Artificial immune surveillance chimeric antigen receptor (AI-CAR) and cells expressing it

Abstract

The present application provides a non-viral vector comprising an artificial immune surveillance chimeric antigen receptor (AI-CAR) expression cassette flanked by two transposons or viral terminal repeats (IRs), wherein the AI-CAR expression cassette comprises an inducible gene expression unit and a CAR expression unit.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application Serial No. 62 / 808,815, filed February 21, 2019, U.S. Provisional Application Serial No. 62 / 808,823, filed February 21, 2019, and U.S. Provisional Application Serial No. 62 / 808,833, filed February 21, 2019, the disclosures of which are incorporated herein by reference in their entireties. [Technical Field]

[0002] The present invention relates to techniques for improving the proliferation, production, survival, and efficacy of chimeric antigen receptor (CAR)-T cells or NK cells. [Background technology]

[0003] Unless otherwise indicated herein, the materials described in this section are not prior art to the claims of this application and are not admitted to be prior art by inclusion in this section.

[0004] CAR cell production is labor-intensive and can be complicated by the need for artificial antigen-presenting cells (aAPCs), antibody stimulation of TCRs, and co-stimulatory receptors and / or multiple cytokines to expand autologous or allogeneic CAR cells prior to administration. For example, electroporation of T cells, NK cells, PBLs, or PBMCs with CAR DNA vectors typically results in cell death of the majority of cells if the electroporation conditions are set to express a high proportion of CARs. Therefore, after electroporation, cells can be co-cultured with irradiated aAPCs, antibodies, and / or growth factors to specifically expand the CAR cell population multiple times to produce a single therapeutic dose. Currently, standard CAR vectors (CARs) do not express genes capable of inducing an anti-tumor immune response in patients, which is often required in the immunosuppressed tumor microenvironment.

[0005] Ideally, the efficacy of CAR cells after administration would correlate with T cells possessing an undifferentiated memory phenotype, characterized by in vivo persistence and maximal therapeutic efficacy. Cytokines such as IL15, IL7, and IL21 have been utilized to selectively expand CAR cells with this phenotype or prevent their terminal differentiation. IL15 and IL7 are known to be important for the generation and support of early memory T cells due to their ability to direct the generation of human memory stem T cells from naive progenitor cells (Cieri et al., 2013; Boyman et al., 2012; Gattinoni L, et al., 2011). IL15 and IL7 can help dedifferentiate T cells, such as the human CD8+ memory T cell subset, in response to antigens and homeostatic cytokines (Geginat 2003). IL15 is required for immune surveillance of innate-like T cells (Dadi S, et al., 2016). Soluble and transfected IL15 / IL-15Ralpha enable sustained IL-15 activity and contribute to the long-term survival of CD8 memory T cells (Sato, et al., 2007). Therefore, CAR-T cells with an undifferentiated memory phenotype exhibit the greatest persistence and therapeutic efficacy in vivo.

[0006] However, all components used to produce and maintain these potentially highly therapeutic CAR cells cannot be co-administered to assess the safety of each component. Furthermore, incorporating these components into a constitutively expressing CAR vector may not be feasible due to the high likelihood of toxicity over time. Instead, if the signaling pathways driving CAR cell proliferation and the undifferentiated memory phenotype were under the control of a single molecule, such as a targeted tumor antigen, this would not only significantly reduce costs but also enable point-of-care treatment. Thus, CAR signaling appears to be sufficient to support CAR cell proliferation without terminal differentiation.

[0007] Clinically, multiple factors contribute to treatment relapse, including insufficient persistence of CAR cells (exhaustion or host anti-CAR), loss of target antigen, insufficient induction of host anti-tumor responses, and / or inability to efficiently localize to lymphoma / solid tumors. Almost all CARs address tumor antigen loss by targeting two or more tumor antigens. Therefore, incorporating additional genes into the CAR vector / RNA to simultaneously achieve one or more CARs and a durable host response is highly desirable. Summary of the Invention

[0008] In one aspect, the present application provides a chimeric antigen receptor complex. In one embodiment, the chimeric antigen receptor complex comprises a first protein having a first extracellular domain linked to a first intercellular domain via a first linker, the first extracellular domain comprising a first scFv with affinity for a first tumor epitope and the first intercellular domain comprising a JAK1-binding domain, and a second protein having a second extracellular domain linked to a second intercellular domain via a second linker, the second extracellular domain comprising a second scFv with affinity for a second tumor epitope and the second intercellular domain comprising a JAK3-binding domain. The first tumor epitope is on a first tumor antigen. The second tumor epitope is on a second tumor antigen.

[0009] In one embodiment, the first intracellular domain comprises IL7Rα (CD127). In one embodiment, the first intracellular domain comprises the intracellular domain of IL15Rβ (CD122), IL21Rα (CD360), or a combination thereof. In one embodiment, the first intracellular domain further comprises a first cytotoxic signaling domain linked to the JAK1 binding domain.

[0010] In one embodiment, the first cytotoxic signaling domain comprises CD28, CD3ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

[0011] In one embodiment, the first scFv domain or the second scFv domain independently have affinity for CD19 or CD22. In one embodiment, the first scFv domain has affinity for CD19. In one embodiment, the second scFv domain has affinity for CD22. In one embodiment, the second intracellular domain comprises γ (CD132).

