Immune cells expressing chimeric antigen receptors and bispecific antibodies and uses thereof

Abstract

The present invention relates to immune cells expressing a chimeric antigen receptor against p95HER2 and a bispecific antibody against HER2 and CD3, and their use in the treatment of cancer, particularly cancers that overexpress p95HER2.

Description

[Technical field]

[0001] The present invention is within the field of biotechnology and biopharmaceuticals, in particular, immune cells expressing a specific chimeric antigen receptor against the p95 fragment of HER2 and a bispecific antibody against HER2 and CD3, and their use in the treatment of cancer. [Background technology]

[0002] Redirection of immune cells against tumor-associated or tumor-specific antigens is an effective therapeutic strategy for certain hematological malignancies. In contrast, it has failed so far against solid tumors. T cells can be redirected using bispecific antibodies, commonly known as bispecific T cell engagers (BiTEs) or T cell bispecific antibodies (TCBs), that simultaneously bind tumor antigens and the invariant subunit of the T cell receptor (TCR), typically CD3. Alternatively, T cells can be engineered to express chimeric antigen receptors (CARs), which are engineered proteins that contain the antigen-binding domain of an antibody and the signaling domain of a TCR and a coactivator receptor.

[0003] In contrast to its success against hematological malignancies, T cell redirection has so far failed against solid tumors, due to down-modulation of the antigen against which the T cells are redirected, heterogeneity in the expression of said antigens, and the immunosuppressive environment in which the tumor is established.

[0004] Although there are inherent limitations in the design of improved bispecific T cell engagers to overcome such evasion mechanisms, CAR T cells can be used as a platform to deliver additional antitumor factors, such as cytokines or antibodies. Cytokines produced by these so-called fourth generation CARs include IL-12 and IL-15. Antibodies delivered by fourth generation CAR T cells include blocking antibodies targeting immune checkpoint inhibitors (and bispecific antibodies targeting tumor antigens (Choi et al., 2019, Nature Publishing Group 37, 1049-1058). Armed CARs represent components of a scalable platform to counter the aforementioned mechanisms of resistance.

[0005] Due to the scarcity of tumor-specific antigens, the majority of CARs developed so far have been directed against tumor-associated antigens. As a result, severe side effects due to on-target off-tumor activity are frequent and have limited their use. Although in principle they should be free of side effects, CARs directed against the few available tumor-specific antigens remain ineffective. This failure is due, at least in part, to low levels and / or heterogeneous expression of these tumor-specific antigens. Tumor cells eventually escape by downmodulating them, or non-expressing tumor cells are rapidly selected. To exploit the specificity of tumor-specific antigens and combine it with the potency of targeting tumor-associated antigens, fourth-generation CAR T cells expressing bispecific T cell engagers have been developed using glioblastoma as a proof of concept.

[0006] The gene encoding the tyrosine kinase receptor HER2 is amplified in approximately 3% of all tumors. In some tumors, such as those affecting the breast and upper gastrointestinal tract, the frequency of HER2 amplification reaches approximately 15%. Approximately one-third of these tumors also express a truncated form of HER2 known as p95HER2. While HER2 is also expressed in normal epithelium, albeit at much lower levels than in HER2-amplified tumors, p95HER2 is a bona fide tumor-specific antigen. Consistently, we have previously shown that specific TCBs effectively redirect lymphocytes to p95HER2-positive breast tumors, but not to target cells expressing normal levels of HER2.

[0007] Several second generation CARs targeting p95HER2 have been developed. Although these CARs have been shown to be highly effective against cell lines in vitro and in vivo, their clinical effectiveness has been limited by their partial efficacy against patient-derived tumor xenografts. Thus, given the limitations of current cell therapies, alternative therapies of CARs or bispecific T cell engagers that exploit the specificity of p95HER2 targeting and the broader efficacy of HER2 targeting are needed. Summary of the Invention

[0008] The present invention discloses an armored CAR immune cell targeting p95HER2, which is further modified to secrete a bispecific T cell engager (BiTE) that recruits immune cells targeting HER2 to enhance its therapeutic antitumor effect. This p95HER2 CAR secreting a HER2 bispecific T cell engager is expected to combine the specificity, and therefore safety, of targeting p95HER2 with the efficacy of targeting HER2 locally.

[0009] In other words, the present invention utilizes a tumor-specific antigen (p95HER2) that is heterogeneously expressed in tumors to safely induce local targeting of a more abundant antigen (HER2) that is also expressed in normal tissues. The present invention significantly increases the antitumor efficacy of previously described second generation p95HER2 CAR immune cells, potentially avoiding the extratumoral side effects typical of HER2-targeted therapy.

[0010] Thus, a first aspect of the present invention is an immune cell comprising: (i) an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A chimeric antigen receptor comprising (ii) a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 and a bispecific antibody comprising are immune cells that express

[0011] A further aspect of the present invention is (i) a first region encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A first region including: (ii) a second region encoding a bispecific antibody, the bispecific antibody comprising: a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 A second region including or (i) a first polynucleotide encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A first polynucleotide comprising: (ii) a second polynucleotide encoding a bispecific antibody, the bispecific antibody comprising: a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 and a second polynucleotide comprising The present invention relates to a nucleic acid composition comprising:

[0012] Another aspect of the invention relates to a vector composition comprising a vector containing a nucleic acid construct according to the invention or a vector wherein the first and second nucleic acids of the composition are found in different vectors.

[0013] Yet another aspect of the present invention relates to a polypeptide encoded by a nucleic acid construct according to the present invention.

[0014] Another aspect is (i) an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A chimeric antigen receptor comprising (ii) a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 and a bispecific antibody comprising A method for obtaining a cell expressing The present invention relates to a method comprising inserting into a cell a nucleic acid construct or a nucleic acid composition according to the invention, or a vector or vector composition according to the invention. [Brief description of the drawings]

[0015] [Figure 1] Effect of p95HER2 CAR T on PDX. Two patient-derived tumor xenografts (PDTX), negative (A) and positive (B) for p95HER2, were orthotopically injected into NSG mice. At the time points indicated by the arrows, 3x106 T cells expressing H1-14 p95HER2 CAR were injected intravenously. Tumor volumes are expressed as mean ± SD (n=6 per arm). UTD, non-targeting CAR control. [Diagram 2] Schematic diagram of three constructs described in this disclosure: (A) h1-214 p95HER2 CAR or "CAR", (B) HER2-CD3 T cell engager or "TECH2", and (C) p95HER2.CAR-TECH2 or "CAR-TECH2". Abbreviations used in the diagram: scFv = single-chain variable fragment, VL = light chain, VH = heavy chain, LS = leader sequence, TM = transmembrane domain, 6xHis = peptide tag consisting of six histidine repeats, T2A = self-cleaving peptide. [Diagram 3] Design and in vitro characterization of HER2-CD3 T cell engagers (TECH2) with different binding affinities to HER2. (A) Simplified scheme of retroviral vectors shown. (B) Binding affinity of scFvs to HER2 as described in (Liu et al., 2015). (C) Comparison of different affinity-tuned scFvs in TECH2 format from the supernatant of HEK 293T producer cells by flow cytometry for both HER2 and CD3. (D) Wild-type MCF7 cells expressing normal levels of HER2 (left), and a PDX-derived cell line overexpressing both HER2 and p95HER2 (right) were co-cultured with transduced T cells for 48 hours to secrete TECH2 with different affinities. Viable target cells were then quantified by flow cytometry using EpCAM as a marker. (E) The same target cells were co-cultured with p95HER2 CAR T secreting high or medium affinity TECH2 for 48 hours and analyzed as in D. [Figure 4]In vivo characterization of CAR-TECH2 Me and Hi. NSG mice were orthotopically injected with MCF7 wild-type cells expressing two different PDTX fragments with (A)(C) normal levels of HER2, or (B)(D) heterogeneous p95HER2 / HER2 expression. At the time points indicated by the arrows, 3x106 T cells expressing the indicated CAR, TECH2 or CAR-TECH2 constructs were injected intravenously. Tumor volumes are presented as mean±SD (n=3-8 per arm). p values ​​are shown for the last time point before sacrificing control mice. UTD, non-targeting T cell control. [Diagram 5] In vitro characterization of p95HER2.CAR-TECH2 NK cells (CAR-TECH2 NK). (A) Transduction efficiency of NK cells 4 days after transduction with the retroviral vectors shown in Figure 3A; the percentage of positive CAR from total NK cells is shown. (B) MCF7 cells expressing an empty vector or the same vector expressing p95HER2 (Parra-Palau et al., 2014) were co-cultured with different ratios of NK cells secreting p95HER2 CAR-TECH2 or non-transduced NK cells for 24 hours. Viable target cells were then quantified by flow cytometry using EpCAM as an epithelial cell marker. Results are presented as the average of three technical replicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Considering the limitations of current cell therapies for the treatment of tumors, the present invention relates to fourth generation CAR immune cells expressing bispecific T cell engagers to exploit the specificity of tumor-specific antigens and combine it with the efficacy of targeting tumor-associated antigens that are uniformly expressed at higher levels in tumor cells. This work discloses immune cells expressing a chimeric antigen receptor specific for p95HER2 and a bispecific antibody targeting HER2 and CD3.

[0017] Unless otherwise defined, all technical terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] All embodiments and definitions disclosed in the context of one aspect of the invention are also applicable to the other aspects of the invention.

[0019] Immune cells according to the present invention Thus, a first aspect of the present invention is an immune cell comprising: (i) an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A chimeric antigen receptor (CAR) comprising (ii) a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 and a bispecific antibody comprising The present invention relates to an immune cell expressing the above-mentioned gene (hereinafter referred to as "an immune cell according to the present invention").

[0020] As used in this disclosure, "immune cell" refers to a cell that plays a role in immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cells are T cells; NK cells; NKT cells; lymphocytes, such as B cells and T cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0021] In certain embodiments of the methods of the invention, the immune cell is a T cell or a natural killer (NK) cell.

