essentially free of other molecules, such as other cellular material. The term "isolated" preferably means that the isolated substance has been separated from its natural environment. The recovered material may be substantially purified. The term "substantially purified" preferably means that the substance is essentially free from other substances with which it is associated in nature or in vivo.

The term "recombinant" in the context of the present invention means "genetically engineered". Preferably, the "recombinant subject", such as a recombinant cell, in the context of the present invention is not of natural origin.

The term "natural origin" as used in this application refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is found in the body (including viruses) and can be isolated from a source in nature and that has not been intentionally modified by humans in a laboratory is of natural origin.

The term "autologous" is used to describe an object that comes from the same subject. For example, "autologous graft" refers to a graft of tissue or organs derived from the same subject. Such techniques are advantageous because they overcome the immunological barrier that would otherwise lead to rejection.

The term "allogeneic" is used to describe an entity that originates from different individuals of the same species. Two or more individuals are considered allogeneic to each other when the genes at the same locus are not identical.

The term "syngeneic" is used to describe an entity that is derived from different individuals or tissues having identical genotypes, ie, identical twins or animals of the same inbred strain or tissues thereof.

The term "heterologous" is used to describe an object that is made up of many different elements. An example is the transplantation of bone marrow from one individual to another individual, which is a heterologous transplant.

A heterologous gene is a gene derived from a source other than the subject.

The terms "reduce" or "inhibit" as used in this application means the ability to cause a complete reduction, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 50% or more. 75% or more of the level. The term "inhibition" or similar terms means complete or substantially complete inhibition, that is, reduction to zero or substantially to zero.

Terms such as "increase" or "enhance" preferably refer to an increase or increase of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

Since the antigen receptor of the present invention can be made to actually target any antigen, including disease-specific antigens, the antigen receptor of the present invention has a broad therapeutic utility. Accordingly, the present invention is directed to the use of the antigen receptor of the invention, its peptide chains, nucleic acids encoding the antigen receptor, and other related molecules in the implementation of therapeutic and prophylactic methods. One such use is the production of antigen-specific immune cells that can be administered to a patient to prevent or treat a disease, the disease being characterized by the expression of one or more antigens that can be bound to an antigen receptor of the invention and expressed in immune cells. The preferred disease is cancer. Further, the antigen receptor of the invention and related molecules can also be used to selectively eradicate cells expressing a given antigen, as well as to immunize or vaccinate against a disease, while the given antigen is expressed, this antigen can be associated with at least one receptor binding site. antigen according to the invention.

According to one embodiment of the invention, a method of treating or preventing a disease comprises administering to a patient an effective amount of a nucleic acid encoding an antigen receptor of the invention, wherein at least one antigen receptor binding site is capable of binding an antigen that is associated with the disease (e.g., a viral or tumor antigen) to be treated or prevented. According to another embodiment, a method for treating or preventing a disease comprises administering to a patient an effective amount of a recombinant immune effector cell or a proliferating population of said immune effector cells, wherein the immune effector cell or population of cells recombinantly expresses an antigen receptor of the invention, wherein at least one antigen receptor binding site capable of binding an antigen that is associated with the disease to be treated or prevented. In preferred embodiments, is a cancer and the antigen is a tumor-associated antigen.

According to another embodiment, the present invention provides a method of immunizing or vaccinating against a disease associated with a specific antigen or against an organism causing the disease and expressing the specific antigen, which method comprises administering to a patient an effective amount of a nucleic acid encoding an antigen receptor of the invention, wherein at least at least one antigen receptor binding site is capable of binding a specific antigen. In another embodiment, the present invention provides a method of immunizing or vaccinating against a disease associated with a specific antigen or against an organism that causes the disease and expresses the specific antigen, the method comprising administering to a patient an effective amount of a recombinant immune effector cell or population, wherein the immune effector cell or population said proliferating immune effector cells, wherein the immune effector cell or population of cells recombinantly expresses an antigen receptor of the invention wherein at least one antigen receptor binding site is capable of binding a specific antigen.

In some embodiments, the immune effector cell population may be a clonogenically proliferating population. Recombinant immune effector cells, or populations thereof, provide therapeutic or prophylactic immune effector function in an antigen-specific manner. Preferably, the antigen receptor of the invention is expressed on the cell surface of immune effector cells.

The cells used in connection with the therapeutic and prophylactic treatments of the present invention are preferably immune effector cells, and the immune effector cells are preferably T cells. In particular, the cells used according to the invention are cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells and lymphokine-activated killer (LAK) cells. Upon activation/stimulation, each of these cytotoxic lymphocytes triggers the destruction of target cells. For example, cytotoxic T cells include destruction of target cells by one or both of the following. First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulosin. Perforin and granulisin lead to the formation of pores in the target cell and granzymes enter the cell and trigger the caspase cascade in the cytoplasm, which initiates apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced through Fas-Fas ligand interaction between T cells and tumor target cells. T cells and other cytotoxic lymphocytes are preferably autologous cells, although heterologous cells or allogeneic cells may be used.

Accordingly, the agents, compositions, and methods described herein can be used to treat a subject having a disease, for example, a disease characterized by the presence of diseased antigen-expressing cells. Particularly preferred diseases are cancerous diseases.

Agents, compositions and methods described in this application can be used for immunization or vaccination in the prevention of the disease described in this application.

The term "disease" refers to an abnormal condition that affects the body of an individual. The disease is often interpreted as a medical condition associated with specific symptoms and signs. The disease may be initially caused by factors from an external source, such as an infectious disease, or may be caused by dysfunctions of internal organs, such as autoimmune diseases. In humans, the term "disease" is often applied more broadly and refers to any condition that causes pain, dysfunction, severe distress (distress), social problems or death of a sick individual, or similar problems for those who are in contact with the individual. In this broad sense, the term sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, abnormal behavior, and atypical variations in structure and function, while in another context and for other purposes, these concepts may be considered as distinct categories. Diseases usually affect individuals not only physically, but also emotionally, as being infected and living with many diseases can change the life outlook and personality of the subject. According to this invention, the term "disease" includes infectious diseases and cancers, in particular those types of cancer that are described in this application. In this application, any reference to cancer or specific types of cancer also includes metastatic cancer.

The disease to be treated according to the invention is preferably an antigen disease. The expressions "a disease involving an antigen", "a disease associated with the expression or overexpression of an antigen" or similar expressions according to the present invention means that the antigen is expressed in the cells of a diseased tissue or a diseased organ. Expression in the cells of a diseased tissue or diseased organ can be enhanced compared to the state of a healthy tissue or healthy organ. In one embodiment, expression is only detected in diseased tissue while expression is not detected in healthy tissue, eg expression can be suppressed. According to the present invention, diseases involving an antigen include infectious diseases and cancers, wherein the antigen associated with the disease is preferably an antigen of an infectious agent and a tumor antigen, respectively. Preferably, the disease involving the antigen is a disease involving cells expressing the antigen, preferably on the cell surface.

The term "healthy" or "normal" refers to non-pathological conditions and preferably means non-infectious or non-cancerous.