[0012] In one embodiment, the second intracellular domain further comprises a second cytotoxic signaling domain linked to the JAK3-binding domain. In one embodiment, the second cytotoxic domain comprises CD28, CD3ζ, CD137, OX40, CD27, ICOS, or a combination thereof. In one embodiment, the second intracellular domain comprises γ (CD132), a JAK3-binding domain, CD28, and CD3ζ in tandem. In one embodiment, the first intracellular domain is configured to dimerize with the second intracellular domain.

[0013] In one embodiment, the first linker and the second linker independently comprise CD8. In one embodiment, the first linker and the second linker independently comprise a stalk and a transmembrane domain.

[0014] In one embodiment, the stalk comprises CD8, Fc hinge, Fc CH2-CH3, TCRα, TCRβ, truncated IL7Rα (CD127), truncated IL15Rβ (CD122), IL15Rα (CD215), truncated gamma (CD132), truncated IL21Rα (CD360), or a combination thereof.

[0015] In one embodiment, the transmembrane domain comprises CD8, CD28, CD3ζ, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα, TCRβ, IL15Rβ (CD122), γ (CD132), IL7Rα (CD127), IL21Rα (CD360), IL15Rα (CD215), or a combination thereof.

[0016] In one embodiment, the tumor antigen comprises CDH17, TROP2, CD19, CD22, CD37, BCMA, CD48, EGFR, HER2, EpCAM, CEACAM5, PSMA, GD2, GPC3, or a combination thereof.

[0017] In another aspect, the present application provides an open reading frame (ORF). In one embodiment, the open reading frame (ORF) consecutively comprises a CD19 scFv, a stalk transmembrane region, and an IL7 alpha endo-domain. In one embodiment, the open reading frame (ORF) consecutively comprises a CD22 scFv, a stalk transmembrane region, a gamma chain endo-domain, a CD28 endo-domain, and a CD3 zeta endo-domain. In one embodiment, the open reading frame (ORF) consecutively comprises a PD-1 scFv, CCL21, and IL7.

[0018] In a further aspect, the present application provides a biomolecular complex. In one embodiment, the biomolecular complex comprises a first protein having a first extracellular domain linked to a first intercellular domain via a first linker, the first extracellular domain comprising a first scFv having affinity for a first tumor epitope and the first intercellular domain comprising a JAK1 binding domain; a second protein having a second extracellular domain linked to a second intercellular domain via a second linker, the second extracellular domain comprising a second scFv having affinity for a second tumor epitope and the second intercellular domain comprising a JAK3 domain; a first tumor antigen; and a second tumor antigen. The first tumor epitope binds to the first tumor antigen. The second tumor epitope binds to the tumor antigen.

[0019] In one embodiment, the first intracellular domain is dimerized with the second intracellular domain, hi one embodiment, JAK1 is dimerized with JAK3.

[0020] In a further aspect, the present application provides a non-viral DNA construct. In one embodiment, the non-viral DNA construct comprises, consecutively from 5' to 3', an inducible promoter followed by a first ORF, the first ORF comprising an anti-PD-1 scFv, CLL21, and IL7, each connected by a single peptide and terminating in a ribosomal skipping peptide; a second ORF comprising at least one constitutive chimeric antigen receptor; and a third promoter followed by at least one RNA sequence.

[0021] In a further aspect, the present application provides a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor comprises, in succession, a cytokine domain, a linker, a truncated CD8 domain, and a signaling domain.

[0022] In one embodiment, the cytokine domain comprises IL7, IL12, IL21, or a combination thereof. In one embodiment, the truncated CD8 domain comprises at least a portion of a hinge, a transmembrane domain, and a cytoplasmic domain. In one embodiment, the cytoplasmic domain comprises CD28 / CD170, CD3ζ, or a combination thereof.

[0023] In one embodiment, the chimeric antigen receptor further comprises a tumor antigen domain that mediates the cytokine domain and the truncated CD8 domain.

[0024] In one embodiment, the present application provides a biomolecular complex comprising a chimeric antigen receptor disclosed herein bound to a tumor antigen.

[0025] In a further aspect, the present application provides a non-viral vector comprising an artificial immune surveillance chimeric antigen receptor (AI-CAR) expression cassette flanked by two transposons or viral terminal repeats (IRs), wherein the AI-CAR expression cassette comprises an inducible gene expression unit and a CAR expression unit.

[0026] In one embodiment, the inducible gene expression unit comprises a STAT, NFAT, or NF-κB inducible promoter, the coding region of one or more genes linked to an IRES or a self-cleaving ribosomal skipping peptide, followed by a first polyA signal sequence. In one embodiment, the self-cleaving ribosomal skipping peptide comprises TA2. In one embodiment, the inducible gene expression unit comprises genes for expressing at least two different cytokine receptors. In one embodiment, the inducible gene expression unit comprises a gene for expressing an antigen binding protein. In one embodiment, the inducible gene expression unit comprises a gene for expressing an anti-PD1 scFv. In one embodiment, the inducible gene expression unit comprises a gene for expressing CCL21. In one embodiment, the inducible gene expression unit comprises a gene for expressing IL7.