[0022] As used in this disclosure, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B lymphocytes, by the presence of T cell receptors on the cell surface. T cells can be isolated or obtained from commercial sources. T cells are of any type that express CD3 and include helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses. The terms "T cell" and "T lymphocyte" are interchangeable and are used interchangeably herein.

[0023] Natural killer cells or "NK cells" are well known in the art. In one embodiment, natural killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG cells, YT cells, NK-YS cells, HANK-1 cells, YTS cells, and NKL cells. NK cells can be detected by specific surface markers in humans, such as CD16, CD56, and CD8. NK cells do not express T cell antigen receptors, the pan-T marker CD3, or surface immunoglobulin B cell receptors.

[0024] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. As such, NKT cells are a subset of T cells that co-express the αβ T cell receptor but also express various molecular markers typically associated with NK cells, such as NK1. Most of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They account for only approximately 0.1% of all peripheral blood T cells. Natural killer T cells should not be confused with natural killer cells.

[0025] Immune cell-chimeric receptor according to the present invention As used in this disclosure, "chimeric antigen receptor" or "CAR", also known as chimeric T cell receptor, T body, artificial T cell receptor and chimeric immune receptor (CIR), is an engineered receptor that grafts any specificity onto immune effector cells. In classical CARs, the specificity of a monoclonal antibody is grafted onto a T cell. Thus, a CAR is a fusion protein that includes at least an extracellular domain or antigen-binding domain that can bind to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular costimulatory domain.

[0026] According to the present invention, the expressions "extracellular domain", "antigen-binding domain", "antigen-binding region", "antigen-binding fragment" or "antibody fragment" are used interchangeably and refer to any oligopeptide or polypeptide capable of binding to a specific antigen. In the present invention, said antigen may be p95HER2, HER2 or CD3, i.e. the antigen-binding domain or antigen-binding region is specific for p95HER2, HER2 or CD3.

[0027] The antigen-binding domain may comprise an antibody fragment, which refers to at least a portion of an intact antibody or a recombinant variant thereof, such as the antigen variable region of an intact antibody, sufficient to allow recognition and specific binding of the antibody fragment to a target. The antigen-binding domain of the present invention comprises at least a VH region and a VL region. Examples of antibody fragments include, but are not limited to, Fab, Fab'-, F(ab')2 and Fv fragments, ScFv antibody fragments, and linear antibodies. Within the context of the present invention, an antigen-binding domain or antibody fragment comprises at least one VH and one VL region, but it may comprise two VL regions and two VH regions. Thus, for example, in a particular embodiment, the antigen-binding domain specific for p95HER2 of the CAR expressed by the immune cell according to the present invention is an ScFv, and therefore it comprises only one VL and one VH region. In another embodiment, the antigen-binding domain specific for p95HER2 of the CAR expressed by an immune cell according to the invention is a Fab fragment, in which case it comprises one VL and VH region (Fab or Fab') or two VH and two VL regions (Fab2, or F(ab')2).

[0028] In certain embodiments, the antigen-binding domain is humanized.

[0029] As used in this disclosure, a "humanized" form of a non-human (e.g., murine) antibody or antigen-binding domain is a chimeric antibody or antigen-binding domain that contains minimal or no sequence derived from a non-human immunoglobulin. For the most part, a humanized antibody or antigen-binding domain is a human immunoglobulin (recipient antibody) in which residues from a recipient hypervariable region are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody or antigen-binding domain may comprise residues that are not found in the recipient antibody or donor antibody. These modifications are generally made to further refine the performance of the antibody or antigen-binding domain. Generally, a humanized antibody or antigen-binding domain will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0030] The terms "HER2" and "HER2 receptor" are used interchangeably herein and refer to ErbB2 protein (also referred to in the literature as HER2 / neu). As used in this disclosure, the term is intended to include variants (e.g., splice variants), isoforms, and homologs (both orthologs and paralogs) of HER2. In some aspects, binding of the anti-HER2 binding molecules disclosed herein to HER2 inhibits the growth of cells expressing HER2 (i.e., typically tumor cells, particularly cancer cells expressing high levels of HER2) by inhibiting the formation of heterodimeric complexes between HER2 and other ErbB family members, for example, by inhibiting heterodimerization with EGFR or HER3.

[0031] HER2 is a receptor tyrosine kinase, composed of an extracellular domain (ECD), which consists of (i) two leucine-rich domains (domain I / L1 and domain III / L2) responsible for ligand binding, and (ii) two cysteine-rich domains (domain II / CR1 and domain IV / CR2) responsible for receptor dimerization; a transmembrane domain; and an intracellular tyrosine kinase domain. Alternative splice variants of HER2 exist and may also be part of the present invention. HER2 is characterized by a sequence according to the database entry of Uniprot, accession number P04626, version of September 29, 2021, composed of 1,255 amino acids.

[0032] The antigen-binding domain of the CAR expressed by the immune cells according to the present invention specifically recognizes the amino-terminal fragment of p95HER2 located outside the cell. In particular, the antigen-binding domain targets the p95HER2 amino acid sequence PIWKFPDE (SEQ ID NO: 5) of human p95HER2, located 34 amino acids from the transmembrane domain that anchors p95HER2 to the membrane.

[0033] The term "p95HER2" as used in this disclosure refers to a polypeptide resulting from a deletion of the N-terminal region of the HER2 receptor protein. As a result, the polypeptide is the carboxy-terminal fragment (CTF) of the HER2 receptor protein, which is also known as "611-CTF" or "p95HER2 100-115 kDa." p95HER2 is characterized by an amino acid sequence corresponding to amino acids 611 to 1,255 of HER2 according to SEQ ID NO:11.

[0034] SEQ ID NO:11 MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDE AYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELTFGAKPYDGIPAREIP DLLEKGERLPQPPICTIDVYMIMVCWIMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMG AAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV

[0035] In a particular embodiment, the antigen-binding domain specific for p95HER2 of the CAR expressed by the immune cell according to the invention is an ScFv.

[0036] As used in this disclosure, "single-chain variable fragment (ScFv)" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA techniques in which the variable (Fv) regions of immunoglobulin heavy (VH) and light (VL) chain fragments are linked via a spacer sequence. Various methods for preparing ScFv are known, including those described in U.S. Pat. No. 4,694,778, Nature, vol. 334, pp. 54454 (1989), and Science, vol. 242, pp. 1038-1041 (1988). In another specific embodiment, the antigen-binding domain specific for p95HER2 of the CAR expressed by the immune cell according to the present invention is an ScFv having a VL region at the N-terminus and a VH region at the C-terminus. In a specific embodiment, the antigen-binding domain specific for p95HER2 of the CAR expressed by an immune cell according to the invention is an ScFv having a VH region at the N-terminus and a VL region at the C-terminus. In another specific embodiment, the antigen-binding domain specific for p95HER2 of the CAR expressed by an immune cell according to the invention is an ScFv having a VL region at the N-terminus and a VH region at the C-terminus.

[0037] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) or complementarity determining regions (CDR). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0038] The terms "CDR", "hypervariable region", "HVR", "complementarity determining region" as used in this disclosure refer to each of the regions of an antibody variable domain that are hypervariable in sequence and / or derived from structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies contain six CDRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). The CDRs therefore determine the affinity (roughly speaking, the strength of binding) of the protein and its specificity for a particular antigen. The CDRs of the two chains of each pair are aligned by the framework regions and acquire the function of binding to a specific epitope. Hence, both the heavy and light variable chains are characterized by three CDRs, respectively VH-CDR1, VH-CDR2, VH-CDR3 and VL-CDR1, VL-CDR2, VL-CDR3.

[0039] As used in this disclosure, the term "functionally equivalent variant of a CDR sequence" refers to a sequence variant of a particular CDR sequence that has substantially similar sequence identity thereto and that substantially retains its ability to bind to its cognate antigen when part of an antibody, antibody fragment or antigen-binding domain, such as an ScFv, as described herein. For example, a functionally equivalent variant of a CDR sequence may be a polypeptide sequence derivative of said sequence that includes the addition, deletion or substitution of one or more amino acids. In one embodiment, the substitution of one amino acid by another amino acid in a functionally equivalent variant is a conservative substitution.

[0040] In certain embodiments, CDR1, CDR2 and CDR3 of the VH region of the ScFv of a CAR expressed by an immune cell according to the present invention comprise the sequences of SEQ ID NOs: 39, 40 and 41, respectively, or functionally equivalent variants thereof, and / or CDR1, CDR2 and CDR3 of the VL region of the ScFv of a CAR expressed by an immune cell according to the present invention comprise the sequences of SEQ ID NOs: 42, 43 and 44, respectively, or functionally equivalent variants thereof. [Table 1]

[0041] As used in this disclosure, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid that has similar chemical properties. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0042] Functionally equivalent variants of CDRs according to the present invention include CDR sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding amino acid sequence shown in one of the above reference sequences. It is also contemplated that functionally equivalent variants of CDR sequences include additions of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids, or more amino acids, at the N-terminus, or at the C-terminus, or at both the N- and C-terminus, of the corresponding amino acid sequence shown in one of the above reference sequences. It is also contemplated that variants include deletions of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids, or more amino acids at the N-terminus, or C-terminus, or at both the N- and C-terminus of the corresponding amino acid sequence shown in one of the above-mentioned sequences.

[0043] Functionally equivalent variants of CDR sequences according to the invention preferably maintain at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200% or more of the ability of the corresponding amino acid sequence shown in one of SEQ ID NOs: 39-44 to bind to its cognate antigen when it is part of an antibody fragment or antigen binding domain, e.g., an ScFv of a CAR expressed by an immune cell according to the invention. This ability to bind to its cognate antigen may be determined as a value of affinity, avidity, specificity and / or selectivity of the antibody or antibody fragment for its cognate antigen.

[0044] The CDR sequences can be determined according to conventional standards, for example according to the IgBLAST standards: http: / / www.ncbi.nlm.nih.gov / igblast / (Ye et al., 2013, Nucleic Acids Res 41 (Web Server Issue: W34-40), by following the numbering provided by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), or by following the numbering provided by Chothia et al. (1989, Nature 342:877-83). This particular region can be determined according to the numbering provided by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Health, Bethesda, Md. (1991) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of an antibody. The CDR sequences provided herein generally follow the Kabat definition.

