RU2792042C2 - Antigen receptors and their applications - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: claimed invention: an antigenic receptor for providing an immune response to cancer cells expressing a tumour antigen, the receptor contains a first peptide chain and a second peptide chain, a recombinant CD8+ T host cell expressing the antigen receptor and a method for its production, a nucleic acid, a pharmaceutical formula and method for treating cancer.

EFFECT: invention allows to use it in the treatment of malignant tumours by targeting cells expressing the antigen on the cell surface, where the tumour antigen is claudin 6.

13 cl, 18 dwg, 5 ex

Description

State of the art of the present invention

The present invention relates to recombinant antigen receptors and their uses. 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 (TCR) expressed on the surface of T cells. TCR T cells are able to interact with immunogenic peptides (epitopes) associated with major histocompatibility complex (MHC) molecules 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.

The TCR is part of a complex signaling apparatus that includes a heterodimeric complex of TCR α- and β-chains, a CD4 or CD8 co-receptor, and a CD3 signal transduction module (FIG. 1). The α/β TCR heterodimer is responsible for antigen recognition and, together with CD3, for transduction of the activation signal across the cell membrane, while the CD3 chains themselves transmit the incoming signal to adapter proteins within the cell. Thus, α/β TCR chain transfer allows T cells to be retargeted to any antigen of interest.

Adoptive cell transfer (ACT) immunotherapy can be broadly defined as a form of passive immunization with pre-sensitized T cells that are transferred into unimmunized recipients or an autologous host after ex vivo expansion from a small number of progenitors to a clinically significant number of cells. Cell types that have been used for 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 (TIL) (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 provide therapeutic activity against human viral infections, such as those caused by CMV. For adoptive immunotherapy of melanoma, Rosenberg and colleagues developed an ACT-based approach based on in vitro infusion of expanded autologous tumor infiltrating lymphocytes (TIL) isolated from resected tumors, combined with non-myeloablative circulating lymphocyte depletion chemotherapy and high doses of IL2. In a clinical study, the proportion of patients with an objective response was ~50% of the total number of treated patients suffering from metastatic melanoma (Dudley, M. E. et al. (2005) J. Clin. Oncol. 23: 2346-2357).

An alternative approach is the adoptive transfer of autologous T cells reprogrammed to express a tumor-reactive immune cell receptor with 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 malignancies even if the patient lacks tumor-reactive T cells. Since the antigenic specificity of T cells is entirely due to the heterodimeric complex of TCR α- and β-chains, the transfer of cloned TCR genes to T-cells makes it possible to redirect them to any antigen of interest. Therefore, gene therapy using TCR provides a promising strategy for developing antigen-specific immunotherapy 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 within a few days and the possibility of introducing specificity properties that are not present in the patient's endogenous TCR repertoire. Several research groups have demonstrated that TCR gene transfer is a promising strategy to redirect antigen 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 feasibility of gene therapy using TCR in humans was first demonstrated in clinical trials for the treatment of malignant melanoma by Rosenberg and his group. Adoptive transfer of autologous lymphocytes transduced using retroviruses specific for melanoma/melanocyte antigens TCR resulted in regression of malignancy in up to 30% of patients treated for melanoma (Morgan, R.A. et al. (2006) Science 314, 126-129; Johnson, L.A. et al (2009) Blood 114, 535-546). Currently, the field of clinical research on gene therapy using TCR has also been extended to malignancies other than melanoma, targeting various tumor antigens (Park, T.S. et al., (2011) Trends Biotechnol. 29, 550-557).

The use of genetic engineering approaches to introduce antigen-targeted receptors with specific specificity into T cells has greatly expanded the potential of ACT. Chimeric antigen receptors (CARs) are a type of antigen-targeted receptor composed of intracellular T-cell signaling domains fused to extracellular antigen-binding domains, typically single chain variable fragments (scFv) from monoclonal antibodies. CARs directly recognize cell surface antigens, independent of MHC-mediated presentation, allowing the use of a single receptor construct specific for any given antigen in all patients. The first CARs fused the antigen recognition domains to the CD3ζ activation chain of the T-cell receptor (TCR) complex (FIG. 2). Subsequent CAR variants included secondary costimulatory signalers along with CD3ζ, including intracellular domains from CD28 or a number of TNF receptor family molecules such as 4-1BB (CD137) and OX40 (CD134). The next third generation receptors, in addition to CD3ζ, include two costimulatory signalers, typically from CD28 and 4-1BB. Second and third generation CARs were characterized by significantly improved antitumor efficacy in vitro and in vivo (Zhao et al., (2009) J. Immunol., (183) 5563-5574), in some cases inducing complete remission in patients with advanced malignancy ( Porter et al., (2011) N. Engl. J. Med., (365) 725-733).

The classic CAR consists of an antigen-specific single chain antibody (scFv) fragment fused to a transmembrane and signaling domain such as CD3ζ. Upon introduction into T cells, it is expressed as a membrane-bound protein and induces immune responses upon binding to its cognate antigen (Eshhar et al., (1993) PNAS, (90) 720-724). The induced antigen-specific immune response leads to the activation of cytotoxic CD8+ T cells, which in turn leads to the elimination of cells expressing the specific antigen, such as tumor cells or virus-infected cells expressing the specific antigen. However, such classical CAR constructs do not activate/stimulate T cells via their endogenous CD3 complex, which is normally required for T cell activation. Through the fusion of the antigen-binding domain to CD3ζ, T cell activation is induced via a biochemical shortcut (Aggen et al., (2012) Gene Therapy, (19) 365-374). Such non-physiological activation of T cells by such a biochemical shortcut is dangerous for the patient treated in this way, since excessive T cell activation can lead to undesirable side effects. For example, a sustained basal activation of recombinant T cells as a result of CAR expression ("background signaling") has been observed in vitro, resulting in increased accumulation of inhibitory molecules such as LAG-3, TIM-3, and PD-1 on the surface of recombinant expressing T cells. CAR T cells, which in turn led to premature depletion of T cells, which subsequently had a strong negative impact on the response against tumor cells in vivo (Long et al., (2015) Nat. Med., (21) 581- 590). This adverse reaction has been associated with irregular clustering of scFv fragments by the backbone residues of this antibody. In addition, although classic CAR constructs of this type have been successfully tested against various forms of neoplasia, such as leukemia (Porter et al., (2011) N.Engl.J.Med., (365) 725-733), they also caused the development of life-threatening autoimmune diseases due to basal expression of the target antigen (target tumor antigen) in normal tissues (on-target/off-tumor reaction; Morgan et al., (2010) Mol Ther., (18) 843 -51).

An alternative approach, in which T cell activation occurs through a mechanism closer to physiological, was to provide a similar single-stranded TCR (scTv) fragment fused to a Cβ constant domain derived from the T cell receptor (TCR) and co-express it with TCR-derived Cα constant domain (Voss et al., (2010) Blood, (115) 5154-5163), the latter of which recruits the required endogenous CD3ζ homodimer (Call et al., (2002) Cell, (111) 967-79 .). However, for such constructs to function as immune system activators, it was necessary that the TCR-derived constant domains originate from mouse TCRs, or that they include mouse sequences (Cohen et al., (2006) Cancer Res., (66 ) 8878-86) to ensure pairing of the scTCR and Cα chains. The fact that such constructs must contain xenogeneic sequences to function increases the risk that the immune system, when administered, will react to them and reduce or impair their therapeutic efficacy.

Thus, there is a need to provide alternative recombinant antigen receptors, wherein, for example, the receptor, after antigen binding, is sufficiently capable of activating the T cell in which it is naturally expressed via the endogenous CD3 complex and optionally without the need for any non-human amino acid sequences, at least in the signal transduction domain of the antigen receptor, that can induce an undesirable immune response against the recombinant antigen receptor itself.

Brief summary of the present invention

The present invention relates to recombinant antigen receptors with at least two antigen binding sites. Antigenic receptors contain two peptide chains. In one aspect, each of the peptide chains contains at least two domains, in addition to an immune cell receptor signaling domain, where each of the two domains on one peptide chain forms an antigen binding site with one of the domains on the other peptide chain. In another aspect, one of the peptide chains contains at least four domains, in addition to the immune cell receptor signaling domain, where two of the four domains form two antigen binding sites with the other two domains on the same peptide chain.

In one aspect, the present invention relates to an antigen receptor, the receptor comprising a first peptide chain and a second peptide chain, wherein the first peptide chain comprises at least first and second domains and an immune cell receptor signal transduction domain; the second peptide chain contains at least the first and second domains and the signal transduction domain from the immune cell receptor; where the first domain from the first peptide chain together with one of the domains from the second peptide chain forms the first antigennegative center, and where the second domain from the first peptide chain together with another domain from the second peptide chain forms the second antigennegative center. In the antigen receptor of this aspect, the domains forming the respective antigen-binding sites are preferably located on different peptide chains. Therefore, antigen-binding centers are formed due to intermolecular interaction of domains.

In one embodiment, each of the first and/or second domains comprises an immunoglobulin chain variable region or a T cell receptor variable chain region or variable region 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 light 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 heavy chain variable region with antigen specificity or a portion thereof.

In one embodiment, the first domain from the first peptide chain provides for 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 first domain from the first peptide chain provides for an immunoglobulin light chain variable region with specificity to an antigen or its site. In one embodiment, the second domain from the first peptide chain provides for 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 provides for an immunoglobulin light chain variable region with specificity to an antigen or its site.

In one embodiment, each of the first and second domains from the first peptide chain comprises an immunoglobulin heavy chain variable region or portion thereof; and each of the first and second domains from the second peptide chain comprises an immunoglobulin light chain variable region or 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 aspect, the present invention relates to an antigen receptor, the receptor comprising a first peptide chain and a second peptide chain, wherein the first peptide chain comprises at least four domains and an immune cell receptor signal transduction domain; the second peptide chain contains the signal transduction domain from the immune cell receptor; where two of the domains from the first peptide chain form the first antigennegative center, and where the other two domains from the first peptide chain form the second antigennegative center. In an antigen receptor according to this aspect, the domains forming the respective antigen binding sites are preferably located on the same peptide chain. Therefore, antigen-binding centers are formed by intramolecular interaction of domains.

In one embodiment, each of the four domains provides for an immunoglobulin chain variable region or a T cell receptor chain variable region or variable region 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 light 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 two N-terminal domains of the four domains together form an antigen binding site; and the two C-terminal domains of the four domains together form an antigen-binding center.

In one embodiment, one of the two N-terminal domains of the four domains provides for an immunoglobulin heavy chain variable region or region thereof, and the other of the two N-terminal domains of the four domains provides for an immunoglobulin light chain variable region or region thereof; and one of the two C-terminal domains of the four domains provides for an immunoglobulin heavy chain variable region or portion thereof, and the other of the two C-terminal domains of the four domains provides for an immunoglobulin light chain variable region or portion thereof.

In one embodiment of the antigen receptors of the present invention, the immune cell receptor signal transduction domain comprises a T cell receptor chain constant or invariant region or an immune cell Fc receptor chain constant or invariant region or a constant or invariant region region. According to one embodiment of the antigen receptors of the present invention (i) the first peptide chain provides a T-cell receptor alpha chain constant region or portion thereof, and the second peptide chain provides a T-cell receptor beta chain constant region or portion thereof, or (ii ) the first peptide chain provides a T-cell receptor beta chain constant region or region, and the second peptide chain provides a T-cell receptor alpha chain constant region or region.

In one embodiment of the antigen receptors of the present invention, the immune cell receptor signal transduction domain is from the human body.

According to one embodiment, the antigen receptor of the present invention comprises (a) linker(s) connecting(s) domains of the antigen receptor. In one embodiment, the antigen receptor of the present invention comprises one or more linkers between antigen-binding site-forming domains and/or between antigen-binding site-forming domains and immune cell receptor signal transduction domains. The linker may be a random amino acid sequence of any length, provided that it does not interfere with the function of the antigen receptor, such as the ability of the antigen receptor to bind an antigen or bind to the endogenous CD3 complex, or does not interfere with the ability of the antigen receptor to induce an immune response upon antigen binding. .

According to one embodiment of the antigen receptors of the present invention, the first and second antigen binding sites bind to the same antigen or to different antigens. According to one embodiment of the antigen receptors of the present invention, the first and second antigen binding sites bind to different epitopes on the same antigen. Therefore, while 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 center originate from the same or from different immunoglobulins, wherein said different immunoglobulins bind to the same or 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.

According to one aspect, the present invention relates to the peptide chain of any of the antigen receptors of the present invention. In one embodiment, the present invention relates to a peptide chain comprising first and second domains, each of which provides for an immunoglobulin heavy chain variable region or a portion thereof, or provides for an immunoglobulin light chain variable region or portion thereof, and wherein the peptide chain further comprises a signal transduction domain from an immune cell receptor. In one embodiment, the present invention relates to a peptide chain that comprises at least four domains and an immune cell receptor signal transduction domain, where two of the domains from the peptide chain form a first antigen-binding site and the other two domains from the peptide chain form a second antigen-binding site . Additional embodiments of the peptide chains of the present invention are as described herein for the antigen receptors of the present invention.

In one aspect, the present invention relates to a cell, in particular an immune effector cell such as a T cell, genetically modified to express an antigen receptor of the present invention. According to one aspect, the present invention relates to a recombinant cell, in particular, 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 present invention, or expressing the peptide chain according to the present invention. Additional embodiments of the cell or recombinant cell of the present invention are as described herein for the antigen receptors of the present invention or the peptide chains of the present invention.

In one aspect, the present invention relates to a method for obtaining a cell expressing an antigen receptor of the present invention, the method comprising: (a) obtaining a cell; (b) obtaining a first genetic construct encoding the first peptide chain of the antigen receptor of the present invention; (c) obtaining a second genetic construct encoding a second antigen receptor peptide chain of the present 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 producing a cell expressing an antigen receptor, wherein the receptor comprises a first peptide chain and a second peptide chain, the method comprising: (a) obtaining a cell; (b) obtaining a first genetic construct encoding a first peptide chain containing at least first and second domains and an immune cell receptor signal transduction domain; (c) obtaining a second genetic construct encoding a second peptide chain containing at least the first and second domains and the signal transduction domain from the immune cell receptor; (d) introducing the first and second genetic constructs into the cell; and (e) allowing expression of the constructs in a cell, wherein the first domain from the first peptide chain, together with one of the domains from the second peptide chain, is capable of forming a first antigen binding site, and where the second domain from the first peptide chain, together with another domain from the second peptide chain, is capable of form a second antigen-binding center. According to one embodiment, the present invention relates to a method for producing a cell expressing an antigen receptor, wherein the receptor comprises a first peptide chain and a second peptide chain, the method comprising: (a) obtaining a cell; (b) obtaining a first genetic construct encoding a first peptide chain containing at least four domains and an immune cell receptor signal transduction domain; (c) obtaining a second genetic construct encoding a second peptide chain containing a signal transduction domain from the immune cell receptor; (d) introducing the first and second genetic constructs into the cell; and (e) allowing expression of the constructs in a cell, wherein two of the domains from the first peptide chain are capable of forming a first antigen binding site, and wherein the other two domains from the first peptide chain are capable of forming a second antigen binding site. According to one embodiment of the methods of the present invention, expression of the antigen receptor takes place on the cell surface. According to one embodiment of the methods of the present invention, the first peptide chain and the second peptide chain are provided in the same genetic construct. In one embodiment of the methods of the present invention, the cell is a human cell. In one embodiment of the methods of the present invention, the cell is an immune effector cell, such as a T cell. In one embodiment of the methods of the present invention, the genetic constructs comprise DNA and/or RNA. Additional embodiments of the methods of the present invention are as described herein for the antigen receptors of the present invention.

According to one aspect, the present invention relates to a recombinant cell, in particular, an immune effector cell, such as a T cell, obtained using the methods for producing an antigen receptor expressing cell of the present invention. Additional embodiments of the recombinant cell of the present invention are as described herein for the antigen receptors of the present invention or methods for producing a cell expressing the antigen receptor of the present invention.

According to one aspect, the present invention relates to a nucleic acid, such as DNA or RNA, encoding the first peptide chain, the second peptide chain, or both the first and second peptide chains of the antigen receptor of the present invention, or encoding the peptide chain of the present invention. Additional embodiments of the nucleic acid of the present invention are as described herein for the antigen receptors of the present invention or the peptide chains of the present invention.

The present invention generally encompasses the treatment of diseases by targeting cells expressing one or more antigens on the cell surface, such as diseased cells expressing one or more disease-specific antigens on the cell surface, in particular cancer cells expressing on the cell surface surface of one or more tumor antigens using the antigen receptors of the present invention. The methods involve selectively killing cells that express one or more antigens on their surface, thereby minimizing adverse effects on normal cells that do not express the antigen(s). In one embodiment, T cells genetically modified to express an antigen receptor of the present invention targeting the cells by binding to the antigen(s) are administered. T cells are able to recognize diseased cells expressing antigen(s) on the cell surface, resulting in the destruction 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 relates to a pharmaceutical composition comprising an antigen receptor of the present invention, a recombinant cell of the present invention, or a nucleic acid of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be used as a drug, in particular in the treatment of a disease, such as a malignant tumor, characterized by the expression of one or more antigens that bind to the antigen receptor of the present invention, such as 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 present 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 with a disease, disorder, or 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 of the present invention targeted at least one antigen. In one embodiment, the disease, disorder, or condition is cancer. In one embodiment, the T cells may be autologous, allogeneic, or syngeneic to the subject.

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

According to one embodiment of all aspects of the present invention, the method of treatment further comprises obtaining a cell sample from a subject, wherein the sample contains T cells or T cell precursors, and transfecting the cells with a nucleic acid encoding an antigen receptor of the present invention to obtain T cells genetically modified to express the antigen receptor. In one embodiment of all aspects of the present 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 or is not integrated into the T cell genome. In one embodiment of all aspects of the present 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 present invention, the T cells and/or cell sample are from a non-mammalian to which T cells genetically modified to express an antigen receptor are administered.