The terms "cancer" or "cancer" refer to or describe the physiological state of an individual, which is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head or neck cancer, skin melanoma or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer. , stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the genital and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system (CNS) neoplasm, neuroectodermal cancer, spinal tumors, glioma, meningioma, and pituitary adenoma. The term "cancer" according to this invention also covers metastatic cancer. Preferably, the "cancer disease" is characterized by the presence of cells expressing a tumor antigen, and the cancer cell expresses the tumor antigen.

In one embodiment, the cancer is a malignant disease that is characterized by signs of anaplasia, invasiveness, and the presence of metastases. A malignant tumor can be contrasted with a non-cancerous benign tumor, since the malignant tumor itself is not limited to growth, is able to invade neighboring tissues, and may be able to spread to distant tissues (metastasize), while a benign tumor has none of these properties.

According to the present invention, the term "tumor" or "tumor disease" refers to a swelling or tumor focus formed by abnormal growth of cells (called neoplastic cells or tumor cells). By "tumor cell" is meant an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after stimulation that initiates arrest of tumor development. Tumors show partial or complete lack of structural organization and functional coordination with normal tissue, and usually form a well-defined mass of tissue that may be either benign, premalignant, or malignant.

According to the invention, a "carcinoma" is a malignant tumor that has developed from epithelial tissue cells. This group of tumors represents the most common types of cancer, including common forms of breast, prostate, lung and colon cancer.

"Adenocarcinoma" is a tumor that grows in glandular tissue. This tissue is also part of the tissue known as epithelial tissue. Epithelial tissue includes the skin, glands, and a variety of other tissues that line the cavities and organs of the body. The epithelium is formed embryologically from the ectoderm, endoderm and mesoderm. When classified as an adenocarcinoma, the cells need not be part of the gland as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they have grown, while poorly differentiated adenocarcinomas may not. By staining cells obtained from a biopsy, the pathologist determines whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinomas can develop in many body tissues due to the ubiquitous nature of glands in the body. Although each gland may not secrete the same substance as long as there is an exocrine function in the cell, it is considered glandular and therefore its malignant form is called adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize if given enough time. Adenocarcinoma of the ovary is the most common type of ovarian carcinoma. It includes serous and mucinous adenocarcinomas, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

Lymphoma and leukemia are malignant diseases that develop from hematopoietic (hematopoietic) cells.

A blastic tumor or blastoma is a tumor (usually malignant) that resembles immature or embryonic tissue. Many of these tumors are common in children.

By "metastasis" is meant the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane into the body cavity and into the vessels, and then, after the transfer of blood, infiltration of target organs. As a result, new tumor growth at the target site is dependent on angiogenesis. Tumor metastases often occur even after removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. According to one embodiment of the present invention, the term "metastasis" refers to "distant metastasis", which means a metastasis remote from the primary tumor and the regional lymph node system. According to one embodiment, the term of this invention "metastasis" refers to metastases in the lymph nodes.

A relapse or relapse occurs when the subject again suffers from a condition that afflicted him in the past. For example, if a patient suffered from a neoplastic disease, received successful treatment for said disease, and developed the disease again, said newly developed disease may be considered a relapse or relapse. However, according to the present invention, the return of the disease or recurrence of the neoplastic disease may, but need not, occur at the site of the original neoplastic disease. Thus, for example, if a patient has suffered from an ovarian tumor and has been successfully treated, the relapse or recurrence may be the occurrence of an ovarian tumor or the occurrence of a tumor at a site other than the ovary. Relapse or tumor recurrence also includes situations where the tumor occurs at a site other than the site of the original tumor, as well as at the site of the original tumor. Preferably, the original tumor for which the patient has been treated is the original tumor and the tumor at a site other than the original tumor is a secondary or metastatic tumor.

The infectious diseases treated or prevented by the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, helminths, and parasites.

Infectious viruses in both humans and non-human vertebrates include retroviruses, RNA viruses, and DNA viruses. Examples of viruses that have been found in humans include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (HIV-1) (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and other isolates such as HIV-LP; Picornaviridae (eg, polioviruses, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (eg, strains that cause gastroenteritis; Togaviridae (eg. , equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue hemorrhagic fever viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. Ebola); Paramyxoviridae (eg parainfluenza viruses, mumps viruses, measles viruses, respiratory syncytial virus); Orthomyxoviridae (eg influenza viruses); Bungaviridae (eg hantaviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (eg reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (mainly adenoviruses); Herpesviridae (herpes simplex virus (HSV) (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (poxviruses, vaccinia viruses, poxviruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g., causative agents of animal spongiform encephalopathy, hepatitis delta virus (considered to be a defective satellite of hepatitis B virus), hepatitis non-A, hepatitis non-B agents (class 1 = transmitted to internal organs; class 2 = parenterally transmitted (i.e., hepatitis C ); Norwalk and related viruses, and astroviruses).

Retroviruses that are embraced by this invention include both simple retroviruses and complex retroviruses. Complex retroviruses include subgroups of lentiviruses, T-cell leukemia viruses, and spumaviruses (foaming viruses). Lentiviruses include HIV-1 and also include HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). T-cell leukemia viruses include HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV). Foam viruses include human foam viruses (HFV), simian foam viruses (SFV), and bovine foam viruses (BFV).

Bacterial infections or diseases that can be treated or prevented according to the present invention are caused by bacteria, including, but not limited to, bacteria that have an intracellular life cycle stage, such as mycobacteria (e.g., Mycobacteria tuberculosis, M. bovis, M. avium, M leprae or M. africanum), rickettsia, mycoplasma, chlamydia and legionella. Other examples of bacterial infections of the invention include, but are not limited to, Gram-positive bacilli (e.g. Listeria, Bacillus such as Bacillus anthracis, Erysipelothrix species), Gram-negative bacilli (e.g. Bartonella, Brucella, Campylobacter, Enterobacter, Escherichia, Francisella , Hemophilus, Klebsiella, Morganella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia species), spirochetes (eg, the genus Borrelia, including Borrelia burgdorferi, which cause Lyme disease), anaerobic bacteria (eg, Actinomyces and Clostridium species), gram-positive and gram-negative coccal bacteria, bacteria of the genus Enterococcus, genus Streptococcus, genus Pneumococcus, genus Staphylococcus, genus Neisseria. Specific examples of infectious bacteria include, but are not limited to: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus viridans, Streptococcus aecalis, Streptococcus bovis, Streptococcus pneumoniae, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneunmoniae, Pasturella multocida, Fusobacterium nucleatuin, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinoyyces israelli.

Fungal diseases that can be treated and prevented according to the present invention include, but are not limited to, aspergillosis, critococcosis, sporotrichosis, coccidioidomycosis, paracoccidioidomycosis, histoplasmosis, blastomycosis, zygomycosis, and candidiasis.

Parasitic diseases that can be treated and prevented according to the present invention include, but are not limited to, amoebiasis, malaria, leishmaniasis, coccidiosis, giardiasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis. The present invention also covers infections caused by various helminths, such as, but not limited to, ascariasis, hookworm, trichuriasis, strongyloidiasis, toxocariasis, trichinosis, onchocircosis, filariasis, and heartworm. Infections caused by various flukes are also covered, such as, but not limited to, schistosomiasis, paragonimiasis, and clonorchiasis.