[0045] The term "identity", "identical" or "percent identity" in the context of two or more amino acid or nucleotide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned (optionally introducing gaps) for maximum correspondence, without considering any conservative substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Publicly available software programs can be used to align sequences. Appropriate parameters for maximum alignment with a particular alignment software can be determined by one of skill in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the identity percentage "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y / Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the second sequence is longer than the first sequence, a global alignment is used that takes into account the entirety of both sequences, and therefore all characters and nulls in each sequence must be aligned. In this case, the same formula as above can be used, except that the Z value is the length of the region where the first and second sequences overlap, said region having a length that is substantially the same as the length of the first sequence.

[0046] For example, 95% identical to a reference sequence according to the present invention, the percentage of identity is calculated over the entire length of the reference nucleotide sequence, with parameters set such that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0047] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0048] As used in this disclosure, the term "functionally equivalent variants of a FR sequence" refers to sequence variants of a particular FR sequence that have substantially similar sequence identity thereto and that, when part of an antibody or antibody binding domain described herein, substantially retain their ability to bind to their cognate antigen. For example, a functionally equivalent variant of a FR sequence may be a polypeptide sequence derivative of said sequence that contains the addition, deletion or substitution of one or more amino acids.

[0049] In certain embodiments, FR1, FR2, FR2 and FR4 of the VH region of a CAR expressed by an immune cell according to the invention comprise the sequences of SEQ ID NOs: 45, 46, 47 and 48, respectively, or functionally equivalent variants thereof, and / or FR1, FR2, FR3 and FR4 of the VL region of a CAR expressed by an immune cell according to the invention comprise the sequences of SEQ ID NOs: 49, 50, 51 and 52, respectively, or functionally equivalent variants thereof. [Table 2]

[0050] Functionally equivalent variants of the FR sequence according to the present invention include FR sequences having at least about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding amino acid sequence shown in one of the above reference sequences. It is also contemplated that functionally equivalent variants of the FR sequence include an addition of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids, or more amino acids, at the N-terminus, or at the C-terminus, or at both the N-terminus and the C-terminus, of the corresponding amino acid sequence shown in one of the above reference sequences. It is also contemplated that variants include deletions of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids, or more amino acids at the N-terminus, or C-terminus, or at both the N- and C-terminus of the corresponding amino acid sequence shown in one of the above-mentioned sequences.

[0051] Functionally equivalent variants of FR sequences according to the invention preferably maintain, when part of an antigen-binding domain of the invention, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200%, or more of the ability of the corresponding amino acid sequence shown in one of SEQ ID NOs: 45-52 to bind to its cognate antigen. This ability to bind to its cognate antigen can be determined as a value of the affinity, avidity, specificity and / or selectivity of the antibody or antibody fragment for its cognate antigen.

[0052] In certain embodiments, the VL of the ScFv of a CAR expressed by an immune cell according to the invention comprises a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 54. In certain embodiments, the VL of the ScFv of a CAR expressed by an immune cell according to the invention comprises the sequence of SEQ ID NO: 54. In certain embodiments, the VL of the ScFv of a CAR ScFv of the invention consists of or consists essentially of the sequence of SEQ ID NO: 54.

[0053] SEQ ID NO:54 DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWFQQKPGKAPKILIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPLTFGQGTKLEIK

[0054] In certain embodiments, the VH of the ScFv of the CAR expressed by an immune cell according to the invention comprises a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 55. In certain embodiments, the VH of the ScFv of the CAR expressed by an immune cell according to the invention comprises the sequence of SEQ ID NO: 55. In certain embodiments, the VH of the ScFv of the CAR expressed by an immune cell according to the invention consists of or essentially consists of the sequence of SEQ ID NO: 55.

[0055] SEQ ID NO:55 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMAWVRQTPDRRLELVATINSNGGKTYHPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREGFDYWGQGTLVTVSS

[0056] In certain embodiments, the VL of the ScFv of a CAR expressed by an immune cell according to the invention comprises a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 54, and the VH of the ScFv of a CAR expressed by an immune cell according to the invention comprises a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 55.

[0057] In a specific embodiment, the VL of the ScFv of the CAR expressed by an immune cell according to the present invention comprises the sequence of SEQ ID NO: 54, and the VH of the ScFv of the CAR expressed by an immune cell according to the present invention comprises the sequence of SEQ ID NO: 55.

[0058] In a specific embodiment, the VL of the ScFv of a CAR expressed by an immune cell according to the invention consists of or essentially consists of the sequence of SEQ ID NO: 54, and the VH of the ScFv of a CAR expressed by an immune cell according to the invention consists of or essentially consists of the sequence of SEQ ID NO: 55.

[0059] In another specific embodiment of the CAR expressed by an immune cell according to the invention, the VH and VL regions of the ScFv of the CAR expressed by an immune cell according to the invention are connected by a linker region.

[0060] The term "flexible polypeptide linker" or "linker" refers to a peptide linker consisting of amino acids such as glycine and / or serine residues used alone or in combination to link together the variable heavy and variable light chain regions or to link any of the CARs or regions thereof expressed by immune cells according to the invention.

[0061] The linker peptide may have any of a variety of amino acid sequences. The proteins may be joined by spacer peptides, typically flexible spacer peptides, although other chemical linkages are not excluded. The linker may be a peptide of about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length. These linkers may be generated by using oligonucleotides that code for synthetic linkers for coupling the proteins. Peptide linkers with a degree of flexibility may be used. The linking peptides may have virtually any amino acid sequence, with the understanding that suitable linkers will generally have sequences that result in flexible peptides. The use of small amino acids, such as glycine and alanine, is of use in creating flexible peptides. Creating such sequences is routine for those skilled in the art.

[0062] Suitable linkers can be readily selected and can be any of different suitable lengths, for example from 1 amino acid (e.g. Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0063] Exemplary flexible linkers include those having the sequence TGSTSGSGKPGSGEGS (SEQ ID NO: 53). Suitable linkers include glycine polymers (G), glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 67) and (GGGS) n(SEQ ID NO:68), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In certain embodiments, the linker comprises a glycine polymer of formula (G4S)3. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and therefore may serve as natural tethers between building blocks. Glycine polymers are of particular interest because glycine has access to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains. Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:69), GGSGG (SEQ ID NO:70), GSGSG (SEQ ID NO:71), GSGGG (SEQ ID NO:72), GGGSG (SEQ ID NO:73), GSSSG (SEQ ID NO:74), and the like. One of skill in the art will recognize that the design of peptides conjugated to any of the elements described above may include linkers that are fully or partially flexible, such that the linker may include not only flexible linkers but also one or more moieties that impart a less flexible structure.

[0064] In a particular embodiment, the linker is located between the VH and VL regions of the ScFv of the CAR expressed by the immune cell according to the invention. In another particular embodiment, the VH and VL regions of the ScFv of the CAR expressed by the immune cell according to the invention are connected by a linker region comprising SEQ ID NO: 53. In one embodiment, the ScFv of the CAR expressed by the immune cell according to the invention comprises the structure VL-linker-VH. In another embodiment, the ScFv of the CAR expressed by the immune cell according to the invention may have the structure VH-linker-VL or VL-linker-VH. In a particular embodiment, the linker is located C-terminal to the VL region and N-terminal to the VH region, i.e., VL-linker-VH.

[0065] In certain embodiments, the ScFv of the CAR expressed by an immune cell according to the present invention comprises a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 56.

[0066] SEQ ID NO:56 DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWFQQKPGKAPKILIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPLTFGQGTKLEIKTGSTSGSGKPG SGEGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMAWVRQTPDRRLELVATINSNGGKTYHPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREGFDYWGQGTLVTVSS

[0067] In another specific embodiment, the ScFv of the CAR expressed by an immune cell according to the invention comprises the sequence of SEQ ID NO: 56. In yet another specific embodiment, the ScFv of the CAR expressed by an immune cell according to the invention consists or consists essentially of the sequence of SEQ ID NO: 56.

[0068] The second element of the CAR expressed by the immune cells according to the present invention is a transmembrane domain that is attached to the extracellular domain of the CAR.

[0069] As used in this disclosure, "transmembrane domain" (TMD) refers to the area of ​​a CAR that crosses the cell membrane. The transmembrane domain of a CAR expressed by an immune cell according to the present invention is a transmembrane domain of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the extracellular region), and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the intracellular region). In one embodiment, the transmembrane domain is one that is associated with one of the other domains of the CAR used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein, e.g., to minimize interaction with other members of the receptor complex. In certain embodiments, the transmembrane domain of the CAR expressed by the immune cell according to the present invention is capable of homodimerization with another CAR on the CAR T cell surface. In different specific embodiments of the CAR expressed by the immune cell according to the present invention, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR T.

[0070] The transmembrane domain may be derived from natural or recombinant sources. If the origin is natural, the domain may be derived from any membrane-associated or transmembrane protein. In one embodiment, the transmembrane domain can transmit a signal to the intracellular domain whenever the CAR binds to the target. Non-limiting examples of transmembrane domains of particular use in the present invention include, for example, the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD3 zeta, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, CTLA4, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4 CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDGA, CDGA, CD103, ITGAL, CDLa, LFA-1, ITGAM, CDllb, ITGAX, CDlc, ITGB1, CD29, ITGB2, CD18, LFA-1, LGA ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT Examples of such a transmembrane domain include at least the transmembrane region of a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A), Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, Kp30, NKp46, e.g., NKG2D, and / or NKG2C.

[0071] In certain embodiments of the CAR expressed by an immune cell according to the invention, the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a 4-1BB transmembrane domain, a CTLA4 transmembrane domain, a CD27 transmembrane domain, and a CD3 zeta transmembrane domain.