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

In one embodiment of all aspects of the present invention, the antigen is expressed in a diseased cell, such as a cancer cell. In one embodiment, the antigen is expressed on the surface of a diseased cell, such as a cancer cell. In one embodiment, the antigen receptor binds to the extracellular domain or an epitope of the extracellular domain of the antigen. In one embodiment, the antigen receptor binds to native epitopes of an antigen present on the surface of living cells. In one embodiment of all aspects of the present invention, the antigen is a tumor antigen. In one embodiment of all aspects of the present 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 present invention, the antigen is a pathogenic antigen. The pathogen may be a fungal, viral, or bacterial pathogen. According to one embodiment of all aspects of the present invention, antigen expression takes place on the cell surface. In one embodiment, the antigen is claudin, in particular claudin 6 or claudin 18.2, wherein said antigen receptor binds to the first extracellular loop of said claudin. In one embodiment, binding of said antigen receptor, which is expressed by T cells and/or present on the surface of T cells, to the antigen present on the cell surface results in immune effector functions of said T cells, such as release of cytokines. In one embodiment, binding of said antigen receptor that is expressed by T cells and/or present on the surface of T cells to an antigen present on the surface of cells, such as antigen presenting cells, results in stimulation, priming and/or propagation of said T -cells. In one embodiment, binding of said antigen receptor that is expressed by T cells and/or present on the surface of T cells to an antigen present on the surface of diseased cells, such as cancer cells, results in cytolysis and/or apoptosis of the diseased cells. , while cytotoxic factors, such as perforins and granzymes, are preferentially released from said T cells.

In one embodiment of all aspects of the present invention, the antigen-binding site-forming domains of an antigen receptor constitute an exodomain of the antigen receptor. According to one embodiment of all aspects of the present invention, the antigen receptor of the present invention comprises a transmembrane domain. In one embodiment, the transmembrane domain is a hydrophobic alpha helix that crosses the membrane.

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

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

In one embodiment of all aspects of the present invention, the antigen receptor of the present invention may be expressed by a T cell and/or present on the surface of a T cell, preferably a cytotoxic T cell. In one embodiment, the T cell is reactive with the antigen(s) targeted by the antigen receptor of the present invention.

In a further aspect, the present invention provides the agents and compositions described herein for use in the methods described herein.

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

Detailed disclosure of the present invention

Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, as they may vary. It should also be understood that the terminology used herein is only intended to describe specific embodiments, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as are commonly understood by one of ordinary skill in the art.

The features of the present invention will be described below. Such features are listed in specific embodiments, however, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described are not to be construed as limiting the present invention to the expressly described embodiments. It is to be understood that the present description is intended to cover and cover embodiments that combine the expressly described embodiments with any number of disclosed and/or preferred elements. In addition, any changes and combinations of all features described in this application should be considered as disclosed by the description of this application, unless the context indicates otherwise.

Preferred terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kölbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

In the practice of the present invention, unless otherwise indicated, methods conventional in the field of biochemistry, cell biology, immunology, and recombinant DNA techniques will be used, which are explained in the literature in this field (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).

In the present description and the following claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be intended to include the claimed element, integer or step or group of elements, integers or steps, but not an exclusion of any other element, integer, or step or group of elements, integers, or steps, although in some embodiments, such other element, integer, or step, or group of elements, integers, or steps may be excluded, i.e., the claimed the object is within the scope of the specified element, integer or stage or group of elements, integers or stages. Singular forms and similar references used in the context of the description of the present invention (in particular in the context of the claims) should be construed as covering both the singular and the plural, unless otherwise indicated herein or clearly contrary to the context. The description of ranges of values in this document is intended to serve only as a shorthand way of individually specifying each individual value that falls within the range. Unless otherwise indicated, in this document, each individual value is included in the present description, as if it were separately described in this document.

All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicts the context. The use of all possible examples or qualifying words (eg, "such as") provided herein is merely intended to better illustrate the present invention, and is not intended to limit the scope of the present invention as otherwise claimed. None of the language in the present description should be construed as indicating any unclaimed element necessary for the implementation of the present invention in practice.

Several documents are cited throughout the text of this specification. Each of the documents referenced in this document (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether cited above or below, are incorporated herein by reference throughout its completeness. Nothing herein should be construed as an admission that the present invention is not entitled to claim primacy in filing the present disclosure by virtue of a prior invention.

The term "immune response" refers to an integrated systemic response to an antigen and preferably refers to a cellular immune response or a cellular as well as a humoral immune response. The immune response may be protective/preventive/prophylactic and/or therapeutic.

“Immune response stimulation” may mean that there was no immune response to a particular target antigen, target cell and/or target tissue before the immune response was stimulated, but it can also mean that there was a certain level of immune response to a specific target antigen before the immune response was stimulated. , target cell and/or target tissue, and after stimulation of the immune response, said immune response was enhanced. Thus, "stimulating an immune response" includes "inducing an immune response" and "enhancing an immune response". Preferably, after stimulating the subject's immune response, the subject is prevented from developing a disease, such as a disease related to cancer, or as a result of stimulating the immune response, the disease state is improved. For example, an immune response to a tumor antigen can be elicited in a patient with a disease related to cancer or in a subject at risk of developing a disease related to cancer. Stimulation of the immune response in such a case may mean that the subject is improving with respect to the disease state, that the subject does not develop metastases, or that the subject at risk of developing a disease related to cancer does not develop a disease related to cancer.

"Cell-mediated immunity" or "cellular immunity" or similar terms is intended to include a cellular response directed to cells expressing an antigen, in particular those characterized by antigen presentation by MHC class I or class II. 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 central role in regulating the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8 ⁺ T cells, or CTLs) destroy diseased cells, such as cancer cells, preventing more diseased cells from forming.

The term "antigen" refers to a substance containing an epitope to which an immune response will be generated and/or directed. Preferably, in the context of the present invention, an antigen is 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 corresponds to, or is derived from, a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasitic organisms and other infectious agents and pathogens, or the antigen may also be a tumor antigen. In accordance with the present invention, the antigen may correspond to a naturally occurring product, for example, a viral protein or a portion thereof.

The term "pathogen" refers to pathogenic microorganisms and includes viruses, bacteria, fungi, unicellular organisms and parasitic organisms. Examples of pathogenic viruses are human immunodeficiency virus (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, amoebae, etc.

In a preferred embodiment, the antigen is a disease-specific antigen or a disease-associated antigen. The term "disease-specific antigen" or "disease-associated antigen" refers to all antigens that have pathological significance. In one particularly preferred embodiment, the antigen is present in diseased cells, tissues, and/or organs, but absent or low in healthy cells, tissues, and/or organs, and thus can be used to target diseased cells, tissues, and/or organs. cells, tissues and/or organs, for example, by means of T cells carrying an antigen receptor targeted to the antigen. In one embodiment, the disease-specific antigen or disease-associated antigen is present on the surface of the cell affected by the disease.

According to a preferred embodiment, the antigen is a tumor antigen or tumor-associated antigen, i.e. a component of cancer cells that can be derived from the cytoplasm, cell surface and cell nucleus, in particular such antigens that are produced, preferably in large quantities, as surface antigens on cancer cells.

In the context of the present invention, the term "tumor antigen" or "tumor-associated antigen" refers to proteins that under normal conditions are specifically expressed in a limited number of tissues and/or organs or at certain stages of development, for example, a tumor antigen under normal conditions can be specifically expressed in gastric tissue, preferably in the gastric mucosa, in reproductive organs, for example, in the testicle, in trophoblast tissue, for example, in the placenta, or in germline cells, and which are expressed or abnormally expressed in one or more tissues of the tumor or malignant tumor. In such a context, "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. proteins that under normal conditions are specifically expressed in cells of a certain type at a certain stage of differentiation, antigens of a malignant tumor / testis, i.e. proteins that under normal conditions are specifically expressed in the testicle and in some cases in the placenta, and germline-specific 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 or rarely expressed in healthy tissues. Preferably, the tumor antigen or abnormal expression of the tumor antigen defines the cancer cells. In the context of the present invention, a tumor antigen that is expressed by a cancer cell in a subject, for example, a patient suffering from a disease related to cancer, is preferably a self protein of said subject's body. According to preferred embodiments, the tumor antigen in the context of the present invention is under normal conditions specifically expressed in a tissue or organ that is not critical, ie. tissues or organs that, when damaged by the immune system, do not result in the death of the subject, or organs or structures of the body that are inaccessible or difficult to access by the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between a tumor antigen that is expressed in healthy tissues and a tumor antigen that is expressed in cancerous tissues.

Examples of tumor antigens that may be useful according to 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 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, TPTE and WT. Particularly preferred tumor antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).

In the context of this document, the term "CLDN" or simply "Cl" means claudin and includes CLDN6 and CLDN18.2. Preferably, the claudin is human claudin. Claudins are a family of proteins that are the most important components of tight junctions where they form a paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Claudins are transmembrane proteins that cross the membrane 4 times, with both the N- and C-terminus located in the cytoplasm. The first extracellular loop, called EC1 or ECL1, consists of an average of 53 amino acids, and the second extracellular loop, called EC2 or ECL2, consists of about 24 amino acids. Such 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 (not expressed in toxically important healthy tissue) and plasma membrane localization.

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

CLDN18.2 is expressed in malignant tumors of various origins such as pancreatic carcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma, and ENT tumors. CLDN18.2 is a valuable 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, malignant colon cancer, liver cancer, head and neck cancer, and gallbladder cancers and their metastases, in particular gastric cancer metastasis such as Krukenberg tumors, peritoneal metastasis and lymph node metastasis. Antigen receptors targeting at least CLDN18.2 are useful in the treatment of such cancer related diseases.

CLDN6 has been found to be expressed, for example, in ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma forms, head and neck cancer, forms sarcomas, malignant tumors of the biliary tract, in renal cell carcinoma and in malignant tumors of the bladder. 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 lung squamous cell carcinoma and adenocarcinoma, 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, malignant tumor of the biliary tract, malignant tumor of the bladder, in particular transitional cell carcinoma and papillary carcinoma, malignant tumor of the kidney, in particular renal cell and carcinomas, including clear cell renal cell carcinoma and papillary renal cell carcinoma, malignant tumor of the colon, malignant tumor of the small intestine, including malignant tumor of the ileum, in particular, adenocarcinoma of the small intestine and adenocarcinoma of the ileum, embryonic testicular carcinoma , choriocarcinomas, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonic testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonic carcinoma, in particular testicular germ cell tumors, and their metastatic forms. Antigenic receptors that target at least CLDN6 are useful in the treatment of such diseases related to malignant tumors.

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 diseased cell. In accordance with the present invention, the antigen, when bound by an antigen receptor, is preferably capable of inducing, optionally in the presence of appropriate costimulatory signalers, stimulation, priming and/or expansion of a T cell bearing an antigen-binding antigen receptor. Recognition of an antigen on the surface of a diseased cell may elicit an immune response to the antigen (or the cell expressing the antigen).

According to various aspects of the present invention, the aim is preferably to stimulate an immune response to an antigen-expressing diseased cell, such as an antigen-expressing cancer cell, e.g. a tumor antigen, in particular CLDN6 or CLDN18.2, and to treat a disease, such as a disease relating to a malignant tumor, involving cells expressing an antigen, such as a tumor antigen. Preferably, the present invention provides for the administration of antigen receptor engineered immune effector cells, such as T cells, targeted to antigen-expressing diseased cells. Cells that express an antigen on the surface can be targeted by immune effector cells that carry an antigen receptor that targets the antigen.

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

The term "extracellular region" or "exodomain" in the context of the present invention refers to the region of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outer surface of said cell, for example, to binding molecules, such as antibodies, located outside cells. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

The terms "section" or "part" are used interchangeably herein and refer to a continuous or discontinuous structural element, such as an amino acid sequence. The term "fragment" refers to a continuous element of the structure, such as an amino acid sequence. The site, part or fragment of the structure preferably provides one or more functional properties of the specified structure, for example, antigenic, immunological properties and/or binding properties. A region or part 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 and/or non-consecutive amino acids of the protein sequence. A protein sequence fragment 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 of the sequence squirrel.

In accordance with the present invention, an antigen is (substantially) not expressed in a cell if the expression level is below the detection threshold and/or if the expression level is too low to allow binding by the antigen-specific antibodies added to the cell. In accordance with the present invention, an antigen is expressed in a cell if the expression level exceeds the detection threshold 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 surface-expressed, ie present on the surface of said cell and thus available for binding by antigen-specific molecules such as antibodies or antigen receptors added to the cell.

By "target cell" is meant 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, the target cell is a cell expressing the target antigen, in particular a disease-specific antigen, which is preferably present on the cell surface.

The term "epitope" refers to an antigenic determinant of a molecule, such as an antigen, i. part or fragment of a molecule that is recognized, i.e. binds to elements of the immune system, for example, is recognized by an antibody or antigen receptor. For example, epitopes are discontinuous, three-dimensional regions on an antigen that are recognized by the immune system. Epitopes usually consist of reactive groups on the surface of molecules, such as amino acids and sugar side chains, and usually have specific three-dimensional structure 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 provides a continuous or discontinuous stretch of said protein, and is preferably 5-100, preferably 5-50, more preferably 8-30, most preferably 10-25 amino acids in length, e.g., the length of 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.

"Antigen processing" refers to the degradation of an antigen to form processing products that are fragments of said antigen (e.g., degradation of a protein to form peptides) and the association of one or more of 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) is a cell that displays an antigen on its surface in the context of a major histocompatibility complex (MHC). T cells can recognize such a complex with their T cell receptor (TCR). Antigen presenting cells process antigens and present them to T cells. In accordance with the present invention, the term "antigen-presenting cell" includes professional antigen-presenting cells and non-professional antigen-presenting cells.

Professional antigen presenting cells are highly efficient in internalizing an antigen, either through phagocytosis or receptor-mediated endocytosis, and subsequently displaying an antigen fragment bound to an MHC class II molecule on their 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 co-stimulatory signal, which leads to the activation of the T cell. Expression of co-stimulatory molecules is a hallmark of professional antigen-presenting cells. The main types of professional antigen presenting cells are dendritic cells, which present the widest range of antigens, and are probably the most important antigen presenting cells, macrophages, B cells, and certain types of activated epithelial cells.

Non-professional antigen-presenting cells do not constitutively express MHC class II proteins required to interact with naive T cells; such proteins are only expressed when nonprofessional antigen-presenting cells are stimulated with certain cytokines, such as IFNγ.

Dendritic cells (DCs) are populations of leukocytes that present peripherally captured antigens to T cells via both class II and class I MHC antigen presentation pathways. It is well known that dendritic cells are effective inducers of immune responses and activation of these cells is a key step for the induction of antitumor immunity. Dendritic and progenitor cells can be obtained from peripheral blood, bone marrow, tumor infiltrating cells, tumor surrounding tissue infiltrating cells, lymph nodes, spleen, skin, cord blood, or any other suitable tissue or body fluid. For example, dendritic cells can be differentiated ex vivo by adding to cultures of monocytes collected from peripheral blood a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFa. Alternatively, CD34-positive cells harvested 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(s) compound(s) that induce differentiation, maturation and proliferation of dendritic cells. Dendritic cells are conventionally divided into "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, such a classification should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with a pronounced 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 low expression of these markers, but high expression of cell surface molecules responsible for T-cell activation, such as MHC class I and class II, adhesion molecules (eg, CD54 and CD11), and costimulatory molecules (eg, , CD40, CD80, CD86 and 4-1 BB). Dendritic cell maturation is considered as a state of activation of dendritic cells, in which such antigen-presenting dendritic cells provide T-cell priming, while presentation by immature dendritic cells leads to tolerance. The maturation of dendritic cells is mainly driven by biomolecules characteristic of microorganisms, detected by natural receptors (bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines (TNF, IL-1, IFN), CD40 binding on the surface of CD40L dendritic cells, and substances released from cells subject to stress-induced cell death. Dendritic cells can be obtained by culturing bone marrow cells in vitro with cytokines such as granulocyte-monocyte colony stimulating factor (GM-CSF) and tumor necrosis factor alpha.

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

The term "immune effector functions" in the context of the present invention includes any functions mediated by components of the immune system that provide, for example, the destruction of diseased cells, such as tumor cells, or the inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor spread and metastasis. Preferably immune effector functions in the context of the present invention are T-cell mediated effector functions. In the case of a helper T cell (CD4 ⁺ T cells), these functions involve the release of cytokines such as interleukin-2 and/or the activation of CD8 ⁺ lymphocytes (CTL) and/or B cells, and in the case of CTL, the elimination of cells those. 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 destruction of antigen-expressing target cells through cytolysis.

The term "immunoreactive cell" or "immune effector cell" in the context of 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 an antigen expressed on the surface of a cell, and mediating an immune response. For example, such cells secrete cytokines and/or chemokines, kill microorganisms, 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 the context of the present invention, "immunoreactive cells" are T cells, preferably CD4 ⁺ and/or CD8 ⁺ T cells. In accordance with the present invention, the term "immunoreactive cell" also includes a cell that, upon suitable stimulation, can mature into an immune cell (such as a T cell, in particular a helper T cell or a cytolytic T cell). Immunoreactive cells include CD34 ⁺ hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of progenitor T cells into a cytolytic T cell, when exposed to an antigen, is similar to clone selection within 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 present on the surface of antigen presenting cells or diseased cells, such as cancer cells. Preferably, said recognition gives the cell that recognizes the antigen a susceptible or reactive status. If the cell is a helper T cell (CD4 ⁺ T cell), such sensitivity or reactivity may involve the release of cytokines and/or activation of CD8 ⁺ lymphocytes (CTL) and/or B cells. If the cell is a CTL, such sensitivity or reactivity may involve elimination of the cells, ie. cells characterized by antigen expression, for example, through apoptosis or perforin-mediated cell lysis. In accordance with the present invention, CTL sensitivity may involve the induction of a continuous calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific killing of antigen-expressing target cells by cytolysis. . CTL sensitivity can also be determined using an artificial reporter that can detect CTL sensitivity with high accuracy. Such a CTL that recognizes an antigen and is sensitive or reactive is also referred to herein as "antigen-responsive CTL".