The term "treatment" or "therapeutic treatment" refers to any type of treatment that improves health and/or prolongs (increases) the life span of a subject. Such treatment can eliminate a disease in a subject, stop or slow the progress of a disease in a subject, inhibit or slow the progress of a disease in a subject, reduce the frequency or severity of symptoms in a subject, and/or reduce relapses in a subject who is ill or has had a previous illness.

The terms "prophylactic treatment" or "preventive treatment" refer to any treatment that is intended to prevent the onset of a disease in a subject. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably in this application.

The terms "individual" and "subject" are used in this application interchangeably. They refer to humans, non-human primates, or other mammals (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate) that may be affected by the disease or disorder. (e.g., cancer) or are susceptible to them, but may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not mean a specific age and therefore cover adults, the elderly, children and infants. According to preferred embodiments of the present invention, "individual" or "subject" is a "patient". The term "patient" according to this invention means a subject to be treated, in particular a sick subject.

By "at risk" is meant a subject, ie a patient, who is identified as having a higher than normal likelihood of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had or is currently ill with a disease, in particular cancer, is a subject that has an increased risk of developing the disease because that subject may continue to develop the disease. Subjects who currently have or have had cancer also have an increased risk of developing cancer metastases.

The term "immunotherapy" refers to treatment involving a specific immune response or immune response.

In the context of the present invention, terms such as "protect", "prevent", "prophylactic", "preventive" or "protective" refer to the prevention or treatment, or the occurrence and/or spread of a disease in a subject, and in particular to minimizing the likelihood that the subject will develop the disease, or delaying the development of the disease. For example, a subject at risk of developing a neoplastic disease as described above would be a candidate for therapy to prevent tumor development.

Prophylactic use of immunotherapy, for example, prophylactic use of an agent or composition of the invention, preferably protects the recipient from the development of the disease. Therapeutic use of immunotherapy, for example, the therapeutic use of an agent or composition of the invention, may result in inhibition of disease progression/escalation. This includes slowing down the development/escalation of the disease, in particular the disruption of the progression of the disease, which preferably leads to the elimination of the disease.

Immunotherapy can be carried out using various methods, in which the agents provided in this application preferably function to remove antigen-expressing cells from a patient. Such removal may take place by enhancing or inducing an immune response in the patient specific for the antigen or a cell expressing the antigen.

The term "immunization" or "vaccination" describes the process of treating a subject to induce an immune response for therapeutic or prophylactic reasons.

The term "in vivo" refers to actions on a living organism.

Antigen receptors, peptide chains, nucleic acids, recombinant cells, immune effector cells, preferably T cells, according to the invention, as well as other compounds and agents described in this application, can be administered in the form of any suitable pharmaceutical composition.

Pharmaceutical compositions of the invention are preferably sterile and contain an effective amount of the agents described in this application, and optionally other agents discussed in this application and generating the desired reaction or desired effect.

Pharmaceutical compositions are usually obtained in the form of a homogeneous dosage form and can be prepared in a manner known per se. The pharmaceutical composition may, for example, be in the form of a solution or suspension.

The pharmaceutical composition may include salts, buffers, preservatives, carriers, diluents and/or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to a non-toxic material that does not interact with the active ingredient of the pharmaceutical composition.

Salts that are not pharmaceutically acceptable can be used to prepare pharmaceutically acceptable salts and are included within the scope of the invention. Pharmaceutically acceptable salts of this kind include, without limitation, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts such as sodium salts, potassium salts or calcium salts.

Suitable buffering agents for use in the pharmaceutical composition include acetic acid and a salt thereof, citric acid and a salt thereof, boric acid and a salt thereof, and phosphoric acid and a salt thereof.

Suitable preservatives for use in the pharmaceutical composition include benzalkonium chloride, chlorobutanol, paraben and thimerosal.

The injectable formulation may include a pharmaceutically acceptable excipient such as Ringer's lactate.

The term "carrier" refers to an organic or inorganic component of natural origin or synthetic, which contains the active component in order to facilitate, improve or provide application. According to this invention, the term "carrier" also includes one or more solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to a patient.

Possible carriers for parenteral administration are, for example, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible polylactides, lactide-glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

The term "excipient" as used in this invention is intended to indicate all substances that may be contained in a pharmaceutical composition that are not active ingredients, such as, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers. , buffer substances, flavoring agents or colorants.

The agents and compositions described in this application may be administered by any conventional route, such as parenteral administration, including injection or infusion. Administration is preferably carried out parenterally, for example intravenously, intraarterially, subcutaneously, intradermally or intramuscularly.

Compositions suitable for parenteral administration generally contain sterile aqueous and non-aqueous preparations of the active compound, which are preferably isotonic with the patient's blood. Examples of compatible vehicles and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as the soluble or suspension medium.

The agents and compositions described in this application are administered in effective amounts. The term "effective amount" refers to an amount that provides the desired response or the desired effect on its own or together with additional agents. In the case of treating a specific disease or a specific condition, the desired response preferably relates to inhibiting the progression of the disease. This includes slowing the progression of the disease and, in particular, interrupting or reversing the progression of the disease. The desired response in the treatment of a disease or condition may also be to delay the onset or prevent the onset of said disease or said condition.

The effective amount of the agent or composition described in this application will depend on the condition being treated, the severity of the disease, individual parameters of the patient, including age, physiological condition, height and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration and the like. Accordingly, the doses of the administered agents described in this application may depend on such various parameters. In the event that the patient's response after the initial dose is insufficient, higher doses (or in fact higher doses obtained from a different, more localized route of administration) may be used.

The agents and compositions described in this application can be administered to patients, for example, in vivo, for the treatment or prevention of a number of diseases, such as those described in this application. Preferred patients include those with pathologies that can be corrected or ameliorated by the administration of the agents and compositions described herein. This includes pathological conditions involving cells characterized by antigen expression.

For example, according to one example in one embodiment, the agents described in this application can be used to treat a patient with a cancer, for example, a cancer, such as described in this application, characterized by the presence of cancer cells expressing an antigen.

Pharmaceutical compositions and methods of treatment according to this invention can also be used for immunization or vaccination to prevent the disease described in this application.

Pharmaceutical compositions of the invention may be administered together with immune enhancing and enhancing agents, such as one or more adjuvants, and may contain one or more immune enhancing agents to further enhance efficacy, preferably to achieve a synergistic immunostimulatory effect. The term "adjuvant" refers to compounds that prolong or enhance or accelerate the immune response. In this regard, various mechanisms are possible depending on the different types of adjuvants. For example, compounds that mediate DC maturation, such as lipopolysaccharides or CD40 ligand, form a first class of suitable adjuvants. Generally, any agent that affects the immune system, such as a "hazard-associated molecular fragment" (LPS, GP96, dsRNA, etc.) or cytokines such as GM-CSF, can be used as an adjuvant that provides an intensification immune response and/or affect in a controlled manner. CpG oligodeoxynucleotides can also be used in this context, although their side effects, which appear under certain circumstances, as explained above, must be taken into account. Particularly preferred adjuvants are cytokines such as monokines, lymphokines, interleukins or chemokines, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFNα, IFNγ, GM-CSF, LT-α, or growth factors such as hGH. Other known adjuvants are aluminum hydroxide, Freund's adjuvant or an oil such as Montanide®, Montanide® ISA51 is most preferred. Lipopeptides such as Pam3Cys are also suitable for use as adjuvants in the pharmaceutical compositions of this invention.