[0027] In one embodiment, the CAR expression unit comprises an anti-CDH17 scFv, an anti-TROP2 scFv, and a gene for expressing a CAR. In one embodiment, the CAR expression unit comprises a promoter, one or two CAR genes, followed by a second polyA signal sequence. In one embodiment, the CAR expression unit further comprises a gene for expressing a safety switch. In one embodiment, the safety switch comprises a truncated EGFR (tEGFR) or a truncated CD20. In one embodiment, the AI-CAR expression cassette is configured to express an shRNA, wherein the shRNA is configured to inhibit an endogenous TCR.

[0028] In a further aspect, the present application provides isolated nucleic acids encoding the biomolecular complexes, biomolecules, antigens, and proteins disclosed herein.

[0029] In a further aspect, the present application provides an expression vector comprising an isolated nucleic acid disclosed herein. In one embodiment, the expression vector comprises an ORF disclosed herein. In one embodiment, the expression vector comprises a non-viral DNA construct disclosed herein. The expression vector may be viral or non-viral. The vector may also be expressible intracellularly.

[0030] In a further embodiment, the present application provides a host cell. In one embodiment, the host cell comprises an isolated nucleic acid and / or an expression vector disclosed herein. In one embodiment, the host cell comprises a non-viral DNA construct disclosed herein. In one embodiment, the host cell comprises a non-viral vector disclosed herein.

[0031] In further embodiments, the present application provides a mammalian cell comprising a chimeric antigen receptor complex, a biomolecule complex, a biomolecule, an antigen, and a protein disclosed herein. In one embodiment, the mammalian cell comprises a chimeric antigen receptor disclosed herein. In one embodiment, the mammalian cell comprises a biomolecule complex disclosed herein.

[0032] In a further embodiment, the present application provides a CAR-T cell or a CAR-NK cell. In one embodiment, the CAR-T cell or the CAR-NK cell expresses a chimeric antigen receptor complex disclosed herein. In one embodiment, the CAR-T cell or the CAR-NK cell expresses a chimeric antigen receptor disclosed herein.

[0033] In a further embodiment, the present application provides a method for treating a tumor in a subject, the method comprising administering to the subject a sufficient amount of a CAR-T or CAR-NK cell disclosed herein.

[0034] In a further aspect, the present invention provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a vector, non-viral vector, CAR-T or CAR-NK cell, protein, biomolecule, or biomolecule complex disclosed herein. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle. [Brief explanation of the drawings]

[0035] These and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which: The present specification will be described with additional specificity and detail through the use of the accompanying drawings, with the understanding that these drawings depict only some embodiments arranged in accordance with the disclosure herein and are therefore not intended to limit its scope. [Figure 1] Figure 1 shows an AI-CAR gene expression cassette containing an inducible gene expression unit and a CAR expression unit in a non-viral vector (pPI) for one or two constitutively expressed CARs to induce gene expression for host anti-tumor response. [Figure 2] Figure 2 shows a typical concept of an AI-CAR. [Figure 3] Figure 3 shows an AI-CAR expression vector encoding a constitutively expressed dual CAR targeting CDH17 and TROP2 in a non-viral vector (pPI) and a cassette of anti-PD1 scFv, CCL21, and IL17 genes under an inducible promoter for CAR-induced host anti-tumor responses. The AI-CAR expression vector encodes a constitutively expressed dual CAR targeting CDH17 and TROP2 in a non-viral vector (pPI) and a cassette of anti-PD1 scFv, CCL21, and IL17 genes under an inducible promoter for CAR-induced host anti-tumor responses. [Figure 4]Figure 4 shows tumor antigen induction of the integrated pPI-anti-CDH17 AI-CAR vector gene. Tumor antigen induction of the integrated pPI-anti-CDH17-AI-CAR vector gene. (A) Expression of the integrated pPI-anti-CDH17-AI-CAR vector was measured by GFP levels in T cells (Jurkat) in response to different concentrations of CDH17. (B) Induction by recombinant CDH17 in colon cancer cells (SW480). (C) Cytotoxicity of pPI-anti-CDH17-AI-CAR-integrated T cells against CDH17-expressing SW480 cells. [Figure 5] Figure 5 shows the expression and binding specificity of pPI-anti-CDH17-TROP2 AI-CAR. [Figure 6] Figure 6 shows iPro mutations to support AI-CAR growth and persistence. iPro mutations to support AI-CAR growth and persistence. (A) Example of iPro7 expression. (B) Induction of proliferation of CD25 T cell population. (C) Increased T cell survival. Detailed Description

[0036] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the disclosure herein, as generally described and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0037] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the disclosure herein, as generally described and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0038] Provided herein, inter alia, are isolated antibodies, methods for producing such antibodies, bispecific or multispecific molecules, antibody-drug conjugates and / or immunoconjugates comprised of such antibodies or antigen-binding fragments, pharmaceutical compositions comprising the antibodies, bispecific or multispecific molecules, antibody-drug conjugates and / or immunoconjugates, methods for producing the molecules and compositions, and methods for treating cancer using the molecules and compositions disclosed herein.