A "lymphoid cell" is a cell which, optionally after suitable modification, for example, after transfer of a T-cell receptor or an antigen receptor, is capable of stimulating an immune response, for example, 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. In one embodiment, the lymphoid cell lacks endogenous expression of the T-cell receptor.

The terms "T cell" and "T lymphocyte" are used interchangeably herein and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), which provide for cytolytic T- cells.

T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They also differ from other types of lymphocytes, such as B cells and natural killer cells, by having special receptors on their cell surface called T-cell receptors (TCRs). The thymus is the main organ responsible for the maturation of T cells. Several distinct subpopulations of T cells have been found, each with a distinct function.

Helper T cells, among other functions, assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. Such cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells become activated upon presentation of peptide antigens by class II MHC molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or participate in an active immune response.

Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. Such 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. This T cell receptor consists of two separate peptide chains that are products of independent T cell receptor alpha and beta genes (TCRα and TCRβ) and are referred to as α- and β-TCR chains. γδ T cells (gamma, delta T cells) are a small subset of T cells that contain 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 much less common (2% of all T cells) than αβ T cells.

All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells colonize the thymus and proliferate by cell division to form a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore classified as double negative (CD4-CD8-) cells. As they develop, they become double positive (CD4+CD8+) thymocytes, and eventually mature into single positive (CD4+CD8- or CD4-CD8+) thymocytes, which then migrate from the thymus to peripheral tissues.

T cells can usually be obtained in vitro or ex vivo using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood, or a cellular 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, non-human animals, cell lines or cultures. The sample containing T cells can be, for example, peripheral blood mononuclear cells (PBMC).

T cells to be used in accordance with the present invention may express an endogenous T cell receptor, or they may lack expression of an endogenous T cell receptor.

Nucleic acids, such as RNA encoding an antigen receptor, can be introduced into T cells or other cells with cytolytic ability, in particular lymphoid cells.

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

The term "antigen-specific T cell" or similar terms refers to a T cell which, in particular when provided 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 exhibits T-cell effector functions as described above. T cells and other cells of the lymphoid lineage are considered specific for an antigen if the cells destroy antigen-expressing target cells. The specificity of T cells can be assessed using any of a variety of standard techniques, such as a chromium release assay or a proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-γ) can be determined.

The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules, and refer to a complex of genes that are found in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting 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 surface of cells, and they expose both their own antigens (peptide fragments from the cell itself) and foreign antigens (eg, fragments of invasive microorganisms) to the T cell.

In accordance with the present invention, the term "antigen receptor" includes engineered receptors that confer on an immune effector cell, such as a T cell, a certain specificity, such as that of a monoclonal antibody. Thus, a large number of antigen-specific T cells can be obtained for adoptive cell transfer. Thus, the antigen receptor according to the present invention may be present on T cells, for example, in addition to or instead of the T cell's own T cell receptor. It is not necessary for such T cells to process and present an antigen in order to recognize the target cell, but rather they may preferentially recognize with some specificity any antigen present on the target cell. Preferably, said antigen receptor is expressed on the cell surface. For the purposes of the present invention, antigen receptor containing T cells are encompassed by the term "T cell" as used herein. In particular, in accordance with 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 center or antigen-binding domain to an antigen expressed on the surface of the target cell), and which can confer specificity on an immune effector cell, such as T a cell that expresses said antigen receptor on the cell surface. 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 components, wherein said protein components comprise one or more of the domains described herein. The term "antigen receptor" preferably does not include T-cell receptors. In accordance with the present invention, the term "antigen receptor" is preferably synonymous with the terms "chimeric antigen receptor (CAR)", "chimeric T-cell receptor" and "artificial T-cell receptor".

In accordance with the present invention, an antigen may be recognized by an antigen receptor through any antigen recognition domains (herein also referred to simply as "domains") capable of forming an antigen-binding site, for example, through antigen-binding regions of antibodies and T-cell receptors, which may be located on the same or on different peptide chains. In one embodiment, the two domains forming the antigen binding site are derived from 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 single chain variable fragments (scFv) derived from monoclonal antibodies, and T-cell receptor variable domains, in particular separate alpha and beta TCR chains. In fact, virtually all entities that bind a given target with high affinity can be used as an antigen recognition domain.

In one embodiment, the antigen receptor of the present invention comprises at least four immunoglobulin variable domains forming at least two binding sites, where the two binding sites may bind to the same or different epitopes, where the epitopes may be on the same the same or on 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 variable domain (or region) (VL) 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. In one embodiment, 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 unable to interact and form an antigen binding site with VL(2), while VH(2) is unable to interact and form an antigen-binding center with VL(1). However, in another embodiment, VH(1) is capable of interacting and forming an antigen binding site with VL(1) as well as VL(2) and VH(2) is capable of interacting and forming an antigen binding site with VL(2) as well as VL. (1). In a 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, the present invention provides an antigen receptor, also referred to herein as a combination antigen receptor, wherein the receptor comprises a first peptide chain and a second peptide chain, wherein the first peptide chain comprises at least first and second domains and an immune cell receptor signal transduction domain. ; the second peptide chain contains at least the first and second domains and the signal transduction domain from the immune cell receptor; where the first domain from the first peptide chain together with one of the domains from the second peptide chain forms the first antigen-binding center, and where the second domain from the first peptide chain together with another domain from the second peptide chain forms the second antigen-binding center.

In one embodiment, the combination antigen receptor of the present invention comprises a heavy chain variable domain coupled to a light chain variable domain on each of both peptide chains, wherein two antigen binding sites are formed by interaction of the heavy chain variable domain with the light chain variable domain on different peptide chains. chains. In one embodiment, the combination antigen receptor of the present invention comprises two peptide chains, where one peptide chain contains VL(1) and VH(2) and the other polypeptide chain contains VH(1) and VL(2). According to another embodiment, the combination antigen receptor of the present invention comprises a heavy chain variable domain coupled to a heavy chain variable domain on one peptide chain and a light chain variable domain coupled to a light chain variable domain on the other peptide chain, where the formation of two antigen binding sites occurs due to the interaction of the heavy chain variable domain with the light chain variable domain on different peptide chains. In one embodiment, the combination antigen receptor of the present invention comprises two peptide chains, where one peptide chain contains VH(1) and VH(2) and the other peptide chain contains VL(1) and VL(2).

In one embodiment, the combination antigen receptor of the present invention comprises a first peptide chain wherein the heavy chain variable region (VH) and the light chain variable region (VL) are preferably located, N-terminus to C-terminus, in the 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 N-terminal to C-terminal in the order VL(1)-VH(2). The immune cell receptor signaling domain is preferably located in the C-terminal direction with respect to the order of the variable regions, and preferably contains the constant region of the alpha chain of the T-cell receptor or its portion located on one of the peptide chains, and the constant region of the beta- T-cell receptor chain located on another of the peptide chains.

In one embodiment, the combination antigen receptor of the present invention comprises a first peptide chain wherein the heavy chain variable region (VH) and the light chain variable region (VL) are preferably located, N-terminus to C-terminus, in the 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 order VL(1)-VL(2). The immune cell receptor signaling domain is preferably located in the C-terminal direction with respect to the order of the variable regions, and preferably contains the constant region of the alpha chain of the T-cell receptor or its portion located on one of the peptide chains, and the constant region of the beta- a chain of a T-cell receptor or its portion located on another of the peptide chains.

In one aspect, the present invention provides an antigen receptor, also referred to herein as a tandem antigen receptor, wherein the receptor comprises a first peptide chain and a second peptide chain, wherein the first peptide chain comprises at least four domains and an immune cell receptor signal transduction domain; the second peptide chain contains the signal transduction domain from the immune cell receptor; where two of the domains from the first peptide chain form the first antigennegative center, and where the other two domains from the first peptide chain form the second antigennegative center.

In one embodiment, the tandem antigen receptor of the present invention comprises a heavy chain variable domain coupled to a light chain variable domain coupled to an additional heavy chain variable domain coupled to a light chain variable domain on the first peptide chain, wherein two antigen binding sites are formed by interactions of a heavy chain variable domain with a light chain variable domain on the same peptide chain. Thus, the first peptide chain contains VH(1) and VL(1), as well as VH(2) and VL(2), on the same peptide chain. It is contemplated that, in one embodiment, the tandem antigen receptor of the present invention may comprise two scFv molecules connected via a linker peptide on the first peptide chain.

In one embodiment, the tandem antigen receptor of the present invention comprises a first peptide chain wherein the heavy chain variable region (VH) and the light chain variable region (VL) are preferably located, N-terminus to C-terminus, in the order VH(1)- VL(1)-VH(2)-VL(2), VL(1)-VH(1)-VH(2)-VL(2), VH(1)-VL(1)-VL(2)- VH(2) or VL(1)-VH(1)-VL(2)-VH(2). The immune cell receptor signaling domain is preferably located in the C-terminal direction with respect to the order of the variable regions, and preferably comprises a T cell receptor alpha chain constant region or portion thereof or a T cell receptor beta chain constant region or portion thereof . The immune cell receptor signal transduction domain located on the second peptide chain preferably comprises a T-cell receptor alpha chain constant region or portion thereof, if the first peptide chain contains a T-cell receptor beta chain constant region or portion thereof, or a beta constant region T-cell receptor α chain or portion thereof, if the first peptide chain contains a T-cell receptor alpha chain constant region or portion thereof.

The antigen receptors of the present invention contain at least two antigen-binding centers, and thus are at least divalent. As mentioned above, the antigen receptor binding sites of the present invention may bind to the same or different epitopes, and the epitopes may be on the same or 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, where such identical or substantially identical domains may occur, for example , 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, where such different domains may come from different immunoglobulins. In the case of such different domains, it is preferred that the domains with specificity for different epitopes do not interact or hardly interact with each other, i. 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 combination antigen receptor of the present invention contains two peptide chains, where one peptide chain contains VH(1) and VL(2), and the other peptide chain contains VH(2) and VL(1), this causes the peptide chains to are not able to form antigen-binding centers through intramolecular interaction of domains.

The two domains of the antigen receptor of the present invention that form the antigen binding site may also be derived from the T cell receptor and may be fragments or regions thereof that provide antigen specific binding, in particular binding to the peptide-MHC complex, such as the variable regions of the T cell receptor.

In accordance with the present invention, the term "variable region of the T-cell receptor" refers to the variable domains of the TCR chains. The variable region of both the α-chain and the β-chain of the TCR contains three hypervariable or complementarity-determining regions (CDRs), while the β-chain variable region contains an additional hypervariable region (HV4) that does not normally come into contact with 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, and the β-chain CDR1 has been shown to interact with the C-terminal portion of the peptide. CDR2 is believed to recognize MHC. The β-chain CDR4 is not believed to be involved in antigen recognition, but has been shown to interact with superantigens.

The above disclosure relating to immunoglobulin variable domains is appropriately applicable to T cell receptor variable domains. The antigenic receptor of the present invention, instead of an immunoglobulin heavy chain (VH) variable domain with a specificity for the first epitope (VH(1)) and an immunoglobulin light chain (VL) variable domain with a specificity for the first epitope (VL(1)) may contain a TCR variable domain -α-chains of TCR with specificity for the first epitope and the variable domain of the TCR-β-chain of TCR with specificity for the first epitope. Alternatively, or additionally, the antigen receptor of the present invention instead of an immunoglobulin heavy chain (VH) variable domain with specificity for a second epitope (VH(2)) and an immunoglobulin light chain variable domain (VL) with a specificity for a second epitope (VL(2 )) may contain a TCR-α TCR chain variable domain with a specificity for a second epitope and a TCR-β TCR chain variable domain with a second epitope specificity.

Because each antigen binding site is formed from two domains, each domain may contain a region or fragment of an immunoglobulin or T-cell receptor, respectively. A single site or fragment may not be able to bind an antigen by itself, but when two separate sites or fragments are associated, together they form or restore the antigen-binding structure of the original immunoglobulin or T-cell receptor and thus are able to bind the same antigen, preferably with the same affinity.

After antigen recognition, receptor clustering and signal transduction into the cell preferably occur. In this regard, an "immune cell receptor signaling domain" or "T cell signaling domain" is a domain that is involved in signaling an activation to a T cell after antigen binding. The possibility of such signaling is provided by the antigen receptors of the present invention, containing a constant or invariant region of the T-cell receptor chain or a constant or invariant region of the Fc receptor chain of an immune cell, or a region of a constant or invariant region, for example, a constant region of the T-cell receptor alpha chain or a portion thereof, on the same peptide chain, and containing the corresponding constant or invariant region of the T-cell receptor chain or the corresponding constant or invariant region of the immune cell Fc receptor chain, or a portion of the constant or invariant region, for example, the constant region of the beta chain of the T-cell receptor or its site, on another peptide chain. In this regard, CD3 complex refers to an antigen that is expressed on mature T cells, thymocytes, and human natural killer cell subpopulations as part of a multimolecular complex that is the T cell receptor (TCR). 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 bind to the T cell receptor (TCR) and the ζ chain to generate an activation signal for T lymphocytes. TCR, ζ-chain, and CD3 molecules together form the TCR complex. CD3 is responsible for TCR signal transduction. As described by Lin and Weiss, Journal of Cell Science 114, 243-244 (2001), activation of the TCR complex by binding to MHC-presented specific antigen epitopes results in phosphorylation of immunoreceptor tyrosine-based (ITAM) activating motifs by Src family kinases, triggering the recruitment of additional kinases. , resulting in T-cell activation, including the release of Ca ²⁺ . Clustering of CD3 on T cells, for example by immobilized anti-CD3 antibodies, results in T cell activation similar to T cell receptor involvement but independent of its clonal specificity.

The antigen receptor signal transduction domain preferably at least serves to interact with the native cellular signal transduction complex, eg the CD3 complex, which is responsible for translating the antigen binding signal to the antigen receptor into the cell, resulting in activation of the immune cell. The specificity of the signal transduction domain is only limited in 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 will form a dimer with the signal transduction domain on the second strand, for example, via disulfide bridges. Preferred signal transduction domains may include a constant or invariant region of the T cell receptor chain, or a constant or invariant region of the immune cell Fc receptor chain, or a portion of the constant or invariant region. Preferred signal transduction domains can include the T cell receptor alpha, beta, gamma or delta chain constant region or portion thereof, as well as the D2 or D3 invariant regions of the immune cell Fc receptor constant domain or portion thereof. In a preferred embodiment, the first peptide chain provides a T cell receptor alpha chain constant region or portion thereof, and the second peptide chain provides a T cell receptor beta chain constant region or portion, or the first peptide chain provides a T beta chain constant region α-cell receptor or portion thereof, and the second peptide chain provides for the constant region of the T-cell receptor alpha chain or portion thereof. In another embodiment, the first peptide chain provides a T cell receptor gamma chain constant region or portion thereof, and the second peptide chain provides a T cell receptor delta chain constant region or portion, or the first peptide chain provides a T cell delta chain constant region α-cell receptor or portion thereof, and the second peptide chain provides for the constant region of the T-cell receptor gamma chain or portion thereof. Optionally, the signal transduction domains can be modified so that additional disulfide bonds are formed between the chains, providing more efficient dimer formation and higher dimer stability.

Without going into a specific mechanism of action, it is believed that the two peptide chains of the antigen receptor of the present invention, when expressed on the surface of an immune cell, through interactions (for example, disulfide bonds) form a dimer between at least separate signal transduction domains from the immune cell receptor. on two chains, and also form a complex with the endogenous CD3 complex involved in physiological signal transduction with the participation of the T-cell receptor. However, the present invention may also include a direct fusion with CD3ζ or any other immune cell signaling domain (CD3, CD3 subunit of FcγR) instead of the Cα and Cβ domains of the TCR. It is believed that after the binding of the antigen, the signal is transmitted to the cell, which leads to the activation of the immune cell and to the development of an antigen-specific immune response. In addition, cross-chain antigen binding is believed to provide a more stable antigen-antigen receptor-endogenous CD3 signal transduction module, while the higher stability in turn provides more effective stimulation of an antigen-specific immune response, compared to monovalent receptors and bivalent receptors capable of only to intrachain antigen binding. This higher stability is also believed to allow the use of exclusively human-derived immune cell receptor signal transduction domains (e.g., substitution of an amino acid sequence originating from a human body with an amino acid sequence originating from another species, such as a mouse, is minimal or missing at all). Thus, any potential unwanted immune response to the antigen receptor itself can be avoided.

The antigen receptors of the present invention, or their peptide chains, may also contain one or more co-stimulatory domains in addition to antigen binding site-forming and immune cell receptor signaling domains, including CD3ζ or any other immune cell signaling domain. Costimulatory domains serve to increase the rate of proliferation and survival of T cells, such as cytotoxic T cells, after antigen receptor binding to a target molecule. The feature of co-stimulatory domains is limited only by the fact that they have the ability to increase the degree of cell proliferation and survival after binding the target molecule to the antigen receptor. Suitable 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 receptor superfamily, 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 can be used without adversely affecting the present invention, and the variants have activity similar or similar to that of the domain on which they are modeled. Such variants will have at least about 80% sequence identity with the amino acid sequence of the domain from which they originate. According to some embodiments of the present invention, antigen receptor constructs or peptide chains thereof contain two co-stimulatory domains. Although certain combinations include all possible variants of the four indicated 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 immune cell receptor signal transduction domains connected in an N-terminal to C-terminal direction. However, the antigen receptors of the present invention or their peptide chains are not limited to such an arrangement, and other arrangements are acceptable and include immune cell receptor signal transduction domains and one or more co-stimulatory domains.

It will be understood that since the domains that form the antigen binding sites must be free to allow antigen binding, the placement of such domains in the fusion protein will typically be such that the region on the outer surface of the cell is exposed. Similarly, since costimulatory and immune cell receptor signaling domains serve to induce T cell activity and proliferation, in a fusion protein these domains will typically be exposed on the outer surface of the cell. Antigenic receptors may include additional elements such as a signal peptide to ensure proper export of the fusion protein 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 domains that form antigen binding sites, and provides strong binding to the antigen.