The pharmaceutical composition may be administered locally or systemically, preferably systemically.

The term "systemic administration" refers to the administration of an agent in such a way that the agent is evenly distributed in the body of the individual in significant amounts and provides the desired effect. For example, such an agent may provide the desired effect in the blood and/or reach the desired site of action via the vascular system. Typical systemic routes of administration include administration by introduction of the agent directly into the vasculature or orally, into the lung or intramuscularly, whereby the agent is adsorbed, enters the vasculature, and is delivered to one or more desired sites of action via the blood.

According to the present invention, it is preferable that the systemic administration is carried out parenterally. The term "parenteral" refers to the introduction of the agent in such a way that the agent does not pass through the intestines. The term "parenteral administration" includes, but is not limited to, intravenous administration, subcutaneous administration, intradermal administration, or intra-arterial administration.

Administration may also be, for example, orally, intraperitoneally or intramuscularly.

The agents and compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, radiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

The present invention is described in detail below with the help of figures and examples, which are used for illustrative purposes only and are not limiting. Through the description and examples, other options are available to a person skilled in the art, which are also included in this invention.

FIGURES

Figure 1 is a schematic representation of all T-cell receptor (TCR)- and chimeric antigen receptor (CAR) constructs used in the experiments. A) and B) The TCR consists of a class I heterodimeric membrane protein, each strand containing a conserved C domain and a variable V domain, where the latter specifically recognizes a truncated MHC restricted peptide. The sequence and structural homology of the mouse (Mu, A) and human (Hu, B) TCR is elevated. The mouse TCR recognizes a human tumor antigen derived from the tight junction protein Claudin 6, while the human TCR recognizes a tumor antigen derived using the melanocytic differentiation antigen gp100. C) The monovalent single chain CAR contains a single chain (sc) Fv fragment attached to the mouse Cβ domain and an autonomous TCR Cα domain preceded by a signal peptide for export to the cell membrane. Optionally, it is linked by an artificial disulfide bond between the TCR C domains to increase cell surface expression and function of this CAR. The scFv Fragment in this and all subsequent exemplary constructs is directed to Claudin 6. D) Classic scCAR Cl6 contains a homodimer, each aligning with the scFv fragment, antibody hinge region, CH2CH3 domains as a spacer region, cell membrane, and domains signal transduction into the cell of the costimulatory molecule CD28 and CD3ζ, respectively. Homodimerization results in recognition of the antigen by the bivalent receptor, with each strand binding one antigen (i.e. an intrachain bond is formed). E) The prototype combined CAR carries allele-related V-domains connected in series (ie, VH-VH or VL-VL), both attached to TCR Cα or Cβ, respectively. Recognition inevitably entails binding of the antigen involving both strands in a combined (ie, interstrand) manner and as a divalent compound. F) Heterodimeric TCR-CAR Cl6 carrying scFv fragments on each full-length TCRα gp100 or TCRβ recognizes the related antigen as a divalent but not combined compound. G) To eliminate the recognition of the residue of the related peptide gp 100(280-288) by the TCR gp100 component of the uncombined TCR-CAR Cl6 (F), a 'silent' (sil) S109Q (according to IMGT nomenclature) point mutation was introduced into the CDR3 loop of the TCRα chain (silCDR3α) . H) Combined TCR-CAR Cl6 was generated by sequencing the allele-associated V domains, either on the full-length TCR α gp100 chain or on the full-length TCR β chain, respectively, as outlined in E). The full-length TCR used here as a binding partner may provide better T-cell signaling under physiological conditions than a truncated TCR containing only TCR C-domains instead of (E). I) To eliminate the recognition of the residue of the related peptide gp100(280-288) by the TCR gp100 component of the combined TCR-CAR Cl6 (H), a 'silent' (sil) S109Q (according to IMGT nomenclature) point mutation was introduced into the CDR3 loop of the TCR α chain (silCDR3 α ), as in G).

Figure 2A/B shows the expression of Claudin 6 on APCs and various CARs on human T cells, respectively, prior to co-cultivation. A) Immature dendritic cells (iDCs) were electroporated with increasing amounts of the related full-length Claudin 6 antigen or with a single high dose of the irrelevant gp100 antigen. In this case, a dose-dependent shift in the Cl6 expression dataset could be observed by flow cytometry, indicating that this donor's iDCs are highly permissive in terms of cellular RNA uptake, protein translation, and export to the cell surface. A high level of CD86 expression indicates efficient differentiation of monocytes into successfully antigen-presenting iDCs. B) Pre-activated CD8+ T cells were electroporated with various CAR-coding RNAs and analyzed for CAR expression by flow cytometry. All CARs, with the exception of monovalent and classic scCAR, moderately recovered on anti-idiotypic staining. Monovalent CAR was the worst expressed, while classic scCAR was found to be the best expressed. Figure 2C shows the performance of CAR Cl6-reprogrammed human T cells in recognizing Claudin 6 expressing iDCs after APC/T cell co-cultivation in IFNγ-ELISA. CAR-electroporated T cells were co-cultured overnight with Cl6-electroporated APCs as explained in Figures 2A/B at an E:T ratio (effector to target cell ratio) of 10:1. Throughout the Cl6 titration spectrum, all CARs showed optimal results at lower doses of Cl6 (0.02 µg). At high levels of Claudin 6 expression, classic scCAR Cl6 showed the best secretion of IFNγ compared to all other CARs, while at the lowest dose of Cl6, combined CARs generally performed better than classic scCAR. In conclusion, combined CARs were superior to classical CARs in their functional efficiencies from high to low antigen expression: They produced very high amounts of IFNγ up to 20-30,000 pg/ml IFNγ for combined CARs and scCAR Cl6, respectively, ending at 12-15,000 pg /ml IFNγ for all constructs at the lowest dose of Cl6. At this dose, the intercombined Cα/Cβ-CAR became most effective.

Figure 3A/B shows the expression of Claudin 6 on APCs and various CARs on human T cells, respectively, prior to co-cultivation. A) Immature dendritic cells (iDCs) were electroporated with increasing amounts of the related full-length Claudin 6 antigen. In this case, only a dose-dependent partial shift in Cl6 expression could be observed by flow cytometry, indicating that this donor's iDCs are undeniably less permissive in terms of cell uptake. RNA, protein translation and export to the cell surface. A high level of CD86 expression indicates efficient differentiation of monocytes into successfully antigen-presenting iDCs. B) Pre-activated CD8+ T cells were electroporated with various RNAs encoding CARs and analyzed for CAR expression by flow cytometry. All CARs, with the exception of monovalent and classic scCAR, were only partially recovered by anti-idiotypic staining, again indicating that this donor's cells are less permissive for RNA uptake and processing. However, poorly antigen-presenting iDCs may represent the state of minimally antigen-positive tumor APCs, as it may mimic the situation of early-stage tumor escape or minimal tumor antigen presentation. Figure 3C shows the efficiency of CAR Cl6-reprogrammed human T cells in recognizing Claudin 6 expressing iDCs after APC/T cell co-culturing in IFNγ-ELISA. CAR-electroporated T cells were co-cultured overnight with Cl6-electroporated APCs, as explained in Figures 3A/B, at an E:T ratio of 10:1. Throughout the Cl6 titration spectrum, all CARs showed optimal results at lower doses of Cl6 (0.02 µg). Given the absolute minimal expression of Claudin 6 as explained above, combined Cl6 CARs showed the best IFNγ secretion compared to classic scCAR Cl6 at all doses, a trend that was even more pronounced at the lowest dose of electroporated Cl6. Finally, for very small amounts of antigen, the combined CARs were even better than the classic scCAR Cl6 in terms of IFNγ secretion. The total amounts of secreted IFNγ fell below 1,000 pg/ml throughout the titration interval due to very low expression of the antigen as well as CARs.