[0039] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal, polyclonal, chimeric, single-chain, bispecific or bipotent, simianized, human, and humanized antibodies, and active fragments thereof. Examples of active fragments of known antigen-binding molecules include Fab, F(ab')2, scFv, and Fv fragments, including the products of a Fab immunoglobulin expression library, and epitope-binding fragments of any of the above antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically binds to an antigen. Immunoglobulins are any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. In one embodiment, antibodies can be whole antibodies and any antigen-binding fragments derived from whole antibodies. A typical antibody typically refers to a heterotetrameric protein having two heavy (H) chains and two light (L) chains. Each heavy chain is composed of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is composed of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions can be further subdivided into hypervariable complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs arranged in the following order: From the amino terminus to the carboxy terminus are FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Within the variable regions of the light and heavy chains are binding domains that interact with antigens.

[0040] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the disclosure herein may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0041] Monoclonal antibodies may also include "chimeric" antibodies (immunoglobulins) in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0042] Monoclonal antibodies can be produced using a variety of methods, including mouse hybridoma or phage display (for a review, see Siegel. Transfus. Clin. Biol. 9:15-22 (2002)), or by molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453-7 (2011)). In this disclosure, antibodies were generated by immunizing rabbits with both human PD-L1 protein and cells transiently expressing human PD-L1 on their surface. Rabbits are known to produce antibodies with high affinity, diversity, and specificity (Weber et al. Exp. Mol. Med. 49:e305). B cells from the immunized animals were cultured in vitro and screened for the production of anti-PD-L1 antibodies. Using recombinant DNA technology, we isolated the antibody variable genes, recombinantly expressed the resulting antibodies, and screened them for desired functions, such as the ability to block PD-L1 binding to PD-1, the ability to bind to PD-L1 in non-human primates, and the ability to promote human T cell activation. This general method of antibody discovery is similar to that described by Seeber et al. PLOS One. 9:e86184 (2014).

[0043] The term "antigen- or epitope-binding portion or fragment" refers to a fragment of an antibody capable of binding to an antigen (in this case, CD19). These fragments may retain the antigen-binding function of the intact antibody as well as additional functions. Examples of binding fragments include, but are not limited to, a single-chain Fv fragment (scFv), consisting of the VL and VH domains of a single antibody arm connected by a synthetic linker in a single polypeptide chain, or a Fab fragment, which is a monovalent fragment consisting of the VL, constant light (CL), VH, and constant heavy chain 1 (CH1) domains. Antibody fragments are even smaller subfragments, and can consist of domains as small as a single CDR domain, particularly the CDR3 region from either the VL and / or VH domain (see, e.g., Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are produced using conventional methods known to those of skill in the art. Antibody fragments can be screened for utility using the same techniques used with intact antibodies.

[0044] "Antigen- or epitope-binding fragments" may be derived from the antibodies disclosed herein by a number of techniques known in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Appropriate fractions containing Fab fragments can then be collected and concentrated by membrane filtration or the like. For further description of general techniques for isolating active fragments of antibodies, see, e.g., Khaw, BA et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0045] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is capable of cross-linking antigen.

[0046] Fab fragments may contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0047] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can also recognize and bind antigen, although with lower affinity than the entire binding site.

[0048] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda (λ), based on the amino acid sequences of their constant domains.

[0049] Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, and IgA-2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, delta, epsilon, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0050] A "humanized antibody" refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one (or more) human immunoglobulins. Additionally, framework support residues may be altered to retain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio / Technology, 9:421 (1991)).

[0051] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

[0052] As used herein, the terms "a," "an," and "the" are defined to mean "one or more" and include the plural unless the context requires otherwise.

[0053] "Isolated" refers to a biological molecule that is free from at least some of the components with which it naturally occurs. "Isolated," when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide is prepared by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen-binding specificities.

[0054] By "recombinant" is meant that the antibody is produced using recombinant nucleic acid techniques in an exogenous host cell.

[0055] The term "antigen" refers to an entity or fragment thereof that is capable of eliciting an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

[0056] Also, as used herein, the term "immunogenic" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against the immunogenic agent and contributes to a human or animal immune response. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells against the administered immunogenic composition disclosed herein to alleviate or ameliorate the disorder being treated.

[0057] "Specific binding" or "binds specifically" or "specific" for a particular antigen or epitope refers to binding that is distinct from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0058] Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 This can be demonstrated by an antibody having a KD for an antigen or epitope of M or greater, where KD refers to the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen may have a KD for the antigen or epitope that is 20-, 50-, 100-, 500-, 1000-, 5000-, 10000-, or greater than that of a control molecule.

[0059] Specific binding to a particular antigen or epitope can also be demonstrated by an antibody having a K A or K A for the antigen or epitope that is at least 20-, 50-, 100-, 500-, 1000-, 5000-, 10000-, or greater than that for a control epitope, where K A or K A refers to the association rate of a particular antibody-antigen interaction.

[0060] The "homology" between two sequences is determined by the sequence identity. When the two sequences compared to each other are different in length, the sequence identity preferably relates to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues in the longer sequence. Sequence identity can be conventionally determined using a computer program. Deviations that appear in the comparison of a given sequence with the above sequences disclosed herein can be caused by, for example, addition, deletion, substitution, insertion or recombination.

[0061] The specification may be understood more readily by reference to the following detailed description of the specific embodiments and examples contained herein. Although the specification has been described with reference to specific details of certain embodiments thereof, it is not intended that such details be deemed limitations on the scope of the disclosure herein.