Optionally, the antigen receptors of the present invention may further comprise a linker, wherein the linker may be a random amino acid sequence or other chemical compound useful as a spacer between amino acid sequences. Typically, the linker is designed to provide flexibility and resistance to proteases. For example, the linker may be located 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 combination antigen receptor of the present invention. In another embodiment, the linker may be located between any two of at least four domains on the first peptide chain, i.e. between the first and second domains, and/or between the second and third domains, and/or between the third and fourth domains of the tandem antigen receptor of the present invention. Optionally, the linker may be between the domains that form the antigen binding sites and the signal transduction domain from the immune cell receptor. 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 present invention. In specific embodiments, the linker may be a random 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, 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 certain embodiments, the linker may be an antibody hinge region or a fragment thereof.

The antigen receptor of the present invention may further comprise another domain that anchors the antigen receptor to the membrane, such as a typical transmembrane domain. Preferably the transmembrane domain is included in the signal transduction domain, or is part of it.

In other embodiments, the antigen receptors or antigen receptor peptide chains of the present invention may further comprise other domains, such as additional domains involved in or enhancing antigen binding, signal sequences for expression of membrane-bound proteins or for secretion capability, domains that provide improved dimerization, and the transmembrane domain if it is not already part of an immune cell receptor signaling domain. In certain embodiments, the transmembrane domain may be a hydrophobic alpha helix that crosses the membrane.

Preferably, the signal sequence or signal peptide is a sequence or peptide that allows proper passage through the secretory pathway and expression on the cell surface so that the antigen receptor, for example, can bind an antigen present in the extracellular space. Preferably, the signal sequence or signal peptide is cleavable and removed from mature peptide chains. The signal sequence or signal peptide is preferably selected based on the cell or organism in which the peptide chains are produced.

According to a particular embodiment, the peptide chain of the combination antigen receptor of the present invention may comprise the following structure: cells-COOH.

In a specific embodiment, the first peptide chain of the tandem antigen receptor of the present invention may have the following structure: NH2 signal peptide - first domain involved in antigen binding - optional linker - second domain involved in antigen binding - optional linker - third domain involved in antigen-binding-optional linker-the fourth domain involved in antigen-binding-optional linker-signaling domain from the immune cell receptor-COOH.

With respect to the tandem antigen receptors of the present invention, herein the first and second (from N-terminus to C-terminus) domains involved in the formation of the antigen-binding center, the first peptide chain, are also referred to as "N-terminal domains", and the third and fourth ( from the N-terminus to the C-terminus) the domains involved in the formation of the antigen-binding center, the first peptide chain, are also referred to as "C-terminal domains".

Exemplary antigen receptors of the present invention include, without limitation, those formed by first and second peptide chains having the structures shown in Table I below (VH is an immunoglobulin heavy chain variable region or region thereof; VL is an immunoglobulin light chain variable region or region thereof ; C1 and C2 are immune cell receptor signaling domains that will form a dimer with each other, e.g. immune cell Fc receptor chain constant or invariant region or T cell receptor chain constant or invariant region or constant or invariant region region ):

Table I

First peptide chain Second peptide chain VH(1)-VL(2)-C1 VL(1)-VH(2)-C2 VH(1)-VH(2)-C1 VL(1)-VL(2)-C2 VH(1)-VH(2)-C1 VL(2)-VL(1)-C2 VH(1)-VL(2)-C1 VH(2)-VL(1)-C2 VH(1)-VL(1)-VH(2)-VL(2)-C1 C2 VL(1)-VH(1)-VH(2)-VL(2)-C1 C2 VH(1)-VL(1)-VL(2)-VH(2)-C1 C2 VL(1)-VH(1)-VL(2)-VH(2)-C1 C2

As defined above, an antigen receptor comprises an immunoglobulin heavy chain (VH) variable domain with a first epitope specificity (VH(1)), an immunoglobulin light chain (VL) variable domain 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. In one embodiment, 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 unable to interact and form an antigen binding site with VL(2), while VH(2) is unable to interact and form an antigen-binding center with VL(1). However, in another embodiment, VH(1) is capable of interacting and forming an antigen binding site with VL(1) as well as VL(2) and VH(2) is capable of interacting and forming an antigen binding site with VL(2) as well as VL. (1). In a 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 C1 and C2 domains of the first and second peptide chains shown in Table I are T-cell receptor alpha and beta chain constant regions, respectively, or portions thereof.

In a preferred embodiment, if two domains on one peptide chain are both an immunoglobulin heavy chain variable region or portion thereof, and two domains on the other chain are both an immunoglobulin light chain variable region or portion thereof, then a linker is present between the first and second domains. on both peptide chains. The linker may be a random amino acid sequence 10-25 amino acids long, more preferably 15 amino acids long. In a specific embodiment, the linker is a 3 repeat amino acid sequence of 5 monomeric units (Gly4Ser).

In certain 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 or an immune cell receptor signal transduction domain, are from a mammalian body, preferably a mouse, and more preferably person. In one embodiment, the amino acid sequences are human-derived, but mouse sequences have been incorporated into them by replacing one or more amino acids in the human sequence with an amino acid at the corresponding position in the mouse sequence. Such a substitution may provide a higher degree of dimerization or stability or the ability to signal into the cell after antigen binding. In yet another embodiment, the amino acid sequences are mouse-derived 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, such as cytotoxic activity, on a cell such as a T cell as described above. However, in contrast to the binding of a T cell receptor to an antigenic peptide-MHC complex as described above, an antigenic receptor according to certain embodiments can bind to an antigen, particularly when it is expressed on a cell surface.

The amino acid sequences of the peptide chains, including any of the domains or linkers, can be modified. For example, and as will be appreciated by those skilled in the art, antibody and T cell receptor variable region sequences can be modified without losing their ability to bind to a target, and therefore the amino acid sequence of antigen binding sites can be similarly modified without losing their ability to bind to a target. For example, the amino acid sequence of the domain that forms the antigen binding center may be identical or substantially homologous to the variable region of the antibody from which it is derived. By "substantially homologous" is meant that 1-5, preferably 1-4, such as 1-3 or 1 or 2 substitutions can be made. According to one embodiment, the peptide chain may contain naturally occurring amino acids and artificial amino acids. According to another embodiment, the peptide chain contains only naturally occurring amino acids. The term "artificial amino acid" refers to an amino acid having a structure different from that of the 20 naturally occurring amino acid molecules. Since artificial amino acids have a structure similar to that of natural amino acids, artificial amino acids can be classified as derivatives or analogues of these natural amino acids.

The present invention also encompasses derivatives of the antigen receptors and peptide chains described herein. In accordance with the present 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 such modified analogs resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, protection/blocking groups, proteolysis, 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 peptide chain thereof has an increased ability to bind or dimerize and/or an increased ability to activate an immune response.

The cells used with the antigen receptor system of the present invention are preferably T cells, in particular cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells and lymphokine-activated killer (LAK) cells. . Once activated, each of these types of cytotoxic lymphocytes triggers the destruction of target cells. For example, cytotoxic T cells trigger destruction of target cells in one or both of the following ways. The first is that, upon activation, cytotoxins such as perforin, granzymes, and granulosin are released from T cells. Perforin and granulosin create pores in the target cell, and granzymes enter the cell and trigger the caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death) of the cell. The second is that apoptosis can be induced through Fas-Fas ligand interaction between T cells and target cells. The cytotoxic lymphocytes will preferably be autologous cells, although heterologous cells or allogeneic cells may also 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, i.e. immunoglobulin domain, with a typical immunoglobulin (Ig) fold. The term encompasses 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 generally 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. Such chains are mainly composed of immunoglobulin domains such as V_L-domain (light chain variable domain), C_L-domain (light chain constant domain) and C_H-domains (heavy chain constant domains) C_H1, C_H2, C_H3 and C_H4. There are five types of heavy chains of mammalian immunoglobulins, ie. α, δ, ε, γ and µ, which correspond to different classes of antibodies, i.e. IgA, IgD, IgE, IgG and IgM. Unlike 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, i.e. lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is predominantly conserved among the various isotypes of immunoglobulins, where the variable region varies greatly 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 linked by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The VH and VL regions can further be divided into hypervariable regions called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged, from amino to carboxy, 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.

In the context of this document, the term "monoclonal antibody" refers to a preparation of antibody molecules of the same molecular composition. A monoclonal antibody has a single binding specificity and affinity. In one embodiment, the monoclonal antibodies are produced by a hybridoma that contains a B cell derived from a non-human animal, such as a mouse, fused to an immortal cell.

As used herein, the term "recombinant antibody" includes all antibodies produced, expressed, generated, or isolated by recombinant methods, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for immunoglobulin genes. , or hybridoma obtained using it, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies obtained expressed, created, or isolated by other means, which involve combining immunoglobulin gene sequences with other DNA sequences.

As used herein, the term "human antibody" is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (eg, in the case of mutations introduced using in vitro random or site-specific mutagenesis or in vivo somatic mutation).

The term "humanized antibody" refers to a molecule with an antigen binding site that is predominantly derived from an immunoglobulin from a non-human species, with the remainder of the immunoglobulin structure of the molecule based on the structure and/or sequence of the human immunoglobulin. The antigen binding site may either contain complete variable domains fused to constant domains or only complementarity determining regions (CDRs) grafted onto the respective variable domain framework regions. Antigen binding sites may be wild-type or modified with one or more amino acid substitutions, for example modified to more closely resemble human immunoglobulins. 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 changed from the original antibody.

The term "chimeric antibody" refers to those antibodies in which one portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to the corresponding sequences of other antibodies. . 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 such chimeric forms is that the variable region can conveniently be derived from currently known sources using readily available B cells or hybridomas of non-human hosts in combination with constant regions derived, for example, from human cell preparations. While the variable region has the advantage of being easy to obtain and having specificity that is independent of source of origin, a human constant region, when administered with antibodies, is less likely to elicit an immune response in a human subject than a constant region from a source other than person. However, the definition is not limited to this particular example.

Antibodies can be from a variety of species, including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

Antibodies described herein 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, more specifically an IgG1, kappa, or IgG1 isotype, 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 herein may contain 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 antigen-binding function of an antibody 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, divalent fragments containing 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 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 methods, with a synthetic linker that allows them to be obtained as a single protein chain in which the VL and VH regions are combined. paired to form monovalent molecules (known as single-stranded 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 intended to be encompassed by the term "antigen binding fragment" of an antibody. A further example are immunoglobulin binding domain-based fusion proteins comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge, and (iii) ) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Fusion proteins based on the immunoglobulin binding domain are further disclosed in US 2003/0118592 and US 2003/0133939. Such antibody fragments are prepared using conventional techniques known to those skilled in the art, and the fragments are screened for utility in a manner similar to that of intact antibodies.

A single chain variable fragment (scFv) is a fusion protein based on the heavy (VH) and light chain (VL) immunoglobulin variable regions linked by a linker peptide. The linker can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. Bivalent (or divalent) single chain variable fragments (di-scFvs, bi-scFvs) can be constructed by joining two scFvs. This can be done by making one peptide chain with two VH and two VL regions to form tandem scFvs.

The term "binding domain" or simply "domain" as used in the present invention means a structure, for example an antibody, that binds/reacts with a given target structure/antigen/epitope, optionally while interacting with another domain. Thus, such domains are referred to as an "antigen binding site" in accordance with the present invention.

Antibodies and antibody derivatives are useful in the production of binding domains such as antibody fragments, in particular in the production of VL and VH regions.

Binding domains for an antigen that may be present in an antigen receptor have the ability to bind to a (target) antigen, i.e., the ability to bind to a (target) epitope present on the antigen, preferably an epitope located within the extracellular domain of the 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 particularly preferred embodiments, the binding domains for an antigen bind to native epitopes of an antigen present on the surface of living cells.

All antibodies and antibody derivatives, such as antibody fragments, as described herein for the purposes of the present invention, are encompassed by the term "antibody".

Antibodies can be generated using a number of techniques, including conventional monoclonal antibody production methodology, such as the standard somatic cell hybridization methodology of Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are preferred, in general, other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B-lymphocytes, or phage display techniques using antibody gene libraries.

A preferred animal system for producing hybridomas that secrete monoclonal antibodies is the mouse system. Obtaining a hybridoma in a mouse is a very well established procedure. Immunization protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (eg mouse myeloma cells) and fusion procedures are also known.

Other preferred animal systems for producing hybridomas that secrete monoclonal antibodies are 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 carrier-conjugated peptides derived from the antigenic sequence, i. the sequence to which the antibodies will be directed, a preparation enriched with recombinantly expressed antigen or fragments thereof, and/or described cells expressing the antigen. Alternatively, mice can be immunized with DNA encoding the antigen or fragments thereof. When antibodies are not generated by immunizations using a purified or enriched antigen preparation, mice can also be immunized with cells expressing the antigen, for example from a cell line, to stimulate immune responses.

The immune response can be monitored during the immunization protocol using plasma and serum samples obtained by blood sampling from the tail vein or retroorbital sinus. Mice with sufficient immunoglobulin titers can be used for fusions. Mice can be restimulated intraperitoneally or intravenously with antigen expressing cells 3 days prior to sacrifice and spleen removal to increase the proportion of hybridomas secreting specific antibodies.

To obtain monoclonal antibody-producing hybridomas from immunized mice, splenocytes and lymph node cells can be isolated and fused with a suitable immortal cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for the presence of antibody-secreting hybridomas. Antibodies with specificity for an antigen can be identified by immunofluorescence assay and FACS using antigen expressing cells. Antibody-secreting hybridomas can be subcultured, screened again and, if still positive for monoclonal antibody production, can be subcloned by serial dilution. The stable subclones can then be cultured in vitro to produce the antibody in tissue culture medium for characterization.

The ability of antibodies and other binding agents to bind an antigen can be determined using standard binding assays (eg, ELISA, Western blot, immunofluorescence, and flow cytometry).

The term "binding" in accordance with the present invention preferably refers to specific binding.

In accordance with the present invention, an agent, such as an antigen receptor, is capable of binding to (targeting) a predefined target if it has a significant degree of affinity for said predefined target and binds to said predefined target in standard assays. "Affinity" or "binding affinity" is often determined by the equilibrium dissociation constant (K _D ). Preferably, the term "significant affinity" refers to binding to a predetermined target with a dissociation constant (K _D ) 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 (practically) unable to bind to (target) a target unless it has a significant degree of affinity for said target and does not bind to a significant extent, in particular does not bind at a detectable level, to said target in standard assays. Preferably, the agent does not bind at a detectable level to said target if it is present at a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg/ml or higher. Preferably, the agent does not have a significant degree of target affinity if it binds to said target with a K _D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times the binding K _D with a predetermined target with which the agent is able to bind. For example, if the K _D of an agent binding to a target for which the agent is able to bind is 10 ^-7 M, then the K _D of binding to a target for which the agent does not have a significant degree of 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 to a predefined target if it is able to bind to said predefined target while being (practically) incapable of binding to other targets, ie. does not have a significant degree of affinity for other targets and does not bind significantly to other targets in standard assays. Preferably, the agent is specific to a predefined target if the degree of affinity and binding to such other targets is not significantly greater than the degree of affinity or binding to proteins that are not related to the predefined target, such as bovine serum albumin (BSA), casein or human serum albumin ( HSA). Preferably, an agent is specific to a predetermined target if it binds to said target with a K _D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times or 10 ⁶ times lower than the K _D binding to the target for which it is specific. For example, if the K _D of an agent binding to a target for which it is specific is 10 ^-7 M, then the K _D of 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 experimentally using any suitable method; see, for example, 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 herein. Affinity values can be readily determined using conventional techniques, such as equilibrium dialysis; using the BIAcore 2000 instrument, using the general procedures described by the manufacturer; using radioimmunoassay using radiolabeled target antigen; or by another method known to the person skilled in the art. Affinity data can be analyzed, for example, using the method of Scatchard et al., Ann NY Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may vary if measurements are made under different conditions, eg salt concentration, pH. Thus, measurements of affinity and other antigen binding parameters, eg K _D , IC ₅₀ , are preferably performed using standardized antibody and antigen solutions and a standardized buffer.

The present invention may include 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 the present invention, the term "transfection" includes the introduction of a nucleic acid into a cell or the absorption of the nucleic acid into a cell, where the cell may be in the body of a subject, such as a patient. Thus, in accordance with the present invention, the cell to be transfected with the nucleic acid described herein 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 the present invention, transfection may be transient or stable. For some transfection applications, it is sufficient that the transfected genetic material is only transiently expressed. Since the nucleic acid included in the transfection process does not usually integrate into the nuclear genome, the foreign nucleic acid will be lost after mitosis or will be destroyed. In cells that allow episomal amplification of nucleic acids, the rate of loss is reduced. If the transfected nucleic acid is to be permanently retained in the cell's genome and its daughter cells, stable transfection must take place. RNA can be transfected into cells for transient expression of the protein it encodes.

In accordance with the present invention, for the introduction of nucleic acids into cells, i. transfer or transfection, any suitable method may be used. Preferably, the nucleic acid, such as RNA, is transfected into cells using standard techniques. Such techniques include electroporation, lipofection and microinjection. According to 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 plasma membrane of a cell due to an externally applied electric field. This technique is usually used in molecular biology as a method of introducing certain substances into cells. In accordance with the present invention, it is preferred that the introduction into cells of a nucleic acid encoding a protein or peptide results in the expression of said protein or peptide.

A number of methods can be used to incorporate antigen receptor constructs into T cells, including transfection based on non-viral DNA, transposon based systems, and virus based systems. Transfection based on non-viral DNA has a low risk of insertional mutagenesis. With transposon-based systems, transgenes can be integrated more efficiently than with 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, transduce T cells efficiently and stably, and have been tentatively shown to be safe in terms of integration into primary human T cells. Lentiviral vectors also efficiently and stably transduce T cells, but are more expensive to manufacture. They are also potentially more secure than retrovirus-based systems.