Figure 4 shows the efficiency of the gp100-CAR Cl6 TCR of reprogrammed human T cells in recognizing Claudin 6 expressing iDCs or gp100 peptide loaded iDCs after co-culture of HLA-A2.1 ⁺ APC/T cells in IFNγ-ELISA. CAR-electroporated T cells were co-cultured overnight with Cl6-electroporated APCs or peptide-loaded gp100(280-288) iDCs at an E:T ratio of 5:1. The main purpose of this experiment was to test whether, firstly (A) bivalent combined TCR-CARs secrete IFNγ more efficiently than bivalent non-combined TCR-CARs, and secondly (B), to what extent they still recognize the gp100 peptide. used in this invention structure TCR gp100. Residual antigen binding was attempted to be eliminated by using a 'silent' mutation S109Q in CDR3 of the TCRα gp100 structure. TCR Cl6 and TCR gp100 served as positive controls for cognate antigen recognition. A) Combined TCR-CARs, whether or not silenced in CDR3α, appeared to be more functional than non-combined CARs, especially at the lowest dose of antigen. B) The non-silent uncombined TCR-CAR Cl6 still recognized gp100 at 10 ^-6 M of the peptide. The introduction of the S109Q mutation completely abolished secretion. For combined TCR-CARs Cl6, no secretion was observed at all, whether functional silencing was performed or not. It is very likely that the interchain binding of the serially connected VH-VH and VL-VL domains on either chain creates steric hindrances for the binding of the gp100 peptide, presented as restricted HLA-A2.1 on APCs.

Figure 5 shows the proliferative ability of various Cl6 CARs upon encounter with Cl6 expressing iDCs following APC/T cell co-culture. CAR-electroporated T cells were co-cultured for 5 days with Cl6-electroporated and - titrated APCs at an E:T ratio of 10:1. A priori, T cells were stained with carboxyfluorescein succinimidyl ether (CFSE) to quantify the CFSE titer at cell division and hence the number of resulting daughter populations indicated to the right of each distribution density plot obtained in the flow cytometry analysis. A) Monovalent antigen-bound CARs showed the weakest proliferation, while combined TCR-CARs and classical CARs showed similar proliferation patterns. B) A histogram of the frequency of T cell populations is shown in A). High frequency T cells 'equipped' with a monovalent CAR did not proliferate (40-60%), while the frequency of non-proliferating T cells carrying combined TCR-CARs and classic CAR was approximately in the range of 10-20 %. Proliferation of T cells bearing the classic CAR Cl6 was highest at the high dose (0.2 µg Cl6) of antigen (90% compared to 80% for combined TCR-CARs), while at the lowest dose (0.002 µg) combined TCR-CARs were at least as effective as the classic CAR (nearly 80%). In conclusion, combined TCR-CARs again outperformed classical CARs in their functional efficiency, in this case in terms of T-cell proliferation, in the range of high to very low doses of antigen. There is a trend towards even higher efficiency than in the case of classical CAR.

Figure 6 shows the upregulation of the CD27 costimulation biomarker on combined TCR-CAR Cl6 electroporated daughter T cells after APC/T cell co-culturing. CAR-electroporated T cells were co-cultured for 6 days with Cl6-electroporated iDCs at an E:T ratio of 3:1. A priori, T cells were stained with CPD-450 to quantify the titer of this fluorophore during cell division and hence the number and frequency of the resulting daughter cell populations indicated to the left of the histograms obtained in the flow cytometry analysis. T cells were stained with CD8-specific antibody to illustrate the moderate and equal upregulation of this co-receptor marker for combined and classic CARs. Simultaneously, they were stained with an antibody specific for the CD27 co-stimulator to assess its regulation in G0-G6 parent/daughter cell populations for all CARs. Although both markers were upregulated, approximately 2-fold, and beyond that, mean CD8 expression was the same for all CARs, mean CD27 expression was much higher for combined CARs, and in particular intercombined TCR-CAR Cl6, than for classical CAR. CD27, a biomarker of long-term T cell resistance in vivo, reached a significantly higher plateau for fading T cells (G2-G4) before expression levels dropped to baseline in G6.

EXAMPLES

The techniques and methods used in the present invention are described in this application or carried out by well-known methods described, for example, in the laboratory practice Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd Edition (1989) Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY All methods, including the use of kits and reagents, are performed according to the information provided by the manufacturers, unless specifically stated otherwise.

Example 1 Analysis of Claudin 6 Expression in iDCs and Antigen Receptors in T Cells by Flow Cytometry.

RNAs for various constructs were generated using in vitro transcription (ivt-RNA) open reading frames (ORFs) cloned into the pST1 RNA vector carrying the T7 promoter at its 5' leader sequence and an optimized polyA 'tail' at its 3 '-end. Claudin 6 expression in human immature dendritic cells was analyzed one day after electroporation of RNA (2-0.002 μg, 300 V, 12 ms, 1 pulse) into GM-CSF/IL-4-treated CD14+ buffy coat monocytes using antibody , specific for Claudin 6, labeled with Dylight-650 fluorophore. Expression of various antigen receptor constructs in autologous human T cells was analyzed one day after RNA electroporation (total 10-30 μg for both strands, 495 V, 9 ms, 1 pulse) in CD8+ T cells preactivated with OKT3 (mouse monoclonal antibody to CD3ε ), using a Cl6 scFv idiotype-specific antibody labeled with the Dylight-649 fluorophore. A detailed description of the production of human iDCs and T cells is given in Example 2. The CAR constructs tested for expression were (i) mouse T-cell receptor TCR Cl6; (ii) human TCR gp100; (iii) monovalent uncombined CAR Cl6; (iv) classic scCAR) (bivalent); (v) inter-combined CAR Cl6 fused to human TCR Cα/β domains (bivalent); (vi) uncombined CAR Cl6 fused to full length TCR gp 100(280-288) (bivalent); (vii) the corresponding uncombined CAR Cl6, also 'silent' in CDR3 of TCRα gp100 to avoid peptide recognition (bivalent); (viii) intercombined CAR Cl6 fused to full length TCR gp 100(280-288) (bivalent) and (ix) corresponding combined CAR Cl6 also 'silent' in CDR3 TCRα gp100 to avoid peptide recognition (bivalent). Various antigen receptor constructs are shown schematically in Figures 1A-I. Staining of cells according to the usual practice was carried out for 0.2×10 ⁶ cells in flow cytometry buffer for 20 min at 4°C, washed and fixed with 1% paraformaldehyde-containing flow cytometry buffer. The results shown in Figure 2A show titrated Claudin 6 expression. In this case, this donor's iDCs were highly permissive for Cl6 RNA uptake and showed a shift in the Claudin 6 expression dataset with increasing amounts of electroporated RNA. Expression of CD86 showed successful differentiation of monocytes into active antigen-presenting iDCs. On FIG. 2B shows the expression of various CARs used in this experiment in human CD8 positive T cells. The classic scCAR Cl6 showed the highest expression, possibly due to its endogenous CD3-independent expression on the surface of T cells. The combined CARs showed somewhat higher expression than the monovalent CAR, with the latter serving as a 'weak control' due to the fact that it only binds antigen as a monovalent compound.