[0072] In certain embodiments, the transmembrane domain is a CD28 transmembrane domain. In certain embodiments, the CD28 transmembrane domain comprises the sequence of SEQ ID NO:57. SEQ ID NO:57 FWVLVVVGGVLACYSLLVTVAFIIFWV

[0073] In another specific embodiment of a CAR expressed by an immune cell according to the invention, the transmembrane domain comprises a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO:57.

[0074] In another specific embodiment of the CAR expressed by an immune cell according to the invention, the transmembrane domain comprises the sequence of SEQ ID NO:57.

[0075] In another particular embodiment of the CAR expressed by an immune cell according to the invention, the transmembrane domain consists or consists essentially of the sequence of SEQ ID NO:57.

[0076] The CAR according to the present invention comprises at least one intracellular signaling domain and / or costimulatory domain.

[0077] "Intracellular signaling domain", as the term is used in this disclosure, refers to any oligopeptide or polypeptide known to function as an intracellular portion of a molecule, more specifically, a domain that transmits a signal that causes activation or inhibition of a biological process in a cell. The intracellular signaling domain produces a signal that stimulates the immune effector function of a CAR-containing cell, e.g., a CAR-T cell. The effector function of a T cell can be, for example, a cytolytic function, or a helper activity, including secretion of cytokines. Thus, the intracellular signaling domain can be a portion of a protein that transmits an effector function signal and instructs a cell (e.g., a T cell) to perform a specialized function.

[0078] Generally, the entire intracellular signaling domain can be used, however, it is recognized that it is not necessary to use the entire domain, provided that the portion of the signaling domain used can still transmit the effector function signal. It is also recognized that variants of such intracellular signaling domains having substantially the same or higher functional capabilities can also be used. By this, we include the meaning that the variants should have substantially the same or higher transmission of the effector function signal. Typically, substantially the same or higher signaling includes at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% or more of the signaling of the unmodified intracellular signaling domain, where the signaling of the unmodified intracellular signaling domain corresponds to 100%. Methods for assessing the transmission of effector function signals are well known to those skilled in the art and include, for example, assessing the amount and / or activity of molecules (e.g., proteins such as cytokines) that indicate the signal transmitted. Thus, if the signal is the cytolytic function of the T cells, the method may include measuring one or more cytokines secreted by the T cells that are known to have cytolytic activity (e.g., IFN-gamma). Another means of assessing cytolytic function is by CFSE staining and counting positive cells by flow cytometry or by chromium release assays, as are well known in the art.

[0079] Examples of intracellular signaling domains for use in CARs expressed by immune cells according to the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signaling following antigen receptor engagement, and any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

[0080] It is known that signals generated by the TCR alone are generally insufficient for full activation of T cells, and that secondary and / or costimulatory signals may also be required. Therefore, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation by the TCR (primary intracellular signaling domains), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary intracellular signaling domains, e.g., costimulatory domains). Costimulatory domains promote the activation of effector functions and may also promote the persistence of effector functions and / or cell survival.

[0081] In certain embodiments of a CAR expressed by an immune cell according to the invention, at least one intracellular signaling domain of the CAR comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

[0082] The primary intracellular signaling domain regulates the primary activation of the TCR complex, either in a stimulatory or inhibitory manner. A primary intracellular signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM (e.g., 2, 3, 4, 5, or more ITAMs). Thus, the intracellular signaling domain may comprise one or more ITAMs. It will be appreciated that one or more ITAMs of an intracellular signaling domain may be modified, for example, by mutation. Modifications may be used to increase or decrease the signaling function of the ITAM compared to the native ITAM domain.

[0083] Examples of ITAMs containing primary intracellular signaling domains of particular use in the present invention include those of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a particular embodiment, at least one intracellular signaling domain of the CAR expressed by the immune cells according to the invention is selected from the group consisting of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a more particular embodiment, at least one intracellular signaling domain of the CAR expressed by the immune cells according to the present invention is the CD3 zeta intracellular domain.

[0084] The term "CD3" refers to the human CD3 protein complex, which is composed of six separate chains (the CD3y chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and one CD3 zeta chain homodimer (SwissProt P20963) (εγ:εδ:ζζ), which associates with the α and β chains of the T cell receptor. The term includes any CD3 variants, isoforms and species homologs naturally expressed by cells, including T cells, or expressed in cells transfected with genes or cDNAs encoding the aforementioned chains.

[0085] The term "zeta", or alternatively "zeta chain", "CD3 zeta" or "TCR zeta" is defined as the protein represented by GenBank entry number BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, primate, etc., and "zeta stimulatory domain", or alternatively "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" is defined as the amino acid residues in the cytoplasmic domain of the zeta chain that are sufficient for functional transmission of a primary signal required to activate a T cell, etc. In one embodiment, the zeta cytoplasmic domain comprises residues 52 to 164, inclusive, of GenBank entry protein BAG36664.1, or the equivalent residues from a non-human species that is a functional orthologist thereof, e.g., mouse, rodent, monkey, primate, etc.

[0086] As noted above, the intracellular signaling domain may comprise a primary intracellular signaling domain alone, or may comprise a primary intracellular signaling domain in combination with one or more secondary intracellular signaling domains, such as one or more costimulatory signaling domains. Thus, the intracellular signaling domain of a CAR may comprise a CD3 zeta signaling domain alone or in combination with one or more other intracellular signaling domains, such as one or more costimulatory signaling domains.

[0087] A costimulatory signaling domain refers to the portion of a CAR that contains the intracellular domain of a costimulatory molecule.

[0088] The term "costimulatory molecule" refers to a recognizable T cell binding partner that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response exhibited by a T cell, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule other than an antigen-specific receptor or its ligand that is necessary for an effective immune response. A costimulatory molecule may be a cell surface molecule other than an antigen receptor or its ligand that is necessary for an effective response of an immune cell (e.g., lymphocyte) to an antigen. A costimulatory molecule may be in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), and NK cell activating receptors. Examples of such molecules include, but are not limited to, OX40, ICOS, DAP10, CD27, CD28, CDS, CD30, CD137 (4-1BB), CD40, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, GITR, NKG2C, SLAMF7, NKp80, BAFFR, HVEM, BTLA, ICAM-1, LFA-1 (CD11a / CD18), B7-H3, and ligands that specifically bind to CD83. For example, CD27 costimulation has been demonstrated to enhance human CAR T cell expansion, effector function, and survival in vitro, and to increase human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012;1 19(3):696-706).

[0089] In another embodiment, the at least one intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

[0090] In a particular embodiment, the CAR expressed by the immune cell according to the invention comprises the intracellular domain of the costimulatory molecule CD28. In a more particular embodiment, the intracellular domain of the costimulatory molecule comprises the sequence of SEQ ID NO:58. SEQ ID NO:58 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0091] In a specific embodiment, at least one intracellular signaling domain of a CAR expressed by an immune cell according to the invention further comprises a CD3 zeta intracellular domain. In another specific embodiment, at least one intracellular signaling domain of a CAR expressed by an immune cell according to the invention comprises the sequence of SEQ ID NO:59. SEQ ID NO:59 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0092] In one particular embodiment, at least one intracellular signaling domain of a CAR expressed by an immune cell according to the invention is positioned N-terminal to the CD3 zeta intracellular domain.

[0093] The intracellular signaling sequences within the intracellular portion of the CAR expressed by the immune cell according to the present invention can be linked to each other randomly or in a defined order. Optionally, a short oligopeptide or polypeptide linker, e.g., 2-10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, can form the link between the intracellular signaling sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In another embodiment, a single amino acid, e.g., alanine or glycine, can be used as a suitable linker.

[0094] In one embodiment, the intracellular signaling domain is designed to include two or more, for example, 3, 4, 5, or more, costimulatory signaling domains. In some embodiments, the two or more, for example, 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, for example, as described herein. In one embodiment, the intracellular signaling domain includes two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0095] In a preferred embodiment, the intracellular portion of the CAR expressed by an immune cell according to the invention comprises: - the signaling domain of CD3 zeta and the signaling domain of CD28, - the signaling domain of CD3 zeta and the signaling domain of 4-1BB, - the signaling domain of CD3 zeta and the signaling domain of OX40, - the signaling domain of CD3 zeta and the signaling domain of ICOS, - the signaling domain of CD3 zeta and the signaling domain of DAP10, - the signaling domain of CD3-zeta, the signaling domain of 4-1BB, and the signaling domain of OX40; - the signaling domain of 4-1BB, and the signaling domain of CD28.

[0096] The intracellular signaling domain can include the entire intracellular portion, or the entire naturally occurring intracellular signaling domain, the molecule from which it originates, or a functional fragment thereof.

[0097] In another embodiment, the CAR expressed by the immune cell according to the present invention further comprises a hinge domain between the antigen binding domain and the transmembrane domain.

[0098] As used in this disclosure, "hinge domain", "hinge region" or "spacer" refers to an amino acid region that allows for separation and flexibility of the binding moiety and the T cell membrane. The length of a flexible hinge also allows for better binding to relatively inaccessible epitopes, e.g., a longer hinge domain allows for optimal binding. One skilled in the art can determine the appropriate hinge for a given CAR target.

[0099] In some cases, the first polypeptide of the CAR expressed by the immune cell according to the present invention comprises a hinge domain, wherein the hinge domain is between the antigen-binding domain and the transmembrane domain.In some cases, the hinge domain is an immunoglobulin heavy chain hinge domain.In some cases, the hinge domain is a domain region polypeptide derived from a receptor (e.g., a CD8-derived hinge domain).

[0100] The hinge domain may have a length of about 10 to about 200 amino acids, preferably 50 to 150 amino acids, and more preferably 75 to 125 amino acids.

[0101] Exemplary hinge domains include glycine polymers (G), glycine-serine polymers (including, for example, (GS), (GSGGS) (SEQ ID NO: 67) and (GGGS) (SEQ ID NO: 68, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used, where both Gly and Ser are relatively unstructured and thus can act as natural tethers between components. Glycine polymers can be used, where glycine has great access to much more phi-psi space compared to alanine and is much less restricted than residues with longer side chains. Exemplary spacers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 69), GGSGG (SEQ ID NO: 70), GSGSG (SEQ ID NO: 701), GSGGG (SEQ ID NO: 712), GGGSG (SEQ ID NO: 73), GSSSG (SEQ ID NO: 734), and the like.