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

In accordance with the present invention, the nucleic acid encoding the antigen receptor is preferably administered in "naked form" or with a carrier. Carriers, such as lipid carriers, for use in the present invention include any substances or delivery vehicles with which a nucleic acid, such as RNA, can be associated, for example, by complexing with the nucleic acid or forming vesicles that enclose or encapsulated nucleic acid. As a result, the stability of the nucleic acid can be increased compared to the "naked" nucleic acid. In particular, the stability of the nucleic acid in the blood can be increased. For example, RNA-based nanoparticle formulations with defined particle size can be used, such as lipoplexes of RNA and liposomes, for example, lipoplexes containing DOTMA and DOPE or DOTMA and cholesterol.

In the context of this document, the term "nanoparticle" refers to any particle with a diameter that makes the particle suitable for systemic, in particular parenteral administration, in particular nucleic acids, usually 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.

In the context of this document, the term "composition based on nanoparticles" or similar terms refers to any substance that contains at least one nanoparticle. In some embodiments, the nanoparticle composition is a homogeneous collection of nanoparticles. In some embodiments, the nanoparticulate compositions are dispersions or emulsions. Typically, a dispersion or emulsion is formed by combining at least two immiscible substances.

The term "lipoplex" or "lipoplex with nucleic acid", in particular "lipoplex with RNA", refers to a complex of lipids and nucleic acids, in particular RNA. Lipoplexes are formed spontaneously by mixing cationic liposomes, which often also contain a neutral "helper" lipid, with nucleic acids.

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

Nucleic acid nanoparticle preparations for use in the present invention can be prepared using a variety of protocols and from any nucleic acid complexing compound. The complexing agents are generally 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 a histone.

In accordance with the present invention, protamine is useful as a cationic carrier. The term "protamine" refers to any of a number of arginine-rich, relatively low molecular weight, strongly basic proteins found in an associated state, particularly DNA, in the spermatozoa of a number of animals (such as fish), instead of somatic histones. In particular, the term "protamine" refers to proteins found in fish milt that are strongly basic, water soluble, do not coagulate when heated, and hydrolyze mainly to form arginine. In their purified form, they are used in formulations based on long-acting insulin and to neutralize the anticoagulant effects of heparin.

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

The protamine used in accordance with the present invention may be sulfated protamine or protamine hydrochloride. In a preferred embodiment, the source of protamine used to make the nanoparticles described herein is Protamine 5000, which provides protamine at a concentration greater than 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, but not limited to, multilayer vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically stabilized liposomes (SSL), multivesicular bodies (MV), and large multivesicular vesicles. body (LMV), as well as other bilayer forms known in this field. The size and number of layers of the liposome will depend on the preparation method, 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 phases and inverse hexagonal phases, cubic phases, micelles, inverted micellar phases consisting of monolayers. Such 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 occur in the nucleic acid nanoparticle formulations of the present invention.

Any suitable method for forming liposomes can be used to form nucleic acid lipoplexes from nucleic acid and liposomes as long as it provides the expected nucleic acid lipoplexes. Liposomes can be formed using standard methods such as the reverse phase evaporation (REV) method, the ethanol injection method, the dehydration-rehydration (DRV) method, sonication, or other suitable methods.

After the formation of liposomes, liposomes can be separated by size to obtain a population of liposomes, characterized by a largely homogeneous range of particles.

Bilayer-forming lipids typically contain two hydrocarbon chains, in particular acyl chains, and an end group, either polar or non-polar. Bilayer-forming lipids are composed of either naturally occurring or synthetic lipids, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, which typically have hydrocarbon chain lengths of approximately 14-22 atoms. carbon, and they are characterized by different degrees of unsaturation. Other suitable lipids for use in the composition of the present invention include glycolipids and sterols such as cholesterol and various analogs thereof, which can also be used in liposomes.

Cationic lipids typically contain a lipophilic moiety such as a sterol, acyl or diacyl chain and have a net positive charge. The end group of a lipid usually carries a positive charge. The cationic lipid preferably has a positive charge corresponding to 1-10 valencies, more preferably a positive charge corresponding to 1-3 valences, and more preferably a positive charge corresponding to 1 valence. Examples of cationic lipids include, without limitation, 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-propane; 1,2-dialkyloxy-3-dimethylammonium-propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate ( DOSPA). Preferred are DOTMA, DOTAP, DODAC and DOSPA. Most preferred is DOTMA.

In addition, the nanoparticles described herein preferably further contain a neutral lipid in view of structural stability, etc. 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. Most preferred is DOPE. In the case where the cationic liposome contains both a cationic lipid and a neutral lipid, the molar ratio of the cationic lipid to the neutral lipid can be determined appropriately in view of the stability of the liposome, etc.

In accordance with one embodiment, the nanoparticles described herein may contain phospholipids. The phospholipids may be a glycerophospholipid. Examples of glycerophospholipid include, without limitation, three types of lipids: (i) zwitterionic phospholipids, which include, for example, phosphatidylcholine (PC), egg yolk phosphatidylcholine derived from PC soybeans in a natural, 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, dimyristoyl-phosphatidylglycerol (DMPG); synthetic derivatives in which the conjugate contains a zwitterionic phospholipid with a negative charge, as in the case of methoxypolyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE); and (iii) cationic phospholipids, which include, for example, phosphatidylcholine or sphingomyelin, the phosphomonoester of which has been O-methylated to form cationic lipids.

The association of the nucleic acid with the lipid carrier can occur, for example, by filling the empty spaces of the carrier with the nucleic acid so that the carrier physically captures the nucleic acid, or by covalent, ionic or hydrogen bonding, or by adsorption through non-specific bonds. Regardless of the mode of association, the nucleic acid must retain its therapeutic, i.e. antigen-coding properties.

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

As used herein, the term "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. The nucleic acid can be single stranded or double stranded. RNA includes in vitro transcribed RNA (IVT-RNA) or synthetic RNA. In accordance with the present invention, the nucleic acid is preferably an isolated nucleic acid.

Nucleic acids may be present in the vector. As used herein, the term "vector" includes any vectors known to those skilled in the art, including plasmid vectors, cosmid vectors, phage-based vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or vectors based on artificial chromosomes, such as artificial bacterial chromosomes (BAC), artificial yeast chromosomes (YAC) or artificial P1 chromosomes (PAC). These vectors include expression vectors as well as cloning vectors. Expression vectors include plasmids as well as viral vectors, and typically contain the required coding sequence and corresponding DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or systems. in vitro expression. Cloning vectors are typically used to construct and amplify a particular desired DNA fragment and may not contain 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 contains ribonucleotide residues and preferably consists entirely or predominantly 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 includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration one or more nucleotides. Such changes may include the addition of a substance other than a nucleotide, for example, to the end(s) of the RNA or inside it, for example, to one or more nucleotides of the RNA. Nucleotides in RNA molecules may also include non-standard nucleotides such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. Such altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

In accordance with the present invention, the term "RNA" includes and preferably refers to "mRNA", which means "messenger RNA", and refers to a "transcript" that can be obtained using DNA as a template and encodes a peptide or protein. mRNA typically contains a 5' untranslated region (5'UTR), a protein or peptide coding region, and a 3' untranslated region (3'UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is obtained by in vitro transcription using a DNA template. According to one embodiment of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. The technique for in vitro transcription is known to the person skilled in the art. For example, there are a number 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 plus-sense RNA. 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 virus genomic RNA or is derived from alpha virus genomic RNA. In one embodiment, the self-replicating RNA is an expression vector based on viral genes. In one embodiment, the virus is a Semliki forest virus. In one embodiment, the self-replicating RNA provides one or more transgenes, wherein at least one of said transgenes encodes the agents described herein. In one embodiment, if the RNA is or is 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 an in vitro transcribed RNA.

In order to increase the level of expression and/or stability of the RNA used in accordance with 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 the RNA used in accordance with the present invention includes any modification of the RNA, which does not naturally occur in the specified RNA.

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

The RNA of the present invention may contain modified naturally occurring or synthetic ribonucleotides to increase its stability and/or reduce cytotoxicity. For example, in one embodiment, in the RNA used in accordance with the present invention, cytidine is replaced by 5-methylcytidine, partially or completely, preferably completely. Alternatively, or additionally, according to one embodiment, in the RNA used in accordance with the present invention, uridine is replaced by pseudouridine, partially or completely, preferably completely.

In one embodiment, the term "modification" refers to the production of RNA with a 5'-cap or 5'-cap analog. The term "5'-cap" refers to a cap structure that is located at the 5'-end of the mRNA molecule and usually consists of a guanosine nucleotide attached to the mRNA through an atypical 5'-5'-triphosphate bond. In one embodiment, such guanosine is methylated at the 7-position. The term "typical 5'-cap" refers to the 5'-cap of a naturally occurring RNA, preferably a cap that is 7-methylguanosine (m7G). In the context of the present invention, the term "5'-cap" includes a 5'-cap analogue that resembles the RNA cap structure and is modified such that it has the ability to stabilize RNA when attached to it, preferably in vivo and/or in a cell.

Providing RNA with a 5'-cap or 5'-cap analog can be accomplished by in vitro transcription of the DNA template in the presence of said 5'-cap or 5'-cap analog, where said 5'-cap is incorporated into the nascent RNA strand during transcription. or RNA can be formed, for example, by in vitro transcription, and the 5'-cap can be attached post-transcriptionally with capping enzymes, for example, vaccinia virus capping enzymes.

The RNA may include further modifications. For example, further modification of the RNA used in the present invention may be an extension or truncation of the naturally occurring poly(A) tail or alteration of the 5' or 3' untranslated regions (UTRs), such as introducing a UTR that is not linked to the coding region of said RNA, e.g. insertion of one or more, preferably two copies of the 3'-UTR derived from a globin gene, e.g. alpha-2-globin, alpha-1-globin, beta-globin, preferably beta-globin, more preferably human beta globin.

Therefore, in order to increase the stability and/or level of expression of the RNA used in accordance with the present invention, it can be modified so that it is present together with a poly-A sequence, preferably 10-500 long, more preferably 30-300, more more preferably 65-200 and especially 100-150 adenosine residues. In a particularly preferred embodiment, the poly-A sequence is about 120 adenosine residues in length. In addition, the inclusion of two or more 3'-untranslated regions (UTRs) in the 3'-untranslated region of the RNA molecule can provide an increase in the efficiency of translation. In a specific embodiment, the 3'UTR is derived from the human β-globin gene.

The term "stability" of RNA refers to the "half-life" of RNA. "Half-life" refers to the period of time it takes to eliminate 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 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 in which the genetic code of a DNA sequence is transcribed into RNA. Subsequently, RNA can be translated into protein. In accordance with the present invention, the term "transcription" includes "in vitro transcription", and the term "in vitro transcription" refers to the process in which RNA, in particular mRNA, is synthesized in vitro in a cell-free system, preferably using appropriate cell extracts. Preferably, cloning vectors are used to obtain transcripts. Such cloning vectors are commonly referred to as transcription vectors and are encompassed by the term "vector" in accordance with the present invention.

The term "translation" in accordance with the present invention refers to the process carried out on the ribosomes of the cell, by which a strand of messenger RNA directs the assembly of an amino acid sequence to form a peptide or protein.

Nucleic acids in accordance with the present invention may be present alone or together with other nucleic acids, which may be homologous or heterologous. In preferred embodiments, the nucleic acid is operably linked to expression control sequences that may be homologous or heterologous to said nucleic acid. The term "homologous" means that, under natural conditions, the nucleic acids are also operably linked, and the term "heterologous" means that, under natural conditions, the nucleic acids are not operably linked.

A nucleic acid and an expression control sequence are "operably" linked to each other if they are covalently linked to each other such that expression or transcription of said nucleic acid is controlled or influenced by said expression control sequence. If the nucleic acid is to be translated into a functional protein, then, together 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 frameshift in the coding sequence or inability to the specified coding sequence to be translated into the desired protein or peptide.

The term "expression control sequence" or "expression control element" in accordance with the present invention includes promoters, ribosome binding sites, enhancers and other control elements that regulate gene transcription or mRNA translation. According to specific embodiments of the present invention, expression control sequences can be regulated. The specific structure of expression control sequences may vary depending on the cell species or type, but generally provides for 5'-untranscribed and 5'- and 3'-untranslated sequences involved in the initiation of transcription and translation, respectively, such as the TATA box, capping sequence, CAAT sequence, etc. More specifically, 5' non-transcribed expression control sequences provide a promoter region that provides a promoter sequence to control transcription of an operably linked nucleic acid. Expression control sequences may also include enhancer sequences or upstream activator sequences.

The term "expression" in accordance with the present invention is used in its most general meaning and it includes the production of RNA and/or peptides or proteins, for example, through transcription and/or translation. With respect to RNA, the term "expression" or "translation" refers in particular to the production of peptides or proteins. It also provides for the partial expression of nucleic acids. In addition, expression may be transient or stable. In accordance with the present invention, the term expression also includes "abnormal expression" or "impaired expression".

"Abnormal expression" or "disturbed expression" in accordance with the present invention means that the expression is altered, preferably increased compared to the reference, for example, the state of the subject, which does not observe a disease associated with abnormal or aberrant expression of a particular protein, for example, tumor antigen. An increase in expression level 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 only found in diseased tissue, while expression in healthy tissue is repressed.

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

In accordance with 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 form the protein or peptide that it encodes.

The term "peptide" in accordance with the present invention includes oligo - and polypeptides and refers to substances containing 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 up to preferably 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 herein.

As mentioned above, the amino acid sequences of the peptide chains and antigen receptors described herein can be modified in order to obtain variants of these amino acid sequences. Accordingly, the present invention includes sequence variants of the peptides and proteins described herein and includes naturally occurring amino acid sequence variants that provide sequences that are functionally equivalent to said sequences, e.g., amino acid sequences having properties identical or similar to those of said sequences. sequences. Important properties are retention of antigen receptor binding to its target, or signal transduction after antigen binding to 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" in accordance with the present invention refers, in particular, to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, in particular those that exist in natural conditions. An allelic variant refers to a change in the normal sequence of a gene, the meaning of which is often not established. Whole gene sequencing typically identifies multiple allelic variants for a given gene. A species homologue is a nucleic acid sequence or amino acid sequence derived from a different species than the given nucleic acid sequence or amino acid sequence. The term "variant" shall encompass 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, has the same or substantially the same immunological properties and/or exhibits 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.

Those skilled in the art will appreciate that, in particular, the sequences of CDR sequences, hypervariable and variable regions can be modified without losing the ability to bind to a target. For example, CDR regions may be either identical or substantially homologous to regions of the original antibodies. By "substantially homologous" is meant that 1-5, preferably 1-4, such as 1-3 or 1 or 2 substitutions per CDR can be made.

For the purposes of the present 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.

Amino acid insertion variants involve the insertion of one or two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants with an insert, one or more amino acid residues are inserted at a specific point in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

Amino acid addition variants provide amino and/or carboxy terminal fusions of one or more amino acids, eg 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 the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can be made at any position on the protein.

Amino acid substitution variants are characterized in that at least one sequence residue 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 among homologous proteins or peptides, and/or substitution of amino acids for other amino acids with similar properties. Preferably, the amino acid changes in protein variants are conservative amino acid changes, ie. substitutions of identically charged or uncharged amino acids. A change in a conservative amino acid involves a substitution of one of a family of amino acids that share similarities in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively referred to as aromatic amino acids.

Preferably, the degree 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 will be 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 considered 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 entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably considered for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. According to preferred embodiments, the degree of similarity or identity is taken into account for the full length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, e.g. using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, fine for opening a gap of 10.0, the penalty for continuing the gap is 0.5.

"Sequence similarity" means the percentage of amino acids that are either identical or are conservative amino acid substitutions. "Sequence identity" between two amino acid sequences means the percentage of amino acids that are identical between sequences.

The term "percent identity" is intended to mean the percentage of amino acid residues that are identical between two compared sequences obtained after the best alignment, such a percentage being purely statistical and the differences between the two sequences distributed randomly and over the entire length of the sequence. Sequence comparisons for two amino acid sequences are typically made by comparing such sequences after they have been optimally aligned, said comparison being done by segment or "comparison window" to identify and compare local areas of sequence similarity. Optimal alignment of sequences for comparison can be performed, in addition to being performed manually, using the local homology search algorithm according to Smith-Waterman, 1981, Ads App. Math. 2, 482, using the Needleman-Wunsch local homology search algorithm, 1970, J. Mol. Biol. 48, 443 using the Pearson-Lipman similarity search method, 1988, Proc. Natl Acad. sci. USA 85, 2444 or by computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI).

Percent identity is calculated by determining the number of identical positions for two compared sequences, dividing this number by the number of compared positions, and multiplying the result by 100, thereby obtaining a percentage identity for the two sequences.

Homologous amino acid sequences in accordance with the present 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% amino acid residue identity.

In accordance with the present invention, a variant, fragment, portion, region or derivative of an amino acid sequence, peptide or protein preferably has a functional property of the amino acid sequence, peptide or protein, respectively, from which it was derived, i. are functionally 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. According to one embodiment, the functional property is a property in relation to antigen binding or signaling after binding to a cell.

The term "derived" in accordance with the present invention means that a particular structure, in particular a particular sequence, is within the entity from which it originates, in particular an organism or molecule. In the case of amino acid sequences, especially specific regions of the sequences, "derived" in particular means that the corresponding amino acid sequence is derived from the amino acid sequence within which it is located.