The results in Figure 3A show titrated expression of Claudin 6 in an independent experiment. In this case, iDCs from this donor were weakly permissive for Cl6 RNA uptake and showed only a partial shift in Claudin 6 expression with increasing amounts of electroporated RNA. But CD86 expression showed successful differentiation of monocytes into active antigen-presenting iDCs. On FIG. 3B shows the expression of various CARs used in this experiment in selected CD8-positive human T cells. Since human T cells were taken from the same donor as differentiated monocytes under autologous parameters, T cells also became weakly permissive for electroporated RNA, and as a result, only weaker expression for CARs was observed than in the experiment shown in Figure 1. Figure 2B. However, the classic scCAR Cl6 again showed the highest level of expression for the same reason mentioned earlier. Consistent with previous observations, the combined CARs showed slightly better expression than the monovalent CAR. Since the order of expression of CARs is preserved compared to the experiment presented in Figure 2A/B, this experiment is suitable for studying the activity of various CARs when only very small amounts of Cl6 are expressed on antigen presenting cells (APCs).

Example 2 IFN-γ Secretion Assay by Antigen Titration

PBMCs клетки CD14+ выделяли, используя метод магнитной сортировки клеток, MACS. Затем поток MACS и остаточные PBMCs сортировали методом MACS, выделяя CD8+ Т-клетки. Клетки CD14+ дифференцировали в незрелые дендритные клетки ("iDCs") посредством введения IL-4 & GM-CSF (1000 ед/мл) на 1, 3, 6 день. CD8+ Т-клетки переносили в 6-луночные планшеты, покрытые антителом OKT3. На 3 день Т-клетки переносили в новые 6- луночные планшеты. На 7 день iDCs подвергали электропорации нерелевантной и Cl6 ivt-РНК в зависимости от дозы в интервале 2-0.002 мкг РНК. Активированные антителом OKT3 Т-клетки электропорировали в один день с контрольными или с конструкциями антигенного рецептора, представленными на отдельных фигурах или описанными в Примере 1. Для обеспечения контроля качества экспрессию Cl6 на клетках iDCs и поверхностную экспрессию антигенного рецептора на Т-клетках анализировали с использованием специфических меченных флуоресцентной меткой антител, описанных ранее, на 8 день. Затем электропорированные Т-клетки и антиген-электропорированные iDCs совместно культивировали в 96-луночном планшете в течение 20 ч при соотношении Е:Т, равном от 3:1 до 10:1, в двойных повторах. Согласно обычной практике 2.5×10⁴ iDCs засевали и совместно культивировали с 7.5×10⁴ - 2.5×10⁵ CAR-электропорированных Т- клеток в объеме 200 мкл среды для Т-клеток. На 9 день отбирали различные количества культуральных супернатантов (10-50 мкл) и анализировали количество секретированного IFNγ методом "сэндвич" - ELISA, используя набор теста IFN-γ Ready Set Go! от кампании eBioscience (#88-7316-88). Оптическую плотность детектировали на ридере Tecan Sunrise ELISA.On day 1 of the experiment, fresh peripheral blood mononuclear cells ("PBMCs") were isolated from the buffy coat of a healthy donor. From

PBMCs of CD14+ cells were isolated using the magnetic cell sorting method, MACS. The MACS stream and residual PBMCs were then sorted by the MACS method to isolate CD8+ T cells. CD14+ cells were differentiated into immature dendritic cells ("iDCs") by administration of IL-4 & GM-CSF (1000 U/ml) on days 1, 3, 6. CD8+ T cells were transferred to 6-well plates coated with OKT3 antibody. On day 3, T cells were transferred to new 6-well plates. On day 7, iDCs were subjected to electroporation of irrelevant and Cl6 ivt-RNA in a dose-dependent manner in the range of 2-0.002 μg of RNA. OKT3 antibody-activated T cells were electroporated on the same day as the control or antigen receptor constructs shown in the individual figures or described in Example 1. For quality control, Cl6 expression on iDCs and antigen receptor surface expression on T cells were analyzed using specific fluorescently labeled antibodies described previously on day 8. The electroporated T cells and antigen-electroporated iDCs were then co-cultured in a 96-well plate for 20 hours at an E:T ratio of 3:1 to 10:1 in duplicate. As per usual practice, 2.5×10 ⁴ iDCs were seeded and co-cultured with 7.5×10 ⁴ - 2.5×10 ⁵ CAR electroporated T cells in a volume of 200 μl of T cell medium. On day 9, various amounts of culture supernatants (10-50 µl) were taken and the amount of secreted IFNγ was analyzed by sandwich ELISA using the IFN-γ Ready Set Go! from the eBioscience Campaign (#88-7316-88). Optical density was detected on a Tecan Sunrise ELISA reader.

Figure 2C shows the amounts of secreted IFNγ for iDCs and T cells from the same donor, which were highly permissive for RNA electroporation and therefore caused massive dose-dependent expression of Cl6 in iDCs and high-level expression of CARs in T cells ( Figure 2A/B). All CAR T cells showed maximum IFNγ secretion at 0.02 µg of electroporated Cl6 RNA, up to 30,000 pg/mL IFNγ for classic scCAR and 20,000 pg/mL for combined CARs fused to either Cα/β TCR or gp100 full-length TCR. According to estimates, T-cells modified with monovalent CAR turned out to be the most "weak" effector cells. Interestingly, at a high level of Cl6 electroporation, the combined CARs showed a reactivity only approximately 50% of that of the classical scCAR Cl6, which increased to 70% at the optimal dose of 0.02 µg of electroporated Cl6. Importantly, at the lowest dose of Cl6 tested in this example, 0.002 μg RNA, the combined CARs were as effective as the classic scCAR Cl6, or, in the case of combined with CAR fused to TCR Cα/β, even more effective. With this very low level of Cl6 present in the mass, the combined CARs were still able to secrete high amounts of IFNγ in the range of 10,000 pg/ml.