[0102] In some cases, the hinge domain in the first polypeptide of the CAR expressed by the immune cell according to the present invention comprises at least one cysteine. For example, in some cases, the hinge domain may comprise the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 75). If present, the cysteine ​​in the hinge domain of the first CAR may be available to form a disulfide bond with the hinge domain in the second CAR.

[0103] Immunoglobulin hinge domain amino acid sequences are known in the art, see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, an immunoglobulin hinge domain may comprise one of the following amino acid sequences: DKTHT (SEQ ID NO: 76); CPPC (SEQ ID NO: 75); CPEPKSCDTPPPCPR (SEQ ID NO: 77) (see, e.g., Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHHT (SEQ ID NO: 78); KSCDKTHTCP (SEQ ID NO: 79); KCCVDCP (SEQ ID NO: 80); KYGPPCP (SEQ ID NO: 81); EPKSCDKTHTCPPCP (SEQ ID NO: 82) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 83) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 84) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 85) (human IgG4 hinge), and the like.

[0104] The hinge domain may comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge domain. The hinge domain may comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge domain. For example, His229 of the human IgG1 hinge may be replaced with Tyr, such that the hinge domain comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 86), see, for example, Yan et al. (2012) J. Biol. Chem. 287:5891.

[0105] The hinge domain may comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0106] In a specific embodiment, the hinge domain comprises, consists of, or consists essentially of a CD8 hinge domain.

[0107] In another specific embodiment, the hinge domain comprises the sequence of SEQ ID NO:60. SEQ ID NO:60 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0108] In a specific embodiment, the hinge domain is a CD8 hinge domain, the transmembrane domain is a CD28 transmembrane domain, and the intracellular signaling domain is a CD28 costimulatory domain.

[0109] In certain embodiments, the CAR expressed by the immune cells according to the invention comprises a CD8 hinge domain, a CD28 transmembrane domain, and a CD3 zeta intracellular signaling domain and a CD28 costimulatory domain.

[0110] In another embodiment, the CAR expressed by an immune cell according to the invention comprises, from N-terminus to C-terminus, an anti-p95HER2 light chain variable domain, a linker domain, an anti-p95HER2 heavy chain variable domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, followed by a CD3 zeta intracellular signaling domain.

[0111] In a particular embodiment, the CAR expressed by the immune cell according to the invention comprises SEQ ID NO:61. SEQ ID NO:61 DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWFQQKPGKAPKILIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPLTFGQGTKLEIKTGSTSGSG KPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMAWVRQTPDRRLELVATINSNGGKTYHPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREGFDYWGQGT LVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0112] Immune cell-bispecific antibodies according to the present invention In addition to the CAR, the immune cells according to the invention further express a bispecific antibody that specifically binds to HER2 and CD3.

[0113] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with an antigen. The constant region of an antibody (Ab) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system. As indicated above, the term antibody in this specification includes fragments of antibodies that are antigen-binding fragments as previously defined, unless otherwise stated or clearly contradicted by the context. It should also be understood that the term antibody, unless otherwise specified, also includes polyclonal antibodies, monoclonal antibodies (mAb), antibody-like polypeptides, such as chimeric and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen provided by any known technique.

[0114] The term "bispecific antibody", in the context of the present invention, refers to an antibody that has two different antigen-binding regions that are defined by different antibody sequences.

[0115] In a particular embodiment of the bispecific antibody expressed by an immune cell according to the invention, the first and second antigen-binding regions of the bispecific antibody are ScFvs.

[0116] In another particular embodiment, the bispecific antibody expressed by the immune cell according to the invention is characterized in that: - the VH region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, or functional equivalents thereof; - the VL region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 4, SEQ ID NO: 100 and SEQ ID NO: 6, respectively, or functional equivalents thereof; - the VH region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, or functional equivalents thereof; and / or the VL region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12, respectively, or functional equivalents thereof.

[0117] This antibody is characterized by exhibiting high affinity for HER2 and is hereinafter referred to as a "high Aff." antibody. In a particular embodiment of the bispecific antibody, the ScFv of the high Aff. antibody has a dissociation constant (K D ).

[0118] In another particular embodiment, the bispecific antibody expressed by the immune cell according to the invention is characterized in that: - the VH region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:102, respectively, or functional equivalents thereof; - the VL region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 4, SEQ ID NO: 103 and SEQ ID NO: 6, respectively, or functional equivalents thereof; - the VH region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, or functional equivalents thereof; and / or the VL region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12, respectively, or functional equivalents thereof.

[0119] This antibody is characterized by exhibiting moderate affinity for HER2 and is hereinafter referred to as the "Medium Aff." antibody. In a specific embodiment of the bispecific antibody, the ScFv of the medium Aff. antibody has a dissociation constant (K D ).

[0120] [Table 3]

[0121] As used in this disclosure, the term "binding" refers in the context of the binding of an antibody / ScFv / FV / CDR to a given antigen or epitope, typically at or above about 10 as determined by surface plasmon resonance (SPR) techniques in a BIAcore 3000 instrument using, for example, an antigen as the ligand and an antibody as the analyte. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11The antibody binds with an affinity, or "binding affinity", corresponding to a KD of at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, such as at least 100,000 times lower, than its affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g. BSA, casein). The amount that affinity is reduced depends on the antibody's KD, such that if the antibody's KD is very low (i.e. the antibody is very specific), the amount that affinity for the antigen is reduced below affinity for a non-specific antigen can be at least 10,000 times.

[0122] The above mentioned methods for determining the binding affinity of antibodies / ScFvs / FVs / CDRs can also be used to compare binding affinities between analytes. In another particular embodiment of the bispecific antibodies expressed by immune cells according to the invention, the bispecific antibody-in-Aff of the immune cells according to the invention has a binding affinity at least 2-fold lower, at least 3-fold lower, at least 4-fold lower, at least 5-fold lower than the bispecific antibody-high Aff of the immune cells according to the invention.

[0123] In further particular embodiments of the bispecific antibody expressed by an immune cell according to the invention, - the VH region of the ScFv of the first antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or functional equivalents thereof; - the VL region of the ScFv of the first antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, respectively, or a functional equivalent thereof; - the VH region of the ScFv of the second antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequences of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, or functional equivalents thereof; and / or the VL region of the ScFv of the second antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, respectively, or a functional equivalent thereof.

[0124] [Table 4]

[0125] In certain embodiments of the bispecific antibodies expressed by immune cells according to the invention, - the VH region of the ScFv of the first antigen-binding region comprises a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 29 or SEQ ID NO: 104, - the VL region of the ScFv of the first antigen-binding region comprises a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 30 or SEQ ID NO: 105, - the VH region of the ScFv of the second antigen-binding region comprises a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 31, and / or - the VL region of the ScFv of the second antigen-binding region comprises a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 32.

[0126] SEQ ID NO:29 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS

[0127] SEQ ID NO:104 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFVAMDVWGQGTLVTVSS

[0128] SEQ ID NO:30 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0129] SEQ ID NO:105 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0130] SEQ ID NO:31 DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE

[0131] SEQ ID NO:32 DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0132] In certain embodiments of the bispecific antibodies expressed by immune cells according to the invention, - the VH region of the ScFv of the first antigen-binding region comprises the sequence of SEQ ID NO: 29 or SEQ ID NO: 104, - the VL region of the ScFv of the first antigen-binding region comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 105, - the VH region of the ScFv of the second antigen-binding domain comprises the sequence of SEQ ID NO: 31, and / or the VL region of the ScFv of the second antigen-binding region comprises the sequence SEQ ID NO: 32.

[0133] In certain embodiments of the bispecific antibodies expressed by immune cells according to the invention, - the VH region of the ScFv of the first antigen-binding region consists or essentially consists of the sequence of SEQ ID NO: 29 or SEQ ID NO: 104, - the VL region of the ScFv of the first antigen-binding region consists or essentially consists of the sequence of SEQ ID NO: 30 or SEQ ID NO: 105, - the VH region of the ScFv of the second antigen-binding region consists or essentially consists of the sequence of SEQ ID NO: 31, and / or the VL region of the ScFv of the second antigen-binding region consists or essentially consists of the sequence of SEQ ID NO: 32.

[0134] It will be clear to the skilled artisan that the organization of the ScFv domains of the first and second antigen binding regions can be altered without affecting their function. Thus, in a particular embodiment, a bispecific antibody expressed by an immune cell according to the invention has the following arrangement of regions: - VL(HER2)-VH(HER2)-VH(CD3)-VL(CD3); - VL(HER2)-VH(HER2)-VL(CD3)-VH(CD3); - VH(HER2)-VL(HER2)-VH(CD3)-VL(CD3); - VH(HER2)-VL(HER2)-VL(CD3)-VH(CD3); - VL(CD3)-VH(CD3)-VH(HER2)-VL(HER2); - VL(CD3)-VH(CD3)-VL(HER2)-VH(HER2); - VH(CD3)-VL(CD3)-VH(HER2)-VL(HER2); or - VH(CD3)-VL(CD3)-VL(HER2)-VH(HER2).

[0135] Here, VH(HER2) is the VH region of the ScFv of an anti-HER2 antibody, VL(HER2) is the VL region of the ScFv of an anti-HER2 antibody, VH(CD3) is the VH region of the ScFv of an anti-CD3 antibody, and VL(CD3) is the VL region of the ScFv of an anti-CD3 antibody.

[0136] In certain embodiments of the bispecific antibodies expressed by immune cells according to the invention, the VH and VL regions of the ScFv of the first antigen-binding domain, the VH and VL regions of the ScFv of the second antigen-binding domain and / or the ScFvs of the first and second antigen-binding domains are connected by a peptide linker.