The term "cell" or "host cell" preferably refers to an intact cell, i. a cell with an intact membrane from which its typical intracellular components such as enzymes, organelles or genetic material have not been released. The intact cell is preferably a viable cell, ie. a living cell capable of carrying out its typical metabolic functions. Preferably, the term in accordance with the present invention refers to any cell that can be transfected with exogenous nucleic acid. Preferably, when transfected with an exogenous nucleic acid and transferred to a recipient, the cell may express the nucleic acid in the recipient. The term "cell" includes bacterial cells; other suitable 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 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 (eg Saccharomyces cerevisiae), Schizosaccharomyces (eg Schizo saccharomyces pombe), Pichia (eg Pichia pastoris and Pichia methanolica) and Hansenula species. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK, etc. However, it is also possible to use amphibian cells, insect cells, plant cells and any other cells used in this field for the expression of heterologous proteins. For adoptive transfer, mammalian cells such as those of humans, mice, hamsters, pigs, goats and primates are particularly preferred. Cells can be derived from a 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. For use in accordance with the present invention, particularly preferred cells 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 propagation" or "propagation" refers to a process where a particular structure increases in number. In the context of the present invention, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated with an antigen, proliferate, and a specific lymphocyte recognizing said antigen is multiplied. Preferably, clonal propagation results in lymphocyte differentiation. The term "priming" refers to the process where a T cell first comes into contact with its specific antigen and causes differentiation into effector T cells.

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

As used herein, the term "isolated" is intended to refer to a structure that is substantially free of other molecules, such as other cellular material. The term "isolated" preferably means that the selected structure has been extracted from its natural environment. The selected structure may be in a practically purified state. The term "substantially pure" preferably means that the structure is substantially free of 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 entity", such as a recombinant cell, in the context of the present invention does not occur in nature.

As used herein, the term "naturally occurring" 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 deliberately modified by humans in a laboratory, is naturally occurring.

The term "autologous" is used to describe anything that comes from a single subject. For example, "autologous transplant" refers to a transplant of tissue or organs originating from the body of the same subject. Such procedures are advantageous because they overcome the immunological barrier that would otherwise lead to rejection.

The term "allogeneic" is used to describe anything that comes from organisms of different individuals of the same species. Two or more individuals are considered allogeneic to each other if the genes at one or more loci are not identical.

The term "syngeneic" is used to describe anything that comes from the organisms of individuals or tissues with identical genotypes, i.e. identical twins or animals of the same inbred line or their tissues.

The term "heterologous" is used to describe anything that consists of several different elements. For example, the transfer of bone marrow from one individual to another individual is a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject.

In the context of this document, "reduce" or "inhibit" means the ability to cause an overall reduction of preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more of the original level. The term "inhibit" or similar phrases includes complete or substantially complete inhibition, ie. decrease to zero or almost 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 at least 50%, even more preferably at least 80% and most preferably at least 100%.

Because the antigen receptor of the present invention can be designed to target virtually any antigen, including disease-specific antigens, the antigen receptor of the present invention has a wide range of therapeutic applications. Accordingly, the present invention relates to the use of the antigen receptor of the present invention, its peptide chains, nucleic acids encoding them, and other related molecules in therapeutic and prophylactic methods. One such application is to provide 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 by the antigen receptor of the present invention expressed on the immune cells. Preferably, the disease is a malignant tumor. In addition, the antigen receptor of the present invention and related molecules can also be used to selectively eliminate cells expressing a predetermined antigen, as well as to immunize or vaccinate against a disease in which a predetermined antigen is expressed, and the antigen can be associated with at least one antigen-binding antigen receptor center of the present invention.

In one embodiment, 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 present invention, wherein at least one antigen-binding site of the antigen receptor is capable of binding an antigen that is associated with the disease (e.g., a viral or tumor antigen) to be treated or prevented. In 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 propagated population of said immune effector cells, wherein the immune effector cell or cell population recombinantly expresses an antigen receptor of the present invention, wherein at least one antigen-binding site of the antigen The receptor is capable of binding an antigen that is associated with the disease being treated or prevented. In preferred embodiments, the disease is a cancer and the antigen is a tumor-associated antigen.

According to another embodiment, the present invention relates to a method of immunizing or vaccinating against a disease associated with a specific antigen, or against a disease-causing organism in which a specific antigen is expressed, the method comprising administering to a patient an effective amount of a nucleic acid encoding an antigen receptor of the present invention, when in this case, at least one antigen-binding center of the antigen receptor is capable of binding a specific antigen. According to another embodiment, the present invention relates to a method of immunizing or vaccinating against a disease associated with a specific antigen, or against a disease-causing organism in which a specific antigen is expressed, the method comprising administering to a patient an effective amount of a recombinant immune effector cell or a propagated population of said immune effector cells wherein the immune effector cell or cell population recombinantly expresses the antigen receptor of the present invention, wherein at least one antigen-binding center of the antigen receptor is capable of binding to a specific antigen.

In certain embodiments, the immune effector cell population may be a clonally propagated 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 present invention is expressed on the cell surface of an immune effector cell.

The cells used in connection with the therapeutic and prophylactic methods of the present invention are preferably immune effector cells, and the immune effector cells are preferably T cells. In particular, the cells used herein 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 types of cytotoxic lymphocytes triggers the destruction of target cells. For example, cytotoxic T cells trigger destruction of target cells in one or both of the following ways. The first is that, upon activation, cytotoxins such as perforin, granzymes, and granulosin are released from T cells. Perforin and granulosin create pores in the target cell, and granzymes enter the cell and trigger the caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death) of the cell. The second is that apoptosis can be induced through Fas-Fas ligand interaction between T cells and target tumor cells. T cells and other cytotoxic lymphocytes will preferably be autologous cells, although heterologous cells or allogeneic cells can also be used.

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

The agents, compositions and methods described herein may also be used for immunization or vaccination to prevent the disease described herein.

The term "disease" refers to an abnormal condition that adversely affects the body of an individual. A disease is often understood as a medical condition associated with specific symptoms and signs. The disease may be caused by factors derived from an external source, such as an infectious disease, or it may be caused by internal functional disorders, such as autoimmune diseases. In the case of humans, "disease" is often used in a broader sense to refer to any condition that causes pain, functional impairment, distress, social impairment or death of the individual suffering from the disease, or similar problems for those who are in contact with the individual. In this broader sense, the term sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, forms of deviant behavior, and atypical variants of structure and function, while in other contexts and for other purposes, these may be considered as separate categories. Diseases usually negatively affect individuals not only physically, but also emotionally, since the occurrence and life with many diseases can change the attitude towards life and character. In accordance with the present invention, the term "disease" includes infectious diseases and diseases related to malignant tumors, in particular, such forms of malignant tumors as described herein. As used herein, any reference to cancer or specific forms of cancer also includes metastasis of the cancer.

The disease to be treated according to the present invention is preferably a disease involving an antigen. "A disease involving an antigen", "a disease associated with the expression or overexpression of an antigen" or similar expressions in accordance with the present invention means that the antigen is expressed in the cells of the tissue or organ affected by the disease. The level of expression in the cells of a diseased tissue or organ may be elevated compared to the level in healthy tissue or organ. In one embodiment, expression is only detected in diseased tissue, while expression is not detected in healthy tissue, eg the expression is repressed. In accordance with the present invention, diseases involving an antigen include infectious diseases and diseases related to malignant tumors, where 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 preferably 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 a non-infectious or non-cancerous condition.

The terms "cancer-related disease" or "cancer" describe or refer to the physiological state of an individual, which is typically characterized by uncontrolled cell growth. Examples of malignant tumors include, without limitation, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancers include bone cancer, hemoblastosis, lung cancer, liver cancer, pancreatic cancer, skin cancer, head or neck cancer, intraocular melanoma or skin melanoma, uterine cancer, cancer ovary, rectal cancer, anal cancer, gastric cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, genital and reproductive organs carcinoma, Hodgkin's disease, esophageal cancer, cancer small intestine, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, mild sarcoma tissue, 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 "malignant tumor" in accordance with the present invention also includes metastases of a malignant tumor. Preferably, the "disease related to cancer" is characterized by the presence of cells expressing a tumor antigen, and the cancer cell expresses the tumor antigen.

According to one embodiment, a cancer related disease is a cancer that is characterized by properties of anaplasia, invasiveness, and metastasis. A malignant neoplasm may differ from a non-cancerous benign tumor in that the malignant tumor is not self-limiting in terms of its growth, is able to invade neighboring tissues, and can spread to distant tissues (metastasize), while a benign tumor does not have any of these properties.

In accordance with the present invention, the term "tumor" or "neoplastic disease" refers to the growth or lesion formed by the 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 cessation of exposure to the stimuli that initiated the neoplasm. Tumors are characterized by a partial or total lack of structural organization and functional coordination with normal tissue, and usually form a discrete mass of tissue that may be benign, precancerous, or malignant.

In accordance with the present invention, "carcinoma" is a malignant neoplasm derived from epithelial cells. This group represents the most common types of cancers, including common forms of breast, prostate, lung and colon cancer.

"Adenocarcinoma" is a malignant tumor that originates in glandular tissue. This tissue is also part of a broader category of tissue known as epithelial tissue. Epithelial tissue includes the skin, glands, and a number of other tissues that line the cavities and organs of the body. The epithelium in terms of embryology comes from the ectoderm, endoderm and mesoderm. To be classified as an adenocarcinoma, cells do not have to be part of a gland, provided that they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Highly differentiated forms of adenocarcinoma often resemble the glandular tissue from which they originate, whereas poorly differentiated forms may not. By staining the biopsy cells, a laboratory diagnostic specialist will determine whether the tumor is an adenocarcinoma or another type of cancer. Forms of adenocarcinoma can occur in many tissues of the body due to the ubiquity of glands in the body. Nevertheless, each of the glands may secrete substances of different composition, but as long as the cell retains its exocrine function, it is considered to be glandular, and its malignant form is thus called adenocarcinoma. Malignant forms of adenocarcinoma invade other tissues and often metastasize if there is enough time to do so. Adenocarcinoma of the ovary is the most common type of ovarian carcinoma. It includes serous and mucinous forms of adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

Lymphoma and leukemia are malignant neoplasms that originate from hematopoietic (blood-forming) cells.

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

By "metastasis" is meant the spread of malignant tumor cells from its primary focus to another part of the body. Metastasis formation is a very complex process and is determined by the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration through endothelial basement membranes with access to the body cavity and blood vessels, and then, after transfer with blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and show metastatic potential. According to one embodiment, the term "metastasis" in accordance with the present invention refers to "distant metastasis", which refers to a metastasis that is distant from the primary tumor and the regional lymph node system. According to one embodiment, the term "metastasis" in accordance with the present invention refers to metastasis to a lymph node.

Relapse or re-emergence occurs when a person is again exposed to a condition that has affected him in the past. For example, if a patient suffering from a neoplastic disease has been successfully treated for said disease and develops said disease again, then said newly developed disease may be considered a relapse or a recurrence. However, in accordance with the present invention, recurrence or recurrence of a tumor disease may, but need not, occur at the site of the original tumor disease. Thus, for example, if a patient has suffered from an ovarian tumor and has been successfully treated, the recurrence or recurrence may be an ovarian tumor or a tumor at a site other than the ovary. Tumor recurrence or recurrence also includes situations where the tumor occurs at a site different from that of the original tumor, as well as at the site of the original tumor. Preferably, the original tumor for which the patient was treated is a primary tumor, and the tumor at a site different from that of the original tumor is a secondary or metastatic tumor.

Infectious diseases that can be treated or prevented using the present invention are caused by infectious agents, including, without limitation, viruses, bacteria, fungi, protozoa, helminths, and parasitic organisms.

Pathogenic 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, without limitation: Retroviridae (for example, human immunodeficiency viruses such as HIV-1 (also called 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 viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. Ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses , mumps virus, measles virus, human respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. hantaviruses, bungaviruses, phleboviruses and nairoviruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (eg reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, poxviruses); and Iridoviridae (e.g. African swine fever virus) and unclassified viruses (e.g., spongiform encephalopathies, delta hepatitis (presumed to be a defective satellite of hepatitis B virus), hepatitis other than A, B (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e. hepatitis C) ); Norwalk and related viruses, and astroviruses).

Retroviruses under consideration include both simple and complex retroviruses. Complex retroviruses include subgroups that include lentiviruses, T-lymphotropic viruses, and 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-lymphotropic viruses include HTLV-1, HTLV-II, simian T-lymphotropic virus (STLV), and bovine leukemia virus (BLV). Foam viruses include human foam virus (HFV), simian foam virus (SFV), and bovine foam virus (BFV).

Bacterial infections or diseases that can be treated or prevented using the present invention are caused by bacteria, including, but not limited to, bacteria that have an intracellular stage in their life cycle, such as mycobacteria (e.g., Mycobacteria tuberculosis, M. bovis, M. avium, M leprae or M. africanum), rickettsia, mycoplasma, chlamydia and legionella. Other examples of contemplated bacterial infections include, but are not limited to, those caused by gram-positive bacilli (e.g. Listeria, Bacillus, such as Bacillus anthracis spp., Erysipelothrix), gram-negative bacilli (e.g. Bartonella spp., Brucella, Campylobacter, Enterobacter, Escherichia, Francisella, Hemophilus , Klebsiella, Morganella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia), bacteria from the spirochete order (for example, Borrelia spp., including Borrelia burgdorferi, which causes Lyme disease), anaerobic bacteria (for example, Actinomyces spp. and Clostridium), gram-positive and gram-negative cocci, Enterococcus spp., Streptococcus spp., Pneumococcus spp., Staphylococcus spp., Neisseria spp. Specific examples of pathogenic bacteria include, without limitation: 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 antracis, Corynebacterium diphtheriae, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani Pasturella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israellii.

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

Parasitic diseases that can be treated or prevented using the present invention include, without limitation, amoebiasis, malaria, leishmaniasis, coccidiosis, giardiasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis. Infections caused by various worms are also covered, such as, but not limited to, ascariasis, hookworm, trichuriasis, strongyloidiasis, toxocariasis, trichinosis, onchocerciasis, filariasis, and heartworm disease. Infections caused by various trematodes are also covered, such as, without limitation, schistosomiasis, paragonimiasis, and clonorchiasis.

The term "treatment" or "therapeutic treatment" refers to any treatment that provides an improvement in health and/or prolongation (increase) life expectancy of an individual. Said treatment may provide for the elimination of a disease in an individual, arresting or slowing the development of a disease in an individual, inhibiting or slowing the development of a disease in an individual, reducing the frequency or severity of symptoms in an individual, and/or reducing the likelihood of recurrence in an individual currently experiencing the disease. or who has been previously diagnosed with the disease.

The terms "prophylactic treatment" or "preventive treatment" refers to any treatment designed to prevent the onset of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

The terms "individual" and "subject" are used interchangeably herein. The terms 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 suffering from a disease or disorder (e.g., malignancy), or be predisposed to develop them, and may or may not have a disease or disorder. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not indicate a specific age, and thus cover adults, the elderly, children, and neonates. According to preferred embodiments of the present invention, "individual" or "subject" is a "patient". The term "patient" in accordance with the present invention means the subject to be treated, in particular, a sick subject.

By "at risk of development" is meant a subject, ie. a patient who is identified as a patient with an increased likelihood of developing a disease, in particular a malignant tumor, compared with the general population. In addition, a subject who has experienced or is currently experiencing a disease, in particular cancer, is a subject at an increased risk of developing the disease, thus the subject may continue to develop the disease. Subjects who currently have, or have experienced, cancer also have an increased risk of developing cancer metastases.

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

In the context of the present invention, terms such as "prevent", "prevent", "prophylactic", "preventive" or "preventive" refer to the prevention or treatment, or both, of the onset and/or development of a disease in a subject and, in particular, to to minimize the likelihood that the subject will develop the disease, or to delay the development of the disease. For example, a person at risk of developing the tumor described above would be a candidate for therapy to prevent tumor development.

Prophylactic use of immunotherapy, eg prophylactic administration of an agent or composition of the present invention, preferably prevents the recipient from developing the disease. The therapeutic use of immunotherapy, for example, therapeutic administration of an agent or composition of the present invention, can provide inhibition of disease progression/development. Such a case involves slowing the progression/development of the disease, in particular, interrupting the progression of the disease, which preferably leads to the elimination of the disease.

Immunotherapy can be carried out using any of a number of techniques in which the agents provided herein preferably function to remove cells expressing a particular antigen from a patient's body. Such removal may occur by enhancing or inducing an antigen-specific or antigen-expressing cell immune response in the patient.

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 an event occurring in a subject.

The antigen receptors, peptide chains, nucleic acids, recombinant cells, immune effector cells, preferably T cells, of the present invention, as well as other compounds and agents described herein, can be administered in the form of any suitable pharmaceutical composition.

Pharmaceutical compositions of the present invention are preferably sterile and contain an effective amount of the agents described herein and optionally discussed herein additional agents to provide the desired reaction or desired effect.

Pharmaceutical compositions are usually provided in unit dosage form and can be prepared in a manner well known in the art. The pharmaceutical composition, for example, may be in the form of a solution or suspension.

The pharmaceutical composition may contain salts, buffers, preservatives, carriers, diluents and/or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interfere with the action of the active (active) component (s) of the pharmaceutical composition.

Salts that are not pharmaceutically acceptable can be used to prepare pharmaceutically acceptable salts and are included in the present invention. Pharmaceutically acceptable salts of this type include, without limitation, those derived from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, etc. 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 as a salt, citric acid as a salt, boric acid as a salt, and phosphoric acid as a salt.

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

The injectable formulation may contain a pharmaceutically acceptable excipient such as lactated Ringer's solution.

The term "carrier" refers to an organic or inorganic component of natural or synthetic origin, with which the active component is combined with the aim of promoting, enhancing its action or enabling use. In accordance with the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating agents that are suitable for administration to a patient.

Possible carrier substances for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible polymers of lactic acid, copolymers of lactic and glycolic acids or copolymers polyoxyethylene and polyoxypropylene.

The term "excipient" as used herein is intended to mean all substances that may be present in a pharmaceutical composition that are not active ingredients, such as, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavors or colors.

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

Compositions suitable for parenteral administration generally provide for a sterile aqueous or anhydrous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of compatible vehicles and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as a solution or suspension medium.