Figure 3C shows the amounts of secreted IFNγ for iDCs and T cells from the same donor, which were weakly permissive to RNA electroporation and therefore resulted in only a partial dose-dependent expression of Cl6 in iDCs, as well as reduced expression of CARs in T -cells (Figure 3A/B). This may create a situation where even less Cl6 is present on iDCs than with low-level Cl6 expression of highly permissive antigen-titratable iDCs (Figure 3A/B versus 2A/B). In this case, for all very small amounts of Cl6 titrated, the combined CARs proved to be more effective in IFNγ secretion than the classic scCAR. This trend becomes even more pronounced for reduced amounts of Cl6 on the surface of iDCs. The amount of IFNγ secreted was low (<1000 pg/mL), but first, it could be increased by better expression of CAR, and second, this could provide good outcome options for clinic patients with (early stage) tumors with very low levels of antigen expression, or to block minimal residual disease.

Figure 4A compares the efficiency of IFNγ secretion for (bivalent) uncombined and combined TCR-CARs against the CAR-specific Cl6 antigen. All combined CARs were more effective in recognizing antigen over a wide range of titratable antigen. For higher doses of antigen, the effector functions of the bivalent CARs were almost equal, except for the 'silent' uncombined TCR-CAR. The S109Q point mutation in CDR3α is known to slightly impair binding of the gp100(280-288) antigen (Knies et al., Oncotarget 2016). Cross-chain binding of V domains and binding of the antigen itself by the 'silent' combined TCR-CAR appears to compensate for the loss of function caused by this mutation. It is important to note that at a low level of Cl6 expression (0.02 μg), combined TCR-CARs, either functionally silent or not, were superior to non-combined TCR-CARs in terms of cytokine secretion.

Figure 4B illustrates the recognition of residual gp100(280-288) antigen in peptide-titrated IFNγ secretion. In this case, TCRαβ gp100 served as a positive control, while TCRαβ Cl6 served as a specificity control to assess background cytokine secretion. The uncombined TCR-CAR is still capable of recognizing the evolutionarily stable A2-1 antigen at a high peptide load. The introduction of the silent mutation S109Q abolished recognition altogether. Importantly, it appears that the interchain combined arrangement of V domains connected as tandem repeats (VH-VH-, VL-VL-) completely prevents recognition of the cognate antigen by the TCR gp100 molecule. Therefore, the introduction of a silent mutation may serve as a protective measure to ensure functional immunological tolerance of the TCR molecule in these CARs and to focus on using its backbone as a scaffold to stabilize domain pairing and as a full-length adapter molecule for T cell signaling. in physiological conditions.

Example 3 Antigen-Titrated Proliferation Assay Combined with Biomarker Phenotyping

of PBMCs выделяли клетки CD14+, используя метод магнитной сортировки клеток MACS, и остаточные PBMCs вымораживали. Клетки CD14+ дифференцировали в iDCs посредством введения IL-4 & GM-CSF (1000 ед/мл) в 1, 3, 6 день. На 7 день iDCs электропорировали с нерелевантной и Cl6 IVT-РНК в зависимости от дозы в интервале 2-0.002 мкг РНК. Замороженные PBMCs подвергали оттаиванию в тот же день и проводили сортировку клеток методом MACS, отбирая CD8+ клетки. Затем без какой- либо предварительной активации (OKT3), наивные Т-клетки, приблизительно 7×10⁶ клеток, электропорировали с использованием классических, одновалентных и интеркомбинированных TCR-CARs, указанных на Фигуре 1.On the first day of the experiment, fresh PBMCs were isolated from the buffy coat of a healthy donor. From

of PBMCs, CD14+ cells were isolated using the MACS magnetic cell sorting method, and residual PBMCs were frozen. CD14+ cells were differentiated into iDCs by administration of IL-4 & GM-CSF (1000 U/ml) on days 1, 3, 6. On day 7, iDCs were electroporated with irrelevant and Cl6 IVT-RNA in a dose-dependent manner in the range of 2-0.002 μg RNA. Frozen PBMCs were thawed the same day and cell sorted by MACS to select CD8+ cells. Then, without any prior activation (OKT3), naive T cells, approximately 7×10 ⁶ cells, were electroporated using the classic, monovalent and intercombined TCR-CARs indicated in Figure 1.

For quality control, CAR-modified T cells were analyzed by flow cytometry staining on day 8. Then, T cells were labeled intracellularly with the fluorescent proliferation marker CFSE (0.8 μM) or CPD-450 (10 μM). Next, electroporated T cells and iDCs were co-cultured in a 96-well plate for 5 days at an E:T ratio of 10:1 (or 3:1 for biomarker phenotyping) in duplicate. As per usual practice, 2.5×10 ⁴ iDCs were co-cultured in 2.5×10 ⁵ CAR electroporated T cells in a volume of 200 μl of T cell medium in a 96-well plate. On day 5, cultured cells were stained in 96-well plates with CD4 or CD8 antibodies labeled with APC-Cy7. T cell proliferation was detected by flow cytometry by a left shift of the fluorophore signal due to dilution in proliferating daughter cells. The frequency of non-proliferating G0 parent population and G1-G7 daughter populations was analyzed using the proliferation program in the FlowJo v7.6.5 flow cytometry software application. The frequency of occurrence for all daughter T cells was calculated from the sum of all proliferating G1-G7 populations. Background T cell proliferation was analyzed for cells cultured with iDCs, electroporated with irrelevant full length gp100 or T cells seeded without APCs. Proliferating cells were stained for CD8 to uniquely identify them as T cells. Alternatively, proliferating T cells were stained with biomarkers such as CD27, CD28, PD-1, CD95, CD45RA, and CCR7 to quantify the differentiation status of initially naive non-proliferating T cells in G0 and emerging G1-G7 daughter populations, respectively, after 5 or 6 days of co-culture with APCs. Antibodies and corresponding isotype controls were titrated to evaluate the optimal signal-to-noise ratio. Quantitative analysis is carried out on a FACS-Canto II-HTS (BD) cytofluorimeter in a 96-well plate.

Figure 5A depicts density plots of proliferating T cells modified with monovalent CAR Cl6 as a 'weak control', combined TCR-CARs with and without the silent mutation S109Q in TCR CDR3α, and classic scCAR Cl6 as a reference CAR. Almost no non-specific proliferation can be observed for iDCs loaded with the irrelevant gp100 antigen. Proliferation towards APCs loaded with a related antigen resulted in six different daughter populations, the frequency of which can be easily distinguished (listed to the right of each graph). All CARs, with the exception of the monovalent CAR Cl6, showed high proliferation rates even at low antigen densities. The maximum frequency of occurrence in almost all cases was observed in G3.

Figure 5B is a histogram showing the numbers of parental G0 T cells and proliferating G1-G7 T cells. At high antigenic load, a high bar for G0 (40%) and a lower bar for G1-G7 (60%) compared to other CARs clearly indicate a weak propensity for univalent CAR-modified T cells to proliferate. Classical scCAR-modified T cells performed slightly better (90%) than combined CARs (80%), according to IFNγ secretion assays. Thus, when the amount of antigen was reduced, T cells modified with combined CAR became at least as efficient as those modified with classical scCAR (almost 80%). Based on this trend, it can be hypothesized that, even in the case of reduced antigenic density, combined CARs may still be superior to classical scCARs in terms of proliferation activity.