[0062] In previous disclosures, CAR exhaustion was addressed by targeting cytokine signaling pathways to promote proliferation without terminal differentiation. Here, the disclosed compositions and methods of use relate to AI-CAR vectors for artificial immune surveillance chimeric antigen receptors. This advancement in AI-CAR technology aims to replace standard CAR production and enable effective "combination" and point-of-care treatments. While other CAR technologies may require the expression of soluble cytokine growth factors and / or multiple administrations for sustained activity to achieve a complete and durable response, AI-CAR vectors incorporate these activities, enabling the production of effective and sustained CAR cells without either constitutively active drivers for proliferation or multiple CAR administrations for durable antitumor responses.

[0063] Second, while retaining the same functionality as standard CARs, AI-CAR signaling improves the production efficiency of CAR cells. AI-CARs require only one target antigen for full proliferation and cytotoxic activity both in vitro and in vivo. In this regard, AI-CARs may enable significant reductions in production costs, as standard CAR-T cell expansion typically requires the use of a combination of growth factors and aAPCs for production.

[0064] Third, when AI-CARs act on tumor cells in vivo, they can induce the expression of multiple antitumor genes encoded in the integrated AI-CAR vector. Expression of these endogenous genes allows patients to mount antitumor responses that broadly target different tumor antigens, such as neoantigens. For example, the STAT5 reporter system is used to induce STAT5-responsive genes in human T cells (Kanai et al., 2014; Zeng et al., 2016; Bednorz et al., 2011; and Fang et al., 2008). This feature is unique because standard CAR constructs typically cannot induce gene expression. Together with these cofactors, AI-CARs could become a platform technology that offers a practical, economical, and effective solution for point-of-care cancer treatment. Many forms of cancer can exist in the immunosuppressive tumor environment. AI-CARs are highly desirable because their vectors are engineered to express additional anti-cancer genes that reduce tumor immunosuppression and activate patients' anti-tumor immune responses. One unique feature of AI-CARs is their ability to control the expression of relevant anti-tumor genes at the tumor site, preventing potentially toxic constitutive expression. Another feature is that AI-CARs are designed for single-dose administration, followed by long-term activity and higher efficacy. With these advantageous features, AI-CARs represent a better solution to unmet market challenges and promise efficacy for treating most, if not all, types of cancer.

[0065] In CAR therapeutics for hematological cancers, targets including CD19, CD22, CD20, CD9, and CD38 can be targeted by dual, bispecific, AI-CARs using non-viral DNA vectors or RNA-CARs (transient). In this way, AI-CARs can be used as transient treatments until transplantation or to enhance durability. Furthermore, AI-CARs can be used as point-of-care treatments to induce host anti-tumor immune responses by targeting neoantigens.

[0066] Furthermore, for certain solid tumors, RNA encoding AI-CAR combination therapy may be applicable. For aggressive tumors that have failed other treatments, the high electroporation efficiency of RNA allows for efficient and rapid production of ready-made RNA CARs. The multiple antitumor mechanisms of transiently expressed RNA AI-CARs, similar to those of induced AI-CAR vector genes, may enable more effective and safe antitumor activity and induce immune responses in patients. Multi-dosing of RNA AI-CARs may serve as a bridge to determining efficacy before treatment with specific, persistent AI-CAR cells. [Example]

[0067] Example 1. Construction of AI-CAR vectors for continuous and combination therapy The purpose of AI-CAR cells is to improve the efficacy of cancer immunotherapy by enabling sustained, long-term immune surveillance in a quiescent state until stimulated by tumor cells. After tumor stimulation, the inducible AI-CAR gene allows for locally safe and more effective "combination" therapy that includes an additional mechanism of antitumor activity. By stimulating patients' antitumor responses, it is hoped that a higher frequency of complete and durable responses can be achieved.

[0068] Typically, AI-CAR constructs are composed of an AI-CAR expression cassette in a non-viral vector (pPI) such as a transposon-based integration system (Ivics and Izsvak, 2010; Z. Cooper et al., US9,629,877; Uckert et al., US20190071484A1). As shown in Figure 1, an AI-CAR vector is composed of an inducible gene expression unit and a CAR expression unit, i.e., an AI-CAR expression cassette flanked by transposon terminal inverted repeats (IRs). The AI-CAR expression cassette may be composed of a STAT, NFAT, or NF-κB inducible promoter, the coding region of one or more genes linked by an IRES or a self-cleaving ribosomal skipping peptide (e.g., P2A or T2A (e.g., SEQ ID NOs: 18-21)), followed by a polyA signal sequence. This may be followed by another promoter for constitutive expression of one or two CARs, followed by another polyA signal. For examples of AI-CAR chain pairs, see SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 7 and 8, and 9 and 10. Both coding regions may be placed between two transposons or viral terminal repeats (IRs) for integration. Alternatively, the coding region of the AI-CAR construct may be integrated into a specific genomic site using zinc finger, TALEN, or CRISPR / Cas9 nucleases (Eyquem 2017).

[0069] As shown in Figure 2, one or two CARs are constitutively expressed on the surface of T cells, allowing them to bind to tumor-associated antigens on tumors. AI-CARs induce the expression of inducible genes, enabling safe and multiple mechanisms of antitumor activity and potent stimulation of host antitumor responses. Ideally, expression of these genes is induced after the AI-CAR binds to tumor antigens or antigens within the tumor microenvironment (TME). To expand the pool of such genes, a second AI-CAR chain may be expressed from a second vector.