[0137] In another particular embodiment of the bispecific antibody expressed by an immune cell according to the invention, the linker is selected from SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35. SEQ ID NO:33 GGGGSGGGGSGGGGS SEQ ID NO:34: GGGGS SEQ ID NO:35 GGSGGSGGSGGSGGVD

[0138] In another embodiment of the bispecific antibody expressed by an immune cell according to the invention, - the ScFv of the first antigen-binding region comprises a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 36 or SEQ ID NO: 106, - the ScFv of the second antigen-binding region comprises a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence of SEQ ID NO: 37.

[0139] SEQ ID NO:36 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS

[0140] SEQ ID NO:37 DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0141] SEQ ID NO:106 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFVAMDVWGQGTLVTVSS

[0142] In another particular embodiment of the bispecific antibody expressed by an immune cell according to the invention, - the ScFv of the first antigen-binding region comprises the sequence of SEQ ID NO: 36 or SEQ ID NO: 106, the ScFv of the second antigen-binding region comprises the sequence SEQ ID NO: 37.

[0143] In another particular embodiment of the bispecific antibody expressed by an immune cell according to the invention, - the ScFv of the first antigen-binding region consists or essentially consists of the sequence of SEQ ID NO: 36 or SEQ ID NO: 106, the ScFv of the second antigen-binding region consists or essentially consists of the sequence of SEQ ID NO: 37.

[0144] In yet another particular embodiment, the bispecific antibody expressed by the immune cell according to the invention has the sequence of SEQ ID NO: 38 or SEQ ID NO: 107.

[0145] SEQ ID NO:38 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSGG GGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSV EGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0146] SEQ ID NO:107 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFVAMDVWGQGTLVTVSSGG GGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSV EGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0147] Uses of immune cells according to the present invention The immune cells according to the invention are used in medicine, in particular in the treatment of cancer. Thus, an aspect of the invention is an immune cell according to the invention for use in medicine.

[0148] Another aspect of the invention is an immune cell according to the invention for use in a method for preventing or treating cancer.

[0149] As used in this disclosure, the terms "treat", "treatment", "treatment" or "amelioration". This term refers to therapeutic treatment, the purpose of which is to reverse, reduce, inhibit, slow or stop the progression or severity of a condition associated with a disease or disorder. The term "treatment" includes reducing or alleviating at least one adverse effect or condition of a condition, disease or disorder, such as cancer. Treatment is usually "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if disease progression is delayed or halted. That is, "treatment" includes not only improvement of symptoms or markers, but also at least prevention of conditions that indicate progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes are reduction of one or more symptoms, whether detectable or not, reduction in the extent of disease, stable (i.e., non-worsening) state of disease. These include, but are not limited to, delay or slowdown of progression, remission or relief of disease state, and remission (partial or total). The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative care). In some embodiments, treating cancer includes reducing tumor volume, reducing the number of cancer cells, inhibiting cancer metastasis, extending life span, reducing cancer cell growth, reducing cell survival, or reducing the cancerous condition. This includes amelioration of various physiological symptoms involved.

[0150] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof, for example, an individual with cancer. The cells then boost the individual's immune system to attack the respective cancer cells. In some cases, an individual is provided with one or more doses of immune cells. In the case where an individual is provided with two or more doses of immune cells, the period between administrations must be sufficient to allow time for propagation in the individual, and in specific embodiments, the period between doses is 1, 2, 3, 4, 5, 6, 7 days or longer.

[0151] In certain embodiments, a growth factor that promotes the growth and activation of immune cells is administered to the subject either simultaneously with the immune cells or after the immune cells. The immune cell growth factor can be any suitable growth factor that promotes the growth and activation of immune cells. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.

[0152] A therapeutically effective amount of the immune cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection or infusion.

[0153] The immune cell population can be administered in a treatment regimen consistent with the disease, such as a single or several doses over one to several days to ameliorate the disease state, or regular doses over a longer period to inhibit disease progression and prevent disease recurrence. The exact dose employed in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and must be determined according to the judgment of the practitioner and each patient's circumstances. The therapeutically effective number of immune cells will depend on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the therapeutically effective number of immune cells can vary from about 5×106 cells / kg body weight to about 7.5×108 cells / kg body weight, such as from about 2×107 cells / kg body weight to about 5×108 cells / kg body weight, or from about 5×107 cells / kg body weight to about 2×108 cells / kg body weight. The exact number of immune cells can be readily determined by one of skill in the art based on the age, weight, sex, and physiological condition of the subject. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0154] In certain embodiments, the compositions and methods of the present invention include the immune cell population in combination with at least one additional therapy, which may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of an adjuvant or neoadjuvant therapy.

[0155] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0156] Various combinations may be used. For the following examples, the pharmaceutical composition or immune cell therapy of the present invention is "A" and the anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0157] Administration of any compound or therapy of the present embodiments to a patient follows general protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0158] A wide variety of chemotherapeutic agents can be used in combination with the pharmaceutical composition or immune cell therapy of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to include compounds or compositions administered in the treatment of cancer. These agents or drugs are classified according to their mechanism of activity within cells, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to induce chromosomal and mitotic abnormalities by directly crosslinking DNA, intercalating into DNA, or affecting nucleic acid synthesis.

[0159] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelanamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic acetogens, such as acetogens, including analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongiostatin; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoenbiquine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma ll and calicheamicin omega ll); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil; uracil) (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal (adrenals) such as mitotane and trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone;Mopidamole; nitracrine; pentostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitra Ctol;pipobroman;gacitosine;arabinoside ("Ara-C");cyclophosphamide;taxoids such as paclitaxel and docetaxel gemcitabine;6-thioguanine;mercaptopurine;platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin;vinblastine;platinum;etoposide (VP-16);ifosfamide;mitoxantrone;vincristine;vinorelbine;novantrone;teniposide;edatrexate;daunomycin;aminopterin;xeloda;ibandronate;irinotecan (e.g. CPT-11);topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above;

[0160] Also included within the definition of "chemotherapeutic agent" are: (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (NOLVADEX®; including tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY 1 17018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestane, fadrozole, R1 (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue); (iv) protein kinase inhibitors, such as ME inhibitors (WO 2007 / 044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf and H-Ras, e.g. oblimersen (GENASENSE®, Genta Inc.);(vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines, such as gene therapy vaccines, such as ALLOVECTIN®, LEUVECTIN®, and VAX1 D®;PROLEUKIN® rlL-2; topoisomerase 1 inhibitors, such as LURTOTECAN®;ABARELIX® rmRH;(ix) antiangiogenic agents, such as bevacizumab (AVASTIN®, Genentech); and pharma- ceutically acceptable salts, acids, and derivatives of any of the above.

[0161] Protein kinase inhibitors include tyrosine kinase inhibitors that inhibit to some extent the tyrosine kinase activity of tyrosine kinases such as ErbB receptors. Examples of tyrosine kinase inhibitors include EGFR-10 targeted agents, such as: (i) MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see US4943533, Mendelsohn et al.) and variants thereof, such as chimeric 225 (C225 or cetuximab; ERBITUX®, Imclone) and reshaped human 225 (H225) (WO96 / 40210, Imclone Systems, Inc.). (ii) antibodies that bind to EGFR, including antibodies that bind to Π-mutant EGFR (US5212290); humanized and chimeric antibodies that bind to EGFR (US5891996); and human antibodies that bind to EGFR, such as ABX-EGF (WO98 / 50433); (ii) anti-EGFR antibodies conjugated to a cyotoxic agent (EP659439A2); and ZD1839 or gefitinib QRESSA™; Astra Zeneca), erlotinib HC1 (CP-358774, TARCEVA™; Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen), small molecules that bind to EGFR, quinazolines such as PD153035, 4-(3-chloroanilino)quinazolines, pyridopyrimidines, pyrimidopyrirnidines, pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-20(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine, curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthaiimide), tyrophostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert) and antisense molecules (such as those that bind to ErbB-encoding nucleic acids).

[0162] In yet another embodiment, the ADC cytotoxic or chemotherapeutic agent of the invention is an antitubulin agent, hi more specific embodiments, the cytotoxic agent is selected from the group consisting of vinca alkaloids, podophyllotoxins, taxanes, baccatin derivatives, cryptophycins, maytansinoids, combretastatins, and dolastatins. In more specific embodiments, the cytotoxic agent is vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, episilon A, episilon B, nocodazole, colchicine, cortisimide, estramustine, semadotine, discodermolide, maytansine DM-1, an auristatin or other dolastatin derivative, such as auristatin E or auristatin F, AEB, AEVB, AEFP, MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), eruterobin, or netropsin.

[0163] In addition, potent chemotherapeutic agents such as CC-1065 analogs, calikiamycin, maytansine, dolastatin 10 analogs, lysoxin, and palytoxin can be linked to ADCs using coordinatively stable linkers to form potent immunoconjugates.

[0164] In certain embodiments, the chemotherapeutic agent is selected from lapatinib, tucatinib, and neratinib.

[0165] The term "cancer" or "tumor" or "tumor disease" as used in this disclosure refers to a broad group of diseases involving unregulated cell growth, also referred to as malignant neoplasms. This term is usually applied to diseases characterized by uncontrolled cell division (or by increased survival or apoptosis resistance) and the ability of the cells to invade other adjacent tissues (invasion) and spread through lymphatic and blood vessels to other areas of the body where the cells are not normally located (metastasis), circulate through the bloodstream, and then invade normal tissues elsewhere in the body. Depending on whether they can spread by invasion and metastasis, tumors are classified as either benign or malignant, benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they only grow locally, while malignant tumors are tumors that can spread by invasion and metastasis. Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration, and metastasis. Cancers usually share some of the following characteristics: sustaining proliferative signaling, evading growth inhibitors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and ultimately metastasis. Cancers can invade nearby parts of the body and spread through the lymphatic system or bloodstream to more distant parts of the body. Cancers are classified by the type of cell that the tumor cells resemble, which is therefore presumed to be the tumor's origin.