Figure 6 on the left shows the frequency of proliferating T cells ‘pulled’ with either classic scCAR (top), combined Cα/β-CAR Cl6 (center) or combined TCR-CAR Cl6 silCDR3α (bottom). Shown on the right is the mean intensity minus non-specific binding (ie, isotype binding) for the CD8 co-receptor and co-stimulatory CD27 receptor across all parent and daughter populations. T cells were stained with CD8-specific antibody to visualize moderate and nearly equal upregulation of this co-receptor marker for combined and classical CARs. Therefore, CD8 staining can serve as a marker of normalization in order to highlight the equal regulation of this 'inert' molecule among all CARs considered in the present invention. In addition, they were stained with an antibody specific for the co-stimulatory molecule CD27 in another fluorescence channel to assess their regulation in G0-G6 parent/daughter populations for all CARs. Although both markers were activated approximately 2-fold and, in addition, mean CD8 expression was nearly the same for all CARs, mean CD27 expression was significantly higher for combined CARs, and in particular for combined TCR-CAR Cl6, than for classical CAR. CD27, a biomarker of long-term T cell resistance in vivo, reached a significantly higher plateau for fading T cells (G2-G4) before expression levels dropped to baseline in G6. Based on this, it is hypothesized that combinatorial CAR-modified T cells may have less terminally differentiated and depleted phenotype (i.e., downregulation of co-stimulatory molecules) in populations with attenuated proliferation and therefore may survive longer in vivo. The size (forward scatter) and granulation (side scatter) of cocultured combined CAR-electroporated T cells were even lower than for classical CAR (data not shown). Therefore, increased CD27 activation is not caused by an increase in cell surface, but by an increased amount of CD27 on a larger surface.

Claims (40)

1. A tumor antigen receptor that provides an immune response to cancer cells expressing a tumor antigen, which includes a first peptide chain and a second peptide chain, while the first peptide chain comprises a first domain with specificity for a tumor antigen, a second domain with specificity for a tumor antigen, a T-cell receptor chain variable region, and an immunoreceptor signal transduction domain; the second peptide chain comprises a first domain with specificity for a tumor antigen, a second domain with specificity for a tumor antigen, a T-cell receptor chain variable region, and an immunoreceptor signal transduction domain; wherein the first domain of the first peptide chain, together with one of the domains of the second peptide chain, forms the first tumor antigen-binding site, and while the second domain of the first peptide chain, together with another domain of the second peptide chain, forms a second tumor antigen-binding site, where (i) the first peptide chain comprises a T cell receptor alpha chain variable region and a T cell receptor alpha chain constant region, and the second peptide chain comprises a T cell receptor beta chain variable region and a T cell receptor beta chain constant region receptor, or (ii) the first peptide chain comprises a T cell receptor beta chain variable region and a T cell receptor beta chain constant region, and the second peptide chain comprises a T cell receptor alpha chain variable region and a T cell receptor alpha chain constant region. receptor where the first peptide chain contains the structure VH(1)-VL(2)-V1-C1 and the second peptide chain contains the structure VL(1)-VH(2)-V2-C2, where the first peptide chain contains the structure VH(1)-VH(2)-V1-C1 and the second peptide chain contains the structure VL(1)-VL(2)-V2-C2, where the first peptide chain contains the structure VH(1)-VH(2)-V1-C1 and the second peptide chain contains the structure VL(2)-VL(1)-V2-C2, or where the first peptide chain contains the structure VH(1)-VL(2)-V1-C1 and the second peptide chain contains the structure VH(2)-VL(1)-V2-C2; and VH(1) denotes an immunoglobulin heavy chain (VH) variable region with specificity for the first epitope; VH(2) denotes an immunoglobulin heavy chain (VH) variable region with specificity for a second epitope; VL(1) denotes an immunoglobulin light chain (VL) variable region with specificity for the first epitope; VL(2) denotes an immunoglobulin light chain (VL) variable region with specificity for a second epitope; V1 and V2 are variable regions of the alpha and beta chains of the T-cell receptor, respectively, or sections thereof, C1 and C2 are immunoreceptor signal transduction domains that form a dimer with each other, with (i) the C1 immunoreceptor signaling domain of the first peptide chain comprises a T cell receptor alpha chain constant region and the second peptide chain C2 immunoreceptor signaling domain comprises a T cell receptor beta chain constant region, or (ii) the C1 immunoreceptor signaling domain of the first peptide chain comprises a T cell receptor beta chain constant region and the C2 immunoreceptor signal transduction domain of the second peptide chain comprises a T cell receptor alpha chain constant region; wherein the first and/or second peptide chains further comprise a linker between the first and second domains and/or between the first and second domains and the variable region of the T-cell receptor chain; and wherein the first and second antigen binding sites bind to different epitopes on the same tumor antigen. 2. The receptor of claim 1, wherein the immunoreceptor signal transduction domain comprises a constant or conserved region of a T cell receptor chain or a constant or conserved region of an immune cell receptor Fc chain or a portion of that constant or conserved region. 3. The receptor according to claim 1 or 2, wherein the linker is an arbitrary amino acid sequence or the linker contains 3 repeats of a 5-mer amino acid sequence (Gly4Ser). 4. The receptor according to any one of paragraphs. 1-3, wherein the C1 and C2 immunoreceptor signal transduction domain is of human origin. 5. The receptor according to any one of paragraphs. 1-4, in which the amino acid sequences of the first and second peptide chains are of human origin, and in which murine sequences are introduced into the domains forming antigen-binding sites by replacing one or more amino acids in the human sequence with an amino acid at the corresponding position in the murine sequence. 6. A method for producing a cell expressing a tumor antigen receptor according to claim 1, which contains a first peptide chain and a second peptide chain, the method comprising: (a) providing a cage; (b) providing a first genetic construct encoding a first peptide chain comprising at least a first domain, a second domain, a T cell receptor chain variable region, and an immunoreceptor signal transduction domain; (c) providing a second genetic construct encoding a second peptide chain comprising at least a first domain, a second domain, a T cell receptor chain variable region, and an immunoreceptor signal transduction domain; (d) introducing the first and second genetic constructs into the cell; And (e) enabling expression of the constructs in the cell; wherein the first domain from the first peptide chain is capable of forming, together with one of the domains from the second peptide chain, the first tumor antigen-binding site, and where the second domain from the first peptide chain is capable of forming, together with another domain from the second peptide chain, a second tumor antigen-binding site: wherein (i) the first peptide chain comprises a T cell receptor alpha chain variable region and a T cell receptor alpha chain constant region, and the second peptide chain comprises a T cell receptor beta chain variable region and a T cell receptor beta chain constant region receptor, or (ii) the first peptide chain comprises a T cell receptor beta chain variable region and a T cell receptor beta chain constant region, and the second peptide chain comprises a T cell receptor alpha chain variable region and a T cell receptor alpha chain constant region. receptor. 7. The method of claim 6 wherein the first peptide chain and the second peptide chain are provided in a single genetic construct. 8. The method according to claim 6 or 7, wherein the cell is a human cell, preferably a T cell. 9. The use of a tumor antigen receptor according to any one of paragraphs. 1-5 for treating a disease, wherein the disease is characterized by the expression of at least one tumor antigen that is associated with a tumor antigen receptor, and wherein the disease is preferably cancer.

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