[0070] There is at least some flexibility in the construction of non-viral vectors for AI-CARs, including piggyBac, Tol2, and Sleeping Beauty (Ivics and Izsvak, 2010). Furthermore, AI-CARs may express safety switches, such as cleaved EGFR (tEGFR), which can be targeted for elimination by the FDA-approved antibody cetuximab. Alternatively, a safety target may be cleaved CD20, which can be targeted for CAR cell elimination by rituximab. AI-CAR vectors may also express, for example, shRNAs that inhibit endogenous TCRs, allowing for the generation of universal AI-CAR cells.

[0071] Instead of an inducible promoter, the AI-CAR may contain a weak promoter, such as a modified PGK promoter, to safely express other antitumor activity mechanisms.

[0072] Example 2. Design of single and dual AI-CAR Single or dual AI-CARs are designed to signal via cytokine receptor pathways to enhance CAR cell persistence and to induce vector-encoded genes for additional antitumor mechanisms and enhanced efficacy. Furthermore, simple conjugation of AI-CARs allows for efficient expansion of AI-CARs and can be used in production to simplify in vitro expansion prior to administration.

[0073] Dual AI-CARs are composed of IL12, IL7, IL21, or IL15 cytokine receptor endodomains, allowing for persistence and induction of vector genes for additional antitumor mechanisms. As shown in Table 2, the cytokine endodomains are fused to one or more TCR or TCR costimulatory cytoplasmic regions (also called costimulatory domains), such as CD3ζ, CD28, CD137, CD27, OX40, or ICOS. For example, a dual AI-CAR may consist of one CAR composed of a tumor antigen-specific scFv fused to a stalk and transmembrane domain and a segment of the intracellular (endo)domain of the IL12 beta1 chain, and CD3ζ. The second CAR may consist of a tumor antigen-specific scFv fused to a stalk and transmembrane domain, a segment of the common beta2 chain endodomain, and a CD137 costimulatory endodomain.

[0074] AI-CARs can induce gene expression through transcription factors such as STAT4. The beta1 chain of IL-12 contains a Ty2k binding site, and the beta2 chain contains a JAK2 binding site. The binding of these two chains can be stabilized by binding to adjacent target antigens or two different but adjacent epitopes on the same tumor target antigen. When CARs bind to tumor antigens, Ty2k and JAK2 phosphorylate the beta chain, ultimately resulting in the phosphorylation of STAT4, which dimerizes and translocates to the nucleus. STAT4 then binds to transcription factor (TF) response elements in promoters, promoting gene expression induction. STAT4-regulated promoters and downstream genes can be integrated into CAR transposons or viral vectors. Once integrated, these genes can be induced after CAR cells bind to tumor or TME antigens. STAT4-induced endogenous genes also support CAR cell persistence (DeRenzo 2019).

[0075] Most of the dual AI-CAR components are independent functional units, i.e., antitumor scFvs, stalks, transmembrane domains, and endodomains, as well as different segments of the IL15, IL7, and IL21 chains, CD3ζ, and costimulatory proteins, that are subject to substitution for any number of specific purposes. As shown in the examples in Tables 1 and 2, there are many possible combinations to produce a specific AI-CAR.

[0076] Monospecific AI-CARs can be constructed to support CAR cell persistence through inducible genes encoded within the CAR vector. AI-CARs can consist of an scFv targeting a tumor- or TME-associated antigen, a stalk, a transmembrane domain, and costimulatory CD137 and CD3ζ endodomains. Upon tumor antigen binding, activated NFAT is generated and binds to response elements within the integrated CAR vector, inducing the expression of one or more genes that support persistence, such as IL15, IL12, and IL7, as well as additional mechanisms of antitumor activity.

[0077] Table 2 lists additional genes encoding proteins or miRNAs with sustained or antitumor activity, such as anti-immune checkpoint inhibitors (ICIs), OX40 agonists, TLR agonists, cytokines, bispecific antibodies, iPros, chemokines, and chemokine receptors, which may be placed under the control of an inducible promoter. Alternatively, mRNAs encoding these proteins, such as those listed in SEQ ID NOS: 18-20, may be co-transfected with an AI-CAR vector for transient expression. Alternatively, CARs and mRNAs encoding these proteins may function as AI-CARs for transient, safe therapy (SEQ ID NOS: 11-17). Several doses of AI-CARs transiently expressing these genes are sufficient to reduce immunosuppression in the tumor microenvironment and activate the patient's antitumor immune response.

[0078] Example 3. AI-CAR Encoding a Bispecific Anti-CDH17 and Anti-TROP2 CAR and an Inducible Gene for Durable and Enhanced Anti-Tumor Responses As shown in Figure 3, an AI-CAR vector may be constructed with an NFAT-inducible promoter, the coding regions of one or more genes, such as anti-PD-1, CCL21, and IL-7, linked by a ribosomal skipping peptide such as T2A, followed by a poly(A) signal sequence. This may be followed by a promoter for a single bispecific CAR targeting CDH17 and TROP2 with CD137 and CD3ζ endodomains, followed by T2A, a signal peptide, tEGFR, and a poly(A) signal. Both coding regions can be integrated between transposons or viral terminal repeats (IRs). Alternatively, the AI-CAR expression cassette can be integrated into specific genomic sites using TALEN or CRISPR / Cas9 (Eyquem 2017).