The agents and compositions described herein are administered in effective amounts. "Effective amount" refers to the amount that allows you to achieve the desired response or the desired effect, alone or together with additional doses. In the case of treating a specific disease or a specific condition, the desired response preferably refers to the suppression of the course of the disease. Such a case involves slowing down 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 delay the manifestation or prevent the manifestation of said disease or said condition.

The effective amount of the agent or composition described herein will depend on the condition being treated, the severity of the disease, individual patient parameters, including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration and similar factors. Accordingly, the doses of agents described herein may be administered depending on a number of such parameters. In the event that the patient's response is insufficient at the initial dose, higher doses (or higher doses that are effective by a different, more localized route of administration) may be used.

The agents and compositions described herein can be administered to patients, for example, in vivo, for the treatment or prevention of a number of disorders, such as those described herein. Preferred patients include human patients with disorders that can be improved or eliminated by administration of the agents and compositions described herein. Such a case includes disorders involving cells characterized by antigen expression.

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

Pharmaceutical compositions and methods of treatment described in accordance with the present invention can also be used for immunization or vaccination to prevent the disease described herein.

The pharmaceutical composition of the present invention may be administered together with complementary immune enhancers, such as one or more adjuvants, and may contain one or more immune enhancers to further enhance its effectiveness, preferably to achieve a synergistic immunostimulatory effect. The term "adjuvant" refers to compounds that prolong or enhance or accelerate the development of an immune response. In this regard, different mechanisms are possible, depending on the different types of adjuvants. For example, compounds that mediate DC maturation, such as lipopolysaccharides or CD40 ligand, constitute a first class of suitable adjuvants. In general, any agent that affects the immune system, such as a danger signal (LPS, GP96, dsRNA, etc.), or cytokines such as GM-CSF, can be used as an adjuvant that allows you to activate the immune response and / or influence it in a controlled way. CpG oligodeoxynucleotides can also optionally be used in this regard, however, their side effects, which occur under certain circumstances, should be taken into account, as explained above. 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. Additional known adjuvants are aluminum hydroxide, Freund's adjuvant or an oil such as Montanide®, most preferably Montanide® ISA51. Lipopeptides such as Pam3Cys are also suitable for use as adjuvants in the pharmaceutical composition of the present invention.

The pharmaceutical composition can be administered topically or systemically, preferably systemically.

The term "systemic administration" refers to the administration of an agent in such a way that the agent is widely distributed throughout the body of the individual in significant amounts and exhibits the desired effect. For example, the agent may exert its desired effect in the blood and/or reach its site of action via the vascular system. Typical systemic routes of administration include administration by administering the agent directly to the vasculature, or oral, inhalation, or intramuscular administration in which the agent is adsorbed, enters the vasculature, and is delivered to one or more desired sites of action via the blood.

In accordance with the present invention, it is preferable that the systemic administration is carried out by parenteral administration. The term "parenteral administration" refers to the administration 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.

The introduction can also be carried out, for example, orally, intraperitoneally or intramuscularly.

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

The present invention is described in more detail with the help of the following drawings and examples, which are used for illustrative purposes only and are not intended to be limiting. Through the description and examples, additional embodiments, which are also included in the present invention, are available to a person skilled in the art.

Blueprints

In FIG. 1 is an image of the TCR-CD3 complex. The TCR and CD3 subunits consist of ectodomains, a stem region, a transmembrane domain, and cytoplasmic domains that contain ITAMs. Expression of the CD4 or CD8 co-receptor determines belonging to the CD4+ or CD8+ subpopulations of T cells. CD3 immunoreceptor tyrosine-based intracytoplasmic activating motifs (ITAM) are shown as cylinders (taken from "The T cell receptor facts book", MP Lefranc, G Lefranc, 2001).

In FIG. 2 shows a diagram of successive generations of CARs. Schematic representation of different generations of CARs (1G, first generation, 2G, second generation, 3G, third generation). First generation members contain extracellular scFv and cytoplasmic CD3ζ chain/ZAP70 mediating immediate effector function such as IFNγ secretion or cytotoxicity, second generation members additionally contain CD28/PI3K to stimulate proliferation, and third generation members also contain 4-1BB or OX40 /TRAF for cell survival (Casucci, M. et al. (2011) 2: 378-382).

In FIG. 3 is a schematic representation of different receptor formats for redirecting T cells to an antigen. Cα/β are TCR constant domains that also function as a signal transduction domain for signaling across the cell membrane from bound antigen to signaling domains of the recruited CD3 complex in the cytoplasm; VH and VL are the variable regions of the immunoglobulin heavy and light chains, respectively, and they are representative domains on two peptide chains that form the antigen binding site.

Left: Second-generation CAR consisting of an antigen-specific scFv fragment derived from the IgG1 spacer domain, CD28 co-stimulatory and CD3ζ signaling domains (classic single-chain CAR); middle: new CAR format based on scFv binding to the mouse TCRß chain constant domain and co-expression of the mouse TCRα chain constant domain (univalent non-combinational antigen receptor); right: Mouse TCR consisting of TCR α/β chains (mu, mice). The CD3δε heterodimer and the CD3ζ ζ homodimer are recruited by the Cα domain, while CD3γε is recruited by the Cβ domain.

Figures 4A and B show the structures of some of the antigen receptors described herein.

The domain names are as described in the explanation of FIG. 3. The tandem antigen receptor (tandem AR) contains a chain containing 4 variable antibody fragments that form 2 antigen-binding centers. Intra-/intercombination AR is capable of intra- and inter-chain antigen binding. Intercombination AR provides antigen binding exclusively through interstrand binding. The monovalent non-combination AR is a monovalent prototype of a new AR format based on the involvement of endogenous CD3, the bivalent non-combination AR is a CAR whose design has already been proposed by Gross et al., 1992 FASEB J., (6) 3370-3378, and in it binding to each antigen is limited to one chain. The univalent Raman AR serves as a reference AR or negative control to demonstrate the improvement in function by providing higher valences in the CAR format with the scFv fragments.

In FIG. 5 is a bar graph showing the relative expression levels of antigen receptors in T cells. Expression on T cells electroporated with CAR RNA was confirmed in flow cytometry analysis using the idiotypic paratope claudin 6-specific antibody IMAB206. The flow cytometry antibody was directly labeled with Dylight-640, expression levels are given in mean fluorescence intensity (MFI).

Figures 6A-6C are bar graphs showing relative induced levels of IFN-γ production, which is an indicator of immune cell activation. Fig. 6A: Detection of IFN-γ production in an ELISA of CD8+ T cells electroporated with different antigen receptor-expressing constructs and controls and co-cultured with Cl6 negative and Cl6 positive immature dendritic cells (iDCs). For bivalent antigen receptors capable of interchain antigen binding, proper production of IFN-γ (cross-combination AR 2GS, 3GS, 4GS) has been shown. Changing the length of the linker among the variable domains did not significantly affect the function of the receptor, however, the 3GS linker was slightly better than the 2 and 4GS linkers. Removal of the N-terminal variable domains of the bivalent antigen receptor to produce a monovalent antigen receptor (univalent combination AR) drastically reduced receptor function, indicating the importance of bivalent antigen binding for better receptor function. Mouse constant domains in the structure of the antigen receptor provide a slightly improved production of IFN-γ (intercomb. AR Mu 3GS). Fig. 6B: In a repeat of the experiment disclosed in FIG. 6A, similar results were shown, but with T cells obtained from a different donor, to prove that the results are independent of the donor. Fig. 6C: Essentially a repetition of the experiments disclosed in figures 6A and 6B. The tandem antigen receptor and the intra/inter-combination antigen receptor (providing antigen recognition involving both intra- and inter-chain combination of VH/VL domains) each provide a higher degree of induction of IFN-γ expression compared to a monovalent antigen receptor (univalent non-combination AR ) and compared to an antigen receptor with only intrachain antigen binding (bivalent non-combinational AR).

In FIG. 7 is a bar graph showing the performance of CAR Cl6-modified T cells against Sk-ov-3 ovarian carcinoma cells. Tumor cells were electroporated with increasing amounts of Cl6 RNA, and CD8+ T cells were electroporated with various antigen receptor expressing constructs. The effector function of T cells, as measured by cell lysis, decreased as the amounts of Cl6 expressed in Sk-ov-3 cells decreased. The cytotoxicity of T cells recombinantly expressing bivalent antigen receptors (tandem AR and intercombinant AR) was less dependent on antigen density on the surface of target cells compared to monovalent noncombinational antigen receptor and classical scCAR.

Figures 8A-8D are histograms showing the results of immune cell activation as measured by cell proliferation in response to iDC as APC. Figures 8A and 8B: Proliferation of CFSE-labeled CD4+ (FIG. 8A) and CD8+ T cells (FIG. 8B), respectively, in response to Cl6 negative immature dendritic cells (iDCs). After cis-costimulation with CD80 and 41BBL molecules (i.e. when administered by electroporation to T cells), the classical single-chain chimeric antigen receptor (classic scCAR) showed background proliferation in the absence of its cognate antigen. This situation was also observed for T cells incubated without any target cells, indicating that non-specific proliferation is an inherent characteristic of T cells expressing classic scCAR (data not shown). This result was also observed for the Cl6 negative Sk-Ov-3 cell line. Figures 8C and 8D. Proliferation of CFSE-labeled CD4+ (FIG. 8C) and CD8+ T cells (FIG. 8D), respectively, in response to Cl6+ iDC. CD4+ T cells proliferated in response to Cl6-loaded iDCs when administered by electroporation with a construct expressing a cross-combination AR. The classic scCAR format provided a lower degree of proliferation. In the case of CD8+ T cells, both classical scCAR and cross-combination AR showed almost the same proper proliferation.

Figures 9A-9B are histograms showing the results of immune cell activation as measured by cell proliferation in response to tumor cells as APC. Proliferation of CFSE-labeled CD4+ (FIG. 9A) and CD8+ (FIG. 9B) T cells in response to Cl6+ ovarian carcinoma cell line Ov-90 electroporated with the costimulatory molecules CD80 and 41BBL (trans costimulation). CD4+ T cells were able to proliferate in response to Ov-90 ^{Cl6+CD80+41BBL+} when administered by electroporation of intercombination AR, similar to iDC ^Cl6+ . Both monovalent and divalent antigen receptors provided for the proliferation of CD8+ T cells. The degree of proliferation was comparable to the classic scCAR. Cys-costimulation of T cells further enhanced T cell proliferation.

In FIG. 10 is a bar graph showing the results of induction of IFN-γ production as a function of dose. Detection of IFN-γ production in an ELISA of CD8+ T cells electroporated with various antigen receptor-expressing constructs and controls, and co-cultured with immature dendritic cells (iDCs) electroporated with the relevant full-length Cl6 RNA at concentrations ranging 0.01 µg to 1 µg and 1 µg of irrelevant gp100. Bivalent antigen receptors were evaluated for cytokine secretion dependent on the expected degree of combinational strand pairing in the order inter->intra/inter->bivalent non-combinational AR. At higher antigen density (1 μg Cl6), classical CAR promoted IFNγ secretion in the greatest amount, followed by intra- and intra/intercombinational AR. The bivalent non-combination AR was less functional than the monovalent combination AR and the monovalent non-combination AR. The bivalent non-combination AR relies on pairing only between invariant human C-domains, while the univalent combination AR has more efficient pairing between human C-domains and mouse V-domains. The monovalent non-combination AR is more functional than the divalent non-combination AR because interstrand pairing is facilitated by mouse C-domains instead of the less stable human C-domains. This is also true for any dose of antigen used in this assay. It is noteworthy that with a decrease in the density of the antigen (cells into which 0.1 μg, 0.01 μg of Cl6 RNA were injected by electroporation), the intercombination AR progressively becomes more reactive with respect to iDC, into which the RNA of the relevant antigen was introduced, when compared with the classical CAR . This is consistent with a similar trend shown in the antigen dose-dependent cytotoxicity assay (FIG. 7) for cells of the Skov-3 tumor cell line electroporated with Cl6.

In FIG. 11 is a bar graph showing the results of induction of IFN-γ production as a function of dose. In FIG. 11A compares the formats of classic and new combination classic CAR. Both CARs are involved in signal transduction via the CD3ζ fusion signaling molecule independently of the endogenous CD3 complex of T cells. In FIG. 11B shows the results of detection of IFN-γ production in an ELISA of CD8+ T cells electroporated with claudin 6-specific classical CAR and novel combination classical CAR constructs, both containing the antibody CH2-CH3 domains of both CD28 and CD3ζ in as a signaling domain. These cells were co-cultured with immature dendritic cells (iDCs), which were electroporated with the relevant full-length Cl6 RNA at a concentration ranging from 0.002 μg to 2 μg and 2 μg of irrelevant gp100.

At higher antigen densities (from 2 µg to 0.2 µg Cl6), both CAR formats promoted IFNγ secretion in the same highest amounts.

In the case of lower antigen density, classical CAR is likely to be somewhat more efficient in terms of IFNγ secretion.

It is important to note that combination classical CAR is functional in the same range of antigen doses as classical CAR.

Examples

The techniques and methods used herein are described herein or performed in known manner and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY All methods involving the use of kits and reagents are performed according to the manufacturer's information, unless specifically noted otherwise.

Example 1 Expression of antigen receptors in T cells.

Expression of the different antigen receptor constructs was assessed one day after administration by electroporation into CD8+ T cells using an idiotype-specific anti-scFv anti-Cl6 antibody. The constructs or combination of constructs tested for expression were (i) mouse T cell receptor alpha chain constant domain alone (mCα); (ii) VH-VL-mCβ alone (scFv-mCβ); (iii) VH-VL-CH2-CH3-CD28-CD3ζ (classic scCAR); (iv) mCα and VH-VL-mCβ (monovalent non-combination AR); (v) VH-VL-mCα and VH-VL-mCβ (bivalent non-combination AR); (vi) VL-VH-mCα and VH-VL-mCβ (intra/intercombination AR); (vii) mCα and VH-VL-VH-VL-mCβ (tandem AR); (viii) VH-hCα and VL-hCβ (monovalent Raman AR); (ix) VH-(GGGGS)3-VH-hCα and VL-(GGGGS)3-VL-hCβ (cross-combination AR 3GS); and (x) VH-(GGGGS)3-VH-mCα and VL-(GGGGS)3-VL-mCβ (cross-combination AR Mu 3GS) (“m” or “mu” indicates murine origin and “h” indicates human origin of the constant domain). These different antigen receptor constructs are shown schematically in Figures 4A-4B. The data in FIG. 5 are the results of two separate measurements for bivalent non-combination, intra/intercombination, and monovalent combination antigen receptors; five measurements for tandem and intercombination antigen receptors; and up to ten measurements for classical scCARs and monovalent non-combinational antigen receptors. The MFI value for each sample was normalized to the respective negative control (mCα or scFv-mCβ taken as 1).

As shown in FIG. 5, the best expression was shown for the classic scCAR format. Monovalent non-combination, bivalent non-combination, intra/intercombination and intercombination human (hC) or mouse (mC) constant domain antigen receptors showed similar expression. The monovalent combination AR showed a low amount of surface staining, while the tandem antigen receptor showed an increased amount of surface staining compared to other constructs except for the classic scCAR.

Example 2 IFN-γ secretion assay.

On day 1 of the experiment, fresh peripheral blood mononuclear cells ("PBMC") were isolated from a buffy coat from a healthy donor. CD14+ cells were isolated from ¾ PBMC using MACS sorting. MACS output cells and PBMC residue were then MACS sorted for CD8+ T cells. CD14+ cells differentiated into immature dendritic cells ("iDC") by administration of IL-4 and GM-CSF (1000 U/ml) on days 1, 3, 6. CD8+ T cells were transferred to OKT3-coated 6-well plates. On day 3, T cells were transferred to new 6-well plates. On day 7, irrelevant and IVT-RNA Cl6 was electroporated into the iDC. The controls or antigen receptor constructs shown in the individual figures and described in Example 1 were then electroporated into OKT3-activated T cells. antigen receptor on the surface of T cells. Electroporated T cells and iDCs electroporated with antigen were then co-cultured in a 96-well plate for 20 hours at an E:T ratio of 10:1 in duplicate. On day 9, culture supernatants were collected and analyzed for the amount of secreted IFN-γ in a sandwich ELISA using the IFN-γ Ready Set Go! from eBioscience (#88-7316-88). Absorbance was detected using a Tecan Sunrise ELISA reader.

As shown in FIG. 6A, mimic electroporation (mCα) T cells in culture with Cl6 positive and negative iDCs did not show non-specific IFN-γ production. When cultured with Cl6 negative target cells, of antigen receptor positive T cells, only classical scCAR positive cells produced the cytokine at a detectable background level. The production of IFN-γ by monovalent antigen receptor positive cells could be observed when cultured with Cl6+ iDC. In this case, classical scCAR produced high amounts of IFN-γ. With the introduction by electroporation of a negative control from only one chain of the combination antigen receptor, in this case VH-VH-Cα, the production of IFN-γ was not observed. With this control, it was shown that both strands are required for antigen recognition and subsequent T cell activation. In contrast, the inter-combination AR (3GS) showed a marked improvement in IFN-γ production over that produced by the monovalent AR (combination and non-combination). For different lengths of linkers between variable domains, 2 or 4 repeats of the Gly4Ser linker were not observed to have a significant effect on function, in contrast to 3 repeats.

It is important to note that in the case of the removal of the N-terminal variable domain of the structure in the form of a combination antigen receptor, a strong decrease in effector function was shown. This observation clearly supports the fact that bivalent antigen binding mediates T cell activation. The marked increase in function of the monovalent combination AR and bivalent combination AR is also a clear indication that, in addition to the improvement in chain pairing due to interchain combination of variable domains, the mere presence of antigen binding within the two chains further stabilizes receptor chain pairing and therefore improves inclusion in the endogenous CD3 complex and subsequent activation/function of T cells. It is well known that in double-stranded T-cell receptors, chain heterodimerization is a necessary condition for efficient incorporation into the CD3 complex.