[0166] Examples of cancers or tumors include, but are not limited to, tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, rectum, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, hepatobiliary system, and liver. Specific tumors / cancers include adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary carcinoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, teratoma, acral lentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bronchial adenocarcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma sarcoma), focal nodular hyperplasia, germ cell tumor, glucagonoma, hemangioblastoma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasm, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant melanoma, malignant mesothelioma, medulloepithelioma, mucoepidermoid carcinoma, neuroepithelial adenocarcinoma, nodular melanoma, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilms' tumor.

[0167] In certain embodiments, the cancer is a p95HER2-positive cancer.

[0168] The terms "p95HER2" and "HER2" have been previously defined within the context of the CAR of the present invention, and said definitions apply equally to this method of diagnosis.

[0169] "Cancer that is p95HER2 positive" refers to a cancer in which at least a portion of the cancer cells contain p95HER2 as determined by immunohistochemistry (IHC), Western blot, VeraTag® assay (Monogram Biosciences), or flow cytometry. In some embodiments, the cancer is determined to be p95HER2 positive by IHC. In some such embodiments, the cancer is determined to be p95HER2 positive using the methods described in Sperinde et al., Clin. Canc. Res., 2010, 16(16):4226-4235, such as using the anti-p95 antibody clone D9 in a VeraTag assay. In some embodiments, the cancer is determined to be p95HER2 positive using the methods described in U.S. Patent No. 8,389,227B2, such as using antibodies produced by hybridoma cell lines deposited by Deutschland Sammlung von Mikroorganismen and Zellen under accession numbers DSM ACC2904 or DSM ACC2980. In some embodiments, the cancer is determined to be p95HER2 positive according to the assay manufacturer's or assay laboratory guidelines. p95HER2 refers to a collection of carboxy-terminal HER2 fragments, which in some embodiments can be divided into a 95-100 kDa fragment and a 100-115 kDa fragment. See, e.g., Arribas et al., Cancer Res., 2011, 71:1515-1519. In some embodiments, the cancer that is p95HER2 positive contains the 100-115 kDa fragment of HER2.

[0170] Nucleic acids and vectors of the invention The immune cells according to the invention can be obtained by the use of nucleic acid constructs and / or nucleic acid compositions encoding the CARs and bispecific antibodies expressed by the T cells of the invention.

[0171] Therefore, another aspect of the present invention is a nucleic acid construct comprising: (i) a first region encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A first region including: (ii) a second region encoding a bispecific antibody, the bispecific antibody comprising: a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 A second region including or a nucleic acid construct (hereinafter "nucleic acid construct according to the present invention") comprising: 1. A nucleic acid composition comprising: (i) a first polynucleotide encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A first polynucleotide comprising: (ii) a second polynucleotide encoding a bispecific antibody, the bispecific antibody comprising: a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 and a second polynucleotide comprising The present invention relates to a nucleic acid composition (hereinafter referred to as "nucleic acid composition according to the present invention") comprising:

[0172] In a particular embodiment of the nucleic acid construct according to the invention or the nucleic acid composition according to the invention, the CAR antigen binding domain specific for p95HER2, the CAR transmembrane domain, the at least one CAR intracellular signalling domain and / or the CAR costimulatory domain, the bispecific antibody anti-HER2 antigen binding region and the bispecific antibody anti-CD3 antigen binding region are as defined in any of the previous aspects and / or embodiments.

[0173] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof, in either single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding capabilities as those of the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also refers to conservatively modified variants (e.g., substitution with degenerate codons), alleles, orthologs, SNPs and complementary sequences, and sequences shown in direct form. In particular, substitution with degenerate codons can be obtained by creating sequences in which the three positions of one or more selected (or all) codons are replaced by mixed-base residues and / or deoxyinosine residues.

[0174] The term "nucleic acid construct" as used in this disclosure refers to one single nucleotide sequence that contains all the elements necessary to encode a chimeric antigen receptor and a bispecific antibody, i.e., a first and a second region of a polynucleotide. The term "nucleic acid composition" as used in this disclosure refers to a composition that comprises at least two nucleotide sequences or polynucleotides, one that encodes a CAR expressed by an immune cell according to the invention and the other that encodes a bispecific antibody expressed by an immune cell according to the invention.

[0175] In certain embodiments of the nucleic acid construct according to the invention or the nucleic acid composition according to the invention, the chimeric antigen receptor encoded by the first polynucleotide of the nucleic acid construct or the nucleic acid composition and the bispecific antibody encoded by the second polynucleotide of the second region of the nucleic acid construct or the nucleic acid composition further comprise a signal sequence.

[0176] The term "signal sequence", also known as "leader peptide", is used herein according to its ordinary meaning in the art and refers to a peptide having a length of about 5 to 30 amino acids. Leader peptides are present at the N-terminus of newly synthesized proteins that form part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to, proteins that reside inside certain cellular organelles (endoplasmic reticulum, Golgi or endosomes), proteins that are secreted from the cell, or proteins that are inserted into the cell membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of the protein.

[0177] In some embodiments, the isolated nucleic acid encodes a protein from the N-terminus to the C-terminus, where a leader peptide is present at the N-terminus of a newly synthesized protein that forms part of the secretory pathway.

[0178] In a particular embodiment of the nucleic acid construct according to the invention or the nucleic acid composition according to the invention, the signal sequence forming part of the chimeric antigen receptor comprises a CD8 signal sequence and / or the signal sequence forming part of the bispecific antibody comprises an IgK signal sequence.

[0179] In a particular embodiment of the nucleic acid construct according to the invention or the nucleic acid composition according to the invention, the signal sequence forming part of the chimeric antigen receptor comprises SEQ ID NO: 62 and / or the signal sequence forming part of the bispecific antibody comprises SEQ ID NO: 63. SEQ ID NO:62ALPVTALLLPLALLLHAARP SEQ ID NO:63 MDFQVQIFSFLLISASVIMSRG

[0180] It will be obvious to the skilled person that a nucleic acid construct according to the invention comprising two regions, one encoding the CAR expressed by the immune cells according to the invention and the other encoding the bispecific antibody expressed by the immune cells according to the invention, makes it possible to obtain two separate polypeptides, i.e. the CAR and the bispecific antibody. Several mechanisms are available for producing two or more polypeptides from the same nucleic acid construct. One such way is the presence in said nucleic acid of different transcriptional facilitating elements, such as promoters and enhancers, for each of the different coding regions (see further below for aspects and embodiments). Another way is the presence of a single facilitating element originating from a unique messenger RNA (mRNA) that codes for both the CAR and the bispecific antibody. In said case, the mRNA may be monocistronic, i.e. both regions are contained in the same open reading frame of the mRNA, or it may be bicistronic, where the mRNA contains two different open reading frames, one encoding a first region corresponding to the CAR expressed by the immune cells according to the invention and the other encoding a second region corresponding to the bispecific antibody expressed by the immune cells according to the invention.

[0181] In a particular embodiment, the nucleic acid construct according to the invention is a monocistronic construct, in which the chimeric antigen receptor encoded by the first region and the bispecific antibody encoded by the second region are found in the same open reading frame and are separated by a self-cleaving peptide region.

[0182] As used in this disclosure, the term "self-cleaving peptide region" refers to a peptide sequence with cleavage activity that exists between two amino acid residues in the peptide sequence itself. Examples of self-cleaving peptides include 2A peptides and 2A-like peptides. For example, in 2A peptides or 2A-like peptides, cleavage occurs between glycine and proline residues in these peptides. This is caused by a "ribosome skipping mechanism" that prevents the normal formation of peptide bonds between glycine and proline residues during translation and does not affect downstream translation. The ribosome skipping mechanism is known in the art and is used for the expression of multiple proteins encoded by a single molecule of mRNA. The self-cleaving peptides used in the present invention can be obtained from 2A peptides or 2A-like peptides of viruses with equivalent functions. Examples of 2A self-cleaving peptides include 2A peptide (F2A) (VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 87)) derived from foot and mouth disease virus (FMDV), 2A peptide (E2A) (QCTNYALLKLAGDVESNPGP (SEQ ID NO: 88)) derived from bovine rhinitis A virus (ERAV), 2A peptide (P2A) (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 89)) derived from porcine teschovirus (PTV-1), and 2A peptide (T2A) (EGRGSLLTCGDVEENPGP (SEQ ID NO: 64)) derived from Thosea signa virus (TaV). Mutations may be introduced into the self-cleaving peptide domain as appropriate, as long as their activity is not significantly impaired.

[0183] In another particular embodiment of the nucleic acid construct according to the invention, the self-cleaving peptide region comprises the sequence of SEQ ID NO:64.

[0184] In another specific embodiment, a nucleic acid construct according to the invention that is a monocistronic construct, the chimeric antigen receptor encoded by the first region and the bispecific antibody encoded by the second region are found in the same open reading frame and are separated by a cis-acting hydrolase element.

[0185] "Cis-acting hydrolase element" or "CHYSEL" refers to a peptide sequence that causes the ribosome to release a growing polypeptide chain being synthesized without dissociation from the mRNA, in which case the ribosome continues translation, thus producing a second polypeptide. FMDV 2A sequence (GSGSRVTELLYRMKRAETYC PRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP, SEQ ID NO: 90); Amphimedon queenslandica 2A sequence (LLCFLLLLLSGDVELNPGP, SEQ ID NO: 91; or HHFMFLLLLLAGDIELNPGP, SEQ ID NO: 92); Saccoglossus kowalevskii (WFLVLLSFILSGDIEVNPGP, SEQ ID NO: 93) 2A sequence; Branchiostoma floridae (KNCAMYMLLLSGDVETNPGP, SEQ ID NO: 94; or MVISQLMLKLAGDVEENPGP, SEQ ID NO: 95) 2A sequence; Porcine tesshouvirus-1 (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 96) 2A sequence; Thoseaa asigna (Thoseaa and the equine rhinitis A virus (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO:98) 2A sequence are CHYSEL for use in the present invention. In some embodiments, the 2A sequence is a naturally occurring or synthetic sequence comprising the 2A consensus sequence DXEX-NPGP (SEQ ID NO:99), where X is any amino acid residue.