[0079] Example 4. Induction of AI-CAR gene by tumor-targeting antigen After transfection into T cell lines, recombinant and cellular tumor-targeting antigens were used to induce the AI-CAR vector gene. The pPI-anti-CDH17-AI-CAR construct, which expresses GFP under the control of an NFAT-inducible promoter, was electroporated into Jurkat T cell lines. This pBac transposon construct was co-electroporated with a transposase expression vector for integration of the AI-CAR vector. Jurkat T cell lines transfected with the pPI-anti-CDH17 AI-CAR vector were incubated (37°C, 5% CO2) for 2 or 14 hours in microtiter wells coated with 0, 1.25, 2.5, 5, 10, or 20 μg / ml of CDH17-Fc. The pPI-anti-CDH17 AI-CAR vector contains GFP under the control of an NFAT-inducible promoter. After 14 hours, GFP expression levels were measured by flow cytometry. GFP expression levels are relative to the maximum induced by 14 hours of immunocytochemical treatment (anti-CD3, CD28, and CD2; StemCell). As little as 2 hours of CDH17 exposure resulted in low levels of GFP detected by flow cytometry, as shown in Figure 4A. After 14 hours, CDH17-exposed GDP increased by up to 70% compared to immunocytochemical treatment in a concentration-dependent manner. In unstimulated cells, GFP levels were negative. CDH17 was expressed at different levels in SW480 cells by electroporation with 0, 1.25, 5, 10, or 20 μg of CDH17 RNA (per 10^7 cells). CDH17 expression was measured by standard flow cytometry, as shown in Figure 4B. Jurkat cells transfected with the pPI-anti-CDH17 AI-CAR vector were incubated for 2 or 14 hours on monolayers of SW480 cells expressing different levels of CDH17. After 14 hours, GFP expression levels were measured by flow cytometry. The GFP expression levels are relative to the maximum induction achieved by 14 hours of immunocytochemistry. As shown in Figure 4C, similar levels of GFP induction (50-80%) were detected when AI-CAR cells were exposed to Sw480 expressing CDH17 for 2 or 14 hours.Thus, tumor cells expressing the target antigen appear to efficiently induce gene expression of the AI-CAR vector, and this AI-CAR construct can therefore be used to express proteins with antitumor activity at tumor sites for safe and enhanced tumor killing.

[0080] Example 5. Expression of dual AI-CAR We demonstrated the expression and binding activity of a dual AI-CAR with two chains. Dual AI-CARs, one with the endodomains of IL15 beta and CD28 and the other with the endodomains of IL15 gamma and CD3 zeta (SEQ ID NOs: 1 and 2), were expressed in CHO cells. As shown in Figure 5, expression of individual chains, pSh3C15b28 and pSh3A4C15g3, or both chains, was measured by staining with biotinylated Protein-L (for scFv). Binding to tumor antigens, CDH17-Fc and TROP2-Fc, was also measured by flow cytometry. Expression and tumor antigen binding were measured using streptavidin-phycoerythrin for Protein-L and anti-human IgG-Alexa647 for CDH17 and TROP2. These results demonstrate the ease of transfection, expression, and ligand-binding function of dual CARs.

[0081] Example 6. Design and expression of iPros that support AI-CAR growth and persistence To support proliferation and persistence, inducers of proliferation (iPro) can be induced in single AI-CARs. As shown in Figure 6A, iPro consists of an N-terminal cytokine or scFv that binds to a TME antigen, followed by a linker, stalk, transmembrane domain, and an endodomain containing JAK family and STAT binding sites. When the N-terminal domain binds to a cytokine receptor or tumor antigen, iPro signals through STATs, inducing the expression of proteins in T cells and NK cells, supporting the survival and maintenance of naive and Tcm stem cell phenotypes. iPro with N-terminal cytokine domains such as IL7, IL15, and IL12 support bidirectional inside-out and outside-in stimulation, activating patient T cells and NK cells in addition to AI-CAR cells. Expression of iPro not only enhances AI-CAR activity in vivo, but also facilitates the transgenic expansion of AI-CARs for production.

[0082] When iPro (iPro7; SEQ ID NO: 19), which carries an IL7 at its N-terminus, was transiently expressed in PBMCs by electroporation of in vitro transcribed mRNA, it was detected by flow cytometry after 24 hours but not after 48 hours (Figure 6B, left panel). On day 2, induced expression of the IL2 receptor CD25 was confirmed by flow cytometry (Figure 6C, center panel). Even without further stimulation, T cell numbers remained stable over 10 days, whereas mock control T cell numbers were significantly reduced (Figure 6D, right panel). These results indicate that variants of AI-CAR-inducible genes, such as iPro, can be engineered to support AI-CAR proliferation and persistence.