The incorporation of mouse residues into the structure of the combinational AR made it possible to further enhance activation, which is explained by the known stronger interaction of the TCR constant Cα/β domains compared to those in humans. Thus, improved heterodimerization of the antigen receptor peptide chains, either by interchain binding of the antigen to variable domains or by dimerization of the individual chain T-cell receptor constant domains, improved incorporation of the antigen receptor into the endogenous CD3 complex, and thus improved function. T cells. These results were highly reproducible with another T cell donor (see Fig. 6B).

Notably, for classical scCAR, this experiment demonstrated non-specific background IFN-γ production against Cl6-negative iDCs. This result is also reproducible and consistent with data published by Long et al. ((2015) Nat. Med., (21) 581-590), which discusses background signaling involving the classic scCAR-CD28-CD3ζ fusion format. Long et al. observed antigen-independent activation for several classic scCARs with different antigen specificities. It is suggested that non-specific background activation of classical scCAR positive T cells is not a specific effect of the T cell donor.

In addition, and as shown in FIG. 6C, bivalent non-combination and intra/intercombination antigen receptors together with the tandem bivalent antigen receptor were tested for IFN-γ production. For comparison, the figure also shows a negative control, a monovalent non-combination AR, and classic scCAR as a positive control. The tandem bivalent antigen receptor has been shown to be improved over the monovalent antigen receptor. The bivalent non-combination antigen receptor VH-VL-Cα + VH-VL-Cβ induced lower IFN-γ production compared to the similar intra/intercombination antigen receptor VL-VH-Cα + VH-VL-Cβ. These observations supported the hypothesis that interchain interactions mediated by both V-domain pairing and antigen binding to variable domains of different chains stabilize the heterodimeric configuration and provide a stronger receptor-dependent signaling response involving T cells.

Example 3 Cytotoxicity Assay.

On day 1 of the experiment, fresh PBMCs were isolated from two buffy coats from healthy donors. PBMCs were MACS sorted for CD8+ T cells. CD8+ T cells were transferred to OKT3-coated 6-well plates. They were cultured in medium containing 50 U/ml IL-2. On day 3, T cells were transferred to new 6-well plates and the culture medium was replaced. On day 7, RNA with various amounts of Cl6 RNA and 10 µg of luciferase RNA were electroporated into Sk-Ov-3 ovarian carcinoma cell line. OKT3-activated T cells were electroporated with the irrelevant classical scCAR, relevant Cl6-specific classical scCAR, monovalent non-combination antigen receptor and tandem antigen receptor, and cross-combination antigen receptor constructs of the present invention, as shown in FIG. 7 and described in Example 1 above. For quality control purposes, on day 8, antigen receptor expression on the surface of T cells was analyzed using a Dylight 650-labeled idiotype-specific antibody to the Cl6 antigen receptor. T cells electroporated with the antigen receptor construct and Sk-Ov-3 electroporated with antigen were then co-cultured in a 96-well plate for 3 hours at an E:T ratio of 30:1 in triplicate . After 3 hours of incubation, luciferin was added to each culture. Specific lysis was detected by a decrease in the intensity of the luciferin signal as a result of its conversion under the action of released luciferase on a TECAN reader.

The Sk-Ov-3 data clearly indicate that the bivalent antigen receptors (cross-combination AR and tandem AR) of the present invention showed a marked improvement over classical scCAR and monovalent non-combination AR. Compared to all other antigen receptor constructs, the classic scCAR format showed the most efficient lysis of approximately 77% for Sk-Ov-3 cells electroporated with Cl6. However, it should be noted that 10 μg of antigen RNA does not correspond to the physiological condition and does not reliably reflect the situation in vivo. At low doses of antigen, the monovalent non-combination AR was unable to mediate tumor cell lysis properly (9.2%). In contrast, cross-combination AR (3GS) still showed proper specific lysis (41.3%) when compared to the classic scCAR format (48.1%) and was clearly less dependent on antigen density for providing a significant cytotoxic effector function.

Example 4 Proliferation Assay.

On day 1 of the experiment, fresh PBMCs were isolated from buffy coat from a healthy donor. CD14+ cells were isolated from ¾ PBMCs using MACS sorting, and the remaining PBMCs were frozen. CD14+ cells differentiated into iDC by administration of IL-4 and GM-CSF (1000 U/ml) on days 1, 3, 6. On day 7, irrelevant and IVT-RNA Cl6 was electroporated into iDC. Frozen PBMCs were thawed the same day and MACS sorted for CD4+ and CD8+ cells. Then, without any prior activation (OKT3), naïve T cells, 6 and 7 x 10 ⁶ cells, respectively, were electroporated with controls, classical, monovalent, and bivalent antigen receptor constructs, as shown in Figures 8A-8D, and the constructs are described in Example 1. In an independent group of immunocompetent T cells, the same antigen receptor constructs were also administered by electroporation along with costimulatory molecules 41BBL and CD80 to provide auto-costimulation, or cis-costimulation, which has been shown to improve effector function.

For quality control purposes on day 8, FACS with staining analyzed the expression of the antigen receptor and 41BBL + CD80 on T cells. T cells were then labeled with a CFSE proliferation assay marker. Electroporated T cells and iDCs and the ovarian carcinoma cell line OV-90 were then co-cultured in a 96-well plate for 5 days at an E:T ratio of 10:1 in duplicate. On day 5, cultured cells were stained in 96-well plates with anti-CD4 or CD8 antibodies labeled with APC-Cy7. T cell proliferation was detected using FACS by a decrease in CFSE signal due to dilution in proliferating daughter cells. With minor adjustments, daughter population sizes were estimated using the proliferation tool implemented in Flowjo. The total number of proliferating cells is designated as daughter generations.

Baseline T cell proliferation was assessed for cells cultured with either the Cl6 negative Sk-Ov-3 cell line or the Cl6 negative iDC. Regardless of the antigen receptor construct that was introduced by electroporation, neither CD4+ nor CD8+ T cells proliferated in response to Cl6 negative cells. After cis-costimulation, only engineered T cells with classical scCAR proliferated nonspecifically in response to Cl6-negative iDC and Sk-Ov-3 (data on Sk-Ov-3 not shown). CD4+ T cells generally proliferated less efficiently in response to Cl6+ cells compared to CD8+ T cells (see Figures 8C-8D). In particular, this was true when targeting Cl6+ tumor cells Ov-90 (Figures 9A-9B).

The results for T cells that were co-cultured with iDC confirmed generally good function of the intercombination antigen receptor. In the case of CD4+ T cells without cis-costimulation, the cross-combination antigen receptor showed better proliferation and even outperformed the classic scCAR format. This effect was not due to a reduced level of surface expression of classical scCAR, as analyzed using CAR idiotype staining. In the case of CD8+ T cells, the cross-combination antigen receptor format showed significant proliferation in response to Cl6-loaded iDCs (70%). Cys-costimulation of T cells could further enhance T cell responses.

Interestingly, cell proliferation in response to Cl6+ cells of the ovarian carcinoma cell line OV-90 was not observed (data not shown). This was explained by the absence of costimulatory molecules on the surface of tumor cells. To compensate for this lack of costimulation, CD80 and 41BBL RNAs were introduced into Ov-90 cells by electroporation. In this case, proliferation of CD4+ and CD8+ T cells could be detected (see Figures 9A and 9B). It is noteworthy that for CD4+ cells, this situation occurred only in the case of the intercombination AR format (15%). In the case of CD8+ cells, the proliferation profile looked different. The responses of the univalent non-combination AR, classic scCAR and intercombination AR were comparable with 60% of proliferating cells. After cis-costimulation, proliferation, in particular of CD4+ T cells, improved.

The data clearly indicate that, upon costimulation, the bivalent antigen receptor construct (intercombinational AR) is capable of proliferating in response to Cl6+ tumor cell lines. In general, the inter-combination AR is significantly superior to the monovalent non-combination AR in providing proliferation in response to iDCs loaded with a cognate target antigen. Classical scCAR tends to mediate non-specific cis-costimulation responses to Cl6 negative iDCs, indicating a higher sensitivity to antigen-independent signaling involving the T cell.

Example 5 IFNγ secretion assay with antigen titration.

On day 1 of the experiment, fresh peripheral blood mononuclear cells ("PBMC") were isolated from buffy coat from a healthy donor. CD14+ cells were isolated from ¾ PBMC using MACS sorting. MACS output cells and PBMC residue were then MACS sorted for CD8+ T cells. CD14+ cells differentiated into immature dendritic cells ("iDC") by administration of IL-4 and GM-CSF (1000 U/ml) on days 1, 3, 6. CD8+ T cells were transferred to OKT3-coated 6-well plates. On day 3, T cells were transferred to new 6-well plates. On day 7, irrelevant and IVT-RNA Cl6 was introduced into the iDC by electroporation in a dose-dependent manner. OKT3-activated T cells were then electroporated with the antigen receptor controls or constructs shown in the individual figures and described in Example 1. For quality control, expression was analyzed on day 8 using a Dylight 650-labeled idiotype-specific antibody to the Cl6 antigen receptor. antigen receptor on the surface of T cells. Electroporated T cells and iDCs electroporated with antigen were then co-cultured in a 96-well plate for 20 hours at an E:T ratio of 10:1 in duplicate. On day 9, culture supernatants were collected and analyzed for the amount of secreted IFN-γ in a sandwich ELISA using the IFN-γ Ready Set Go! from eBioscience (#88-7316-88). Absorbance was detected using a Tecan Sunrise ELISA reader. The results are shown in FIG. 10.

Bivalent antigen receptors were evaluated for the amount of cytokine secreted, depending on the perceived propensity to form pairs in a combinational manner, which contributes to stable expression, and, subsequently, signaling with the participation of the T cell. The inventors of the present invention hypothesized that for the inter-combination AR, only the combination of inter-strand V-domain pairing was observed, while the intra/inter-combination AR may simultaneously be in a ratio of less favorable intra-strand pairing. At higher antigen density (1 µg Cl6), classical CAR promoted IFNγ secretion in the greatest amount, followed by intra- and intra/intercombination AR as expected. Bivalent non-combination AR was less functional than monovalent combination AR and monovalent non-combination AR as standards. The bivalent non-combination AR relies on pairing only between invariant human C-domains, while the univalent combination AR has more efficient pairing between human C-domains and mouse V-domains. As expected, also the univalent non-combinational AR is more functional than the bivalent non-combinational AR because interstrand pairing, despite being limited to C domains in this case, is facilitated by mouse C domains instead of the less stable C domains. person. This is also true for any dose of antigen used in this assay.

It is noteworthy that with a decrease in the density of the antigen (cells into which 0.1 μg, 0.01 μg of Cl6 RNA were injected by electroporation), the intercombination AR progressively becomes more reactive with respect to iDCs, into which the relevant RNA was introduced, when compared with the classical CAR. This is consistent with a similar trend shown in the antigen dose-dependent cytotoxicity assay (FIG. 7) for cells of the Skov-3 tumor cell line electroporated with Cl6.

The classic CAR was also compared to the novel combination classic AR in an IFNγ secretion assay with antigen titration (FIG. 11). Both chimeric antigen receptors contain the CH2-CH3 domains of the antibody and CD28 and CD3ζ as a signal transduction domain. These cells were co-cultured with immature dendritic cells (iDCs), which were electroporated with the relevant full-length Cl6 RNA at a concentration ranging from 0.002 μg to 2 μg and 2 μg of irrelevant gp100. At higher antigen densities (from 2 µg to 0.2 µg Cl6), both CAR formats promoted IFNγ secretion in the same highest amounts. In the case of lower antigen density, classical CAR is likely to be somewhat more efficient in terms of IFNγ secretion. It is important to note that the combination classic CAR is functional over the same antigen dosage range as the classic CAR, down to very low amounts of endogenously expressed claudin 6. However, this combination AR format, in contrast to the Cα/Cβ TCR-based combination AR format, appears to provide the same, but no more efficient T-cell-mediated signaling than classical CAR, presumably due to exogenous CD3ζ-dependent but endogenous CD3-independent signaling. It is assumed that the first mechanism does not depend on the recruitment of endogenous CD3 to TCR or CAR, respectively, which, in turn, will regulate the degree of T-cell activation.

Claims (49)

1. An antigenic receptor for providing an immune response to cancer cells expressing a tumor antigen, wherein the receptor comprises a first peptide chain and a second peptide chain, where the first peptide chain contains a first tumor antigen specific domain and a second tumor antigen specific domain and an immune cell receptor signal transduction domain; the second peptide chain contains a first tumor antigen specific domain and a second tumor antigen specific domain and an immune cell receptor signal transduction domain; where the first domain of the first peptide chain together with one of the domains of the second peptide chain forms the first antigennegative center, and where the second domain of the first peptide chain together with another domain of the second peptide chain forms the second antigennegative center; where the first peptide chain is characterized by the structure VH(1)-VL(2)-C1 and the second peptide chain is characterized by the structure VL(1)-VH(2)-C2, where the first peptide chain is characterized by the structure VH(1)-VH(2)-C1 and the second peptide chain is characterized by the structure VL(1)-VL(2)-C2, where the first peptide chain is characterized by the structure VH(1)-VH(2)-C1 and the second peptide chain is characterized by the structure VL(2)-VL(1)-C2, or where the first peptide chain is characterized by the structure VH(1)-VL(2)-C1 and the second peptide chain is characterized by the structure VH(2)-VL(1)-C2; Where VH(1) is an immunoglobulin heavy chain (VH) variable domain with specificity for the first epitope, VH(2) is an immunoglobulin heavy chain (VH) variable domain with specificity for a second epitope, VL(1) is an immunoglobulin (VL) light chain variable domain with specificity for the first epitope, VL(2) is an immunoglobulin (VL) light chain variable domain with specificity for a second epitope, C1 and C2 are immune cell receptor signaling domains that will form a dimer with each other, where (i) the C1 immune cell receptor signaling domain of the first peptide chain comprises the T cell receptor alpha chain constant region and the second peptide chain C2 immune cell receptor signaling domain comprises the T cell receptor beta chain constant region, or (ii) the C1 immune cell receptor signaling domain of the first peptide chain comprises a T cell receptor beta chain constant region, and the C2 immune cell receptor signaling domain of the second peptide chain comprises a T cell receptor alpha chain constant region; where the linker is present between the first and second domains on both peptide chains; where the first and second antigen binding sites bind to different epitopes on the same tumor antigen, and where the tumor antigen is claudin; where the receptor is selected from the group consisting of: VL-VH-mCα and VH-VL-mCβ (intra/intercombination AR); VH-(GGGGS) 3-VH-hCα and VL-(GGGGS) 3-VL-hCβ (cross-combination AR 3GS); And VH-(GGGGS) 3-VH-mCα and VL-(GGGGS) 3-VL-mCβ (cross-combination AR Mu 3GS). 2. The receptor according to claim 1, where the tumor antigen is claudin 6 or claudin 18.2. 3. The receptor according to claim 1 or 2, wherein the immune cell receptor signal transduction domain comprises a constant or invariant region of the T cell receptor chain or a constant or invariant region of the immune cell Fc receptor chain or a portion of the constant or invariant region. 4. The receptor according to any one of paragraphs. 1-3, where the linker between the first and second domains on both peptide chains is a 3 repeat amino acid sequence of 5 monomeric units (Gly4Ser). 5. The receptor according to any one of paragraphs. 1-4, wherein the immune cell receptor signaling domains C1 and C2 originate from the human body. 6. The receptor according to any one of paragraphs. 1-5, where each of the first and second domains from the first peptide chain contains an immunoglobulin heavy chain variable region or a portion thereof; and each of the first and second domains from the second peptide chain contains an immunoglobulin light chain variable region or portion thereof. 7. The receptor according to any one of paragraphs. 1-6, where the first peptide chain is characterized by the structure VH-(GGGGS)3-VH-hCα and the second peptide chain is characterized by the structure VL-(GGGGS)3-VL-hCβ; where hCα is the human constant domain of the human T cell receptor alpha chain and hCβ is the human constant domain of the human T cell receptor beta chain, which act as immune cell receptor signal transduction domains. 8. The receptor according to any one of paragraphs. 1-7, wherein the amino acid sequences of the first and second peptide chains are from the human body, and wherein the domains that form the antigen binding sites are murinated by replacing one or more amino acids in the human sequence with an amino acid at the corresponding position in the mouse sequence. 9. A method for producing a CD8+ T cell expressing an antigen receptor, wherein the receptor comprises a first peptide chain and a second peptide chain as defined in claim 1, the method comprising: (a) providing CD8+ T cells; (b) providing a first genetic construct encoding a first peptide chain comprising at least first and second domains and an immune cell receptor signal transduction domain as defined in paragraph 1; (c) providing a second genetic construct encoding a second peptide chain comprising at least first and second domains and an immune cell receptor signal transduction domain as defined in paragraph 1; (d) introducing the first and second genetic constructs into a CD8+ T cell; And (e) enabling the expression of the constructs in a CD8+ T cell; wherein the first domain from the first peptide chain, together with one of the domains from the second peptide chain, is capable of forming a first antigen binding site, and wherein the second domain from the first peptide chain, together with another domain from the second peptide chain, is capable of forming a second antigen binding site. 10. Recombinant CD8+ T host cell obtained by the method of claim 9 and expressing a first peptide chain and a second peptide chain as defined in claim 1. 11. A nucleic acid encoding a first peptide chain and a second peptide chain as defined in any one of claims 1-8. 12. Pharmaceutical composition for the treatment of a patient with a malignant tumor characterized by the presence of cells expressing a tumor antigen, containing (a) an effective amount of a recombinant CD8+ T host cell according to claim 10, (b) an effective amount of the nucleic acid according to claim 11; or (c) an effective amount of a nucleic acid encoding a first peptide chain and an effective amount of a nucleic acid encoding a second peptide chain, where said chains are defined in any of paragraphs. 1-8; and a pharmaceutically acceptable carrier, and where the tumor antigen is claudin 6. 13. A method of treating cancer, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition according to claim 12, wherein the disease is characterized by the expression of at least one antigen that binds to an antigen receptor, where the antigen is a tumor antigen, and where the tumor antigen is claudin 6.

Images