[0083] References Rodriguez-Galan A, Fernandez-Messina L, Sanchez-Madrid F. Control of Immunoregulatory Molecules by miRNAs in T Cell Activation. Front Immunol. 2018 Sep 25;9:2148. Lykken EA, Li QJ. The MicroRNA miR-191 Supports T Cell Survival Following Common γ Chain Signaling. J Biol Chem. 2016 Nov 4;291(45):23532-23544. Epub 2016 Sep 15. Ivics, Z., Izsvak, Z. The expanding universe of transposon technologies for gene and cell engineering. Mobile DNA 1, 25 (2010). Cooper Lj, Torikai H, Zhang L, Huls H, Wang-Johanning F, Hurton L, Olivares S, Krishnamurthy J. Human application of engineered chimeric antigen receptor (CAR) T-cells. US9629877B2. Uckert W, Bunse M, Clauss J, Izsvak Z. A transposon-based transfection system for primary cells US20190071484A1.

[0084] table Table 1 shows examples of AI-CAR configurations. AI-CARs can be constructed using fragments of different genes encoding different functional segments, including anti-tumor-associated antigen (TAA) scFv, stalk, transmembrane domain, and endodomain. Different classes of endodomains can function, for example, in signaling proliferation and survival responses of cytokine receptors or tumor cytotoxicity responses. Cytokine receptor, CD3ζ, and costimulatory endodomains can be fused in various tandem configurations. [Table 1]

[0085] Table 2 shows examples of inducible genes that can be incorporated into AI-CAR vectors. These genes can be selected to enhance CAR localization to tumors (e.g., lymphomas), reverse tumor immunosuppression, stimulate host immune responses, or for direct anti-tumor cell activity. These genes can be placed downstream of a STAT5-inducible promoter to avoid toxicity that can result from long-term constitutive expression. Alternatively, certain chemokine and chemokine receptor genes and cytokines (e.g., IL7) can be placed downstream of weak promoters. Low-level expression can avoid toxicity. [Table 2]

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Claims

1. 1. A chimeric antigen receptor complex comprising: a first protein having a first extracellular domain linked to a first intracellular domain via a first linker, wherein the first extracellular domain comprises a first scFv having affinity for a first tumor epitope, and the first intracellular domain comprises a JAK1 binding domain; and a second protein having a second extracellular domain linked to a second intracellular domain via a second linker, wherein the second extracellular domain comprises a second scFv having affinity for a second tumor epitope, and the second intracellular domain comprises a JAK3 binding domain; Including, the first tumor epitope is on a first tumor antigen and the second tumor epitope is on a second tumor antigen; A chimeric antigen receptor complex, wherein the first tumor antigen and the second tumor antigen comprise CDH17 and TROP2, respectively.

2. 2. The chimeric antigen receptor complex of Claim 1, wherein the first intracellular domain further comprises a first cytotoxic signaling domain linked to a JAK1-binding domain.

3. 3. The chimeric antigen receptor complex of Claim 2, wherein the first cytotoxic signaling domain comprises CD28, CD3ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

4. 2. The chimeric antigen receptor complex of claim 1, wherein the second intracellular domain comprises gamma (CD132).

5. 2. The chimeric antigen receptor complex of claim 1, wherein the second intracellular domain further comprises a second cytotoxic signaling domain linked to a JAK3-binding domain.

6. 6. The chimeric antigen receptor complex of claim 5, wherein the second cytotoxic signaling domain comprises CD28, CD3ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

7. 2. The chimeric antigen receptor complex of claim 1, wherein the second intracellular domain comprises gamma (CD132), a JAK3 binding domain, CD28, and CD3ζ in tandem.

8. 2. The chimeric antigen receptor complex of claim 1, wherein the first intracellular domain is configured to dimerize with a second intracellular domain.

9. 2. The chimeric antigen receptor complex of claim 1, wherein the first and second linkers independently comprise CD8.

10. 2. The chimeric antigen receptor complex of claim 1, wherein the first and second linkers independently comprise a stalk and a transmembrane domain.

11. 11. The chimeric antigen receptor complex of claim 10, wherein the stalk comprises CD8, Fc hinge, Fc CH2-CH3, TCR alpha, TCR beta, truncated IL7R alpha (CD127), truncated IL15R beta (CD122), IL15R alpha (CD215), truncated gamma (CD132), truncated IL21R alpha (CD360), or a combination thereof.

12. 11. The chimeric antigen receptor complex of claim 10, wherein the transmembrane domain comprises CD8, CD28, CD3ζ, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα, TCRβ, IL15Rβ (CD122), γ (CD132), IL7Rα (CD127), IL21Rα (CD360), IL15Rα (CD215), or a combination thereof.

13. A biomolecular complex, a first protein having a first extracellular domain linked to a first intracellular domain via a first linker, wherein the first extracellular domain comprises a first scFv having affinity for a first tumor epitope, and the first intracellular domain comprises a JAK1 binding domain; a second protein having a second extracellular domain linked to a second intracellular domain via a second linker, wherein the second extracellular domain comprises a second scFv having affinity for a second tumor epitope, and the second intracellular domain comprises a JAK3 binding domain; and a first tumor antigen, and a second tumor antigen, Including, the first tumor epitope binds to a first tumor antigen and the second tumor epitope binds to the tumor antigen; A biomolecular complex, wherein the first tumor antigen and the second tumor antigen comprise CDH17 and TROP2, respectively.

14. 14. The biomolecular complex of claim 13, wherein the first intracellular domain is dimerized with the second intracellular domain.

15. The biomolecular complex of claim 13, wherein the JAK1 binding domain dimerizes with the JAK3 binding domain.

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