[0186] In a particular embodiment, a nucleic acid construct according to the invention that is a polycistronic construct, wherein the chimeric antigen receptor encoded by the first region and the bispecific antibody encoded by the second region are found in different open reading frames and are separated by an internal ribosome entry site or a cis-acting hydrolase element.

[0187] The term "internal ribosome entry site" or "IRES" defines a sequence motif that promotes the attachment of ribosomes to that motif on an internal mRNA sequence. Thus, an mRNA containing an IRES sequence motif results in two translation products, one that initiates from the 5' end of the mRNA and the other by an internal translation mechanism mediated by the IRES. Many IRES have been described and can be used in the nucleic acid constructs according to the invention. See, for example, US8,192,984; WO2010 / 119257; and US2005 / 0112095.

[0188] In addition, the nucleic acid construct according to the invention or the nucleic acid composition according to the invention may further contain a "marker", "label" or "tag" that may improve or enhance certain protein properties, i.e., protein stability or resistance to degradation, or may provide technical advantages during production, i.e., facilitate expression and / or purification. Examples of such tags are biotin, His-tag, FLAG-tag, Halo-tag, MBP-tag, HA-tag, Myc-tag, V5-tag, PA-tag, fluorescent protein tag, etc. Said tag may be fused or conjugated to the amino or carboxyl terminus of a polypeptide, e.g., a CAR expressed by an immune cell according to the invention or a bispecific antibody expressed by an immune cell according to the invention. The His-tag or "polyhistidine" sequence of histidine amino acids is linked by a peptide bond, which is fused or conjugated to the amino terminus of the protein of the invention and / or to the carboxyl terminus of the protein of the invention. In a preferred embodiment, the polyhistidine region contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 histidine residues.

[0189] In certain embodiments of the nucleic acid construct according to the invention or the nucleic acid composition according to the invention, the chimeric antigen receptor encoded by the first polynucleotide of the nucleic acid construct or the nucleic acid composition and / or the bispecific antibody encoded by the second polynucleotide of the second region of the nucleic acid construct or the nucleic acid composition further contain a tag.

[0190] In another particular embodiment of the nucleic acid construct according to the invention or the nucleic acid composition according to the invention, the tag is located at the C-terminus of the bispecific antibody.

[0191] In another particular embodiment of the nucleic acid construct according to the invention or the nucleic acid composition according to the invention, the tag is a hexahistidine tag.

[0192] Another aspect of the present invention relates to a polypeptide encoded by a nucleic acid construct according to the present invention.

[0193] The terms "polypeptide" or "peptide" or "protein (when single chain)" are used interchangeably herein and generally refer to amino acid polymers of any length. The polymers may be linear or branched, may contain modified amino acids or may be interrupted by non-amino acids. The terms also include amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component). Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or may be artificially synthesized products, so long as they are expressed.

[0194] In particular embodiments of the polypeptide encoded by the nucleic acid construct according to the invention comprises the sequence of SEQ ID NO:65, or SEQ ID NO:66, or SEQ ID NO:108 or SEQ ID NO:109.

[0195] SEQ ID NO:65

[0196] SEQ ID NO:66

[0197] SEQ ID NO:108

[0198] SEQ ID NO:109

[0199] A further aspect of the invention relates to a vector composition comprising a vector containing a nucleic acid construct according to the invention (hereinafter the "vector of the composition") or a vector in which the first and second nucleic acids of the composition are found in different vectors (hereinafter the "vector composition of the invention").

[0200] As used in this disclosure, a "vector," "cloning vector," and "expression vector" are vehicles by which a host can be transformed and expression (e.g., transcription and translation) of an introduced sequence can be achieved. The term "vector" is also meant to include plasmids, phages, viruses, and the like.

[0201] The nucleotide sequence encoding either the CAR expressed by the immune cell according to the invention and / or the bispecific antibody expressed by the immune cell according to the invention may be present in an expression vector and / or a cloning vector. The expression vector may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, etc.

[0202] Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for making the subject recombinant constructs. The following vectors are provided by way of example: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0203] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may also be present. Suitable expression vectors include, but are not limited to, viral vectors, particularly lentiviruses (e.g., vaccinia virus; poliovirus; adenovirus; adeno-associated virus; SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus; retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0204] In certain embodiments of the vector of the invention or the vector composition of the invention, the vector is a lentiviral vector.

[0205] The term "promoter" or "regulatory sequence" as used in this disclosure refers to a DNA sequence recognized by the transcriptional machinery of a cell / host necessary to initiate specific transcription of a polynucleotide sequence and / or necessary for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements necessary for expression of the gene product. The promoter / regulatory sequence may be one that expresses the gene product, for example, in a tissue-specific manner.

[0206] Examples of promoter / regulatory sequences suitable for the present invention include, but are not limited to, human cytomegalovirus promoter (CMV) (Hosseini Rad et al., PLoS One, 2020, 15(7):e0232915), Chinese hamster elongation factor-1 alpha (CHEF-1) (Ebadat et al., PLoS One, 2017, 12(10):e018596), human elongation factor-1 alpha (EF-1) (Hosseini Rad et al., PLoS One, 2020, 15(7):e0232915), human phosphoglycerate kinase (hPGK) (Hosseini Rad et al., PLoS One, 2020, 15(7):e0232915), RPBSA (Hosseini Rad et al., PLoS One, 2020,15(7):e0232915), MND promoter (WO2018085690), murine stem cell virus (MSCV) long terminal repeat (LTR) (Hughes, M. et al., Hum Gene Ther. 2005 April;16(4):457-472) and the NFAT-minimal IL2 promoter (Hoojberg et al., Blood, 2000 July;96(2):459-466).

[0207] In a particular embodiment of the vector of the invention or the vector composition of the invention, the nucleic acid construct, the first polynucleotide and / or the second polynucleotide are under the operational control of one or more promoters, where the promoters may be the same or different. In a particular embodiment of the vector of the invention or the vector composition of the invention, the nucleic acid construct, the first polynucleotide is under the operational control of the LTR promoter and the second polynucleotide is under the operational control of the NFAT-minimal IL2 promoter.

[0208] In another particular embodiment of the vector of the invention or the vector composition of the invention, the nucleic acid construct, the first polynucleotide and / or the second polynucleotide are under the operative control of the murine stem cell virus (MSCV) long terminal repeat.

[0209] The term "operably linked" or alternatively "transcriptional control" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence resulting in subsequent expression. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other, e.g., in the same reading frame when necessary to join two protein coding regions.

[0210] As mentioned above, in some embodiments, the nucleic acid comprising any of the CARs of the present invention is, in some embodiments, RNA, for example, RNA synthesized in vitro. Methods for in vitro synthesis of RNA are known in the art, and any known method can be used to synthesize RNA comprising a nucleotide sequence encoding the first and / or second polypeptide of the heterodimeric conditionally active CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. The introduction of RNA comprising a nucleotide sequence encoding the first and / or second polypeptide of the heterodimeric conditionally active CAR of the present disclosure into a host cell can be performed in vitro or ex vivo or in vivo. For example, a host cell (e.g., NK cell, cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding the first and / or second polypeptide of the heterodimeric conditionally active CAR of the present disclosure.

[0211] To assess the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into the cells may also contain a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that are desired to be transfected or infected with the viral vector; in other embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cells. Useful selection markers include, for example, antibiotic resistance genes, such as neomycin. Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that encode a polypeptide whose expression is manipulated by some easily detectable property, such as an enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0212] Uses of the nucleic acid constructs, nucleic acid compositions, vectors and / or vector compositions according to the present invention Another aspect of the present invention relates to a nucleic acid construct according to the invention or a nucleic acid composition according to the invention or a vector of the invention or a vector composition of the invention for use in medicine.

[0213] A further aspect of the present invention relates to a nucleic acid construct according to the invention or a nucleic acid composition according to the invention or a vector of the invention or a vector composition of the invention for use in a method for preventing or treating cancer.

[0214] In certain embodiments of the nucleic acid construct according to the invention, the nucleic acid composition according to the invention, the vector of the invention or the vector composition of the invention for use in a method for preventing or treating cancer, the cancer is p95HER2 positive.

[0215] All previous definitions and embodiments described are equally valid for this aspect and their embodiments.

[0216] Method for Obtaining Immune Cells According to the Invention The present invention further comprises a method that makes it possible to obtain the cells of the invention, which express the CAR and the bispecific antibody. Thus, another aspect of the present invention comprises: (i) an antigen-binding domain specific for p95HER2; A transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A chimeric antigen receptor (CAR) comprising (ii) a first antigen-binding region that specifically binds to HER2, and A second antigen-binding domain that specifically binds to CD3 and a bispecific antibody comprising A method for obtaining a cell expressing The present invention relates to a method (hereinafter "method of the invention") comprising inserting into a cell a nucleic acid construct according to the invention or a nucleic acid composition according to the invention or a vector of the invention or a vector composition of the invention.

[0217] The term "cell" or "engineered cell" refers to any cell of any organism that has been modified, transformed, or engineered by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. It also refers to the progeny of such a cell. The cells or engineered cells of the present invention include isolated immune cells, such as T cells, NK cells, or NKT cells, that contain DNA or RNA sequences encoding a CAR or CAR complex specific for p95HER2 and a bispecific antibody specific for HER2, express said CAR on the cell surface, and secrete said bispecific antibody. The isolated and engineered cells can be used, for example, to enhance the activity of NK cells or NKT cells, or the activity of T lymphocytes, the treatment of cancer, and the treatment of infectious diseases.

[0218] In certain embodiments of the methods of the present invention, the cell is a mammalian cell.

[0219] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0220] In some instances, the cells are not immortalized cell lines, but instead are cells (e.g., primary cells) obtained from an individual. For example, in some cases, the cells are immune cells obtained from an individual.

[0221] The engineered cells may be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor infiltrating lymphocytes, lymph node tissue, or thymus tissue. Host cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. Cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. Cells may be obtained from established cell lines.

[0222] The cells may be obtained by any known means. The cells may be autologous, syngeneic, allogeneic, or xenogeneic to the recipient of the engineered cells. The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0223] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual to which the material is introduced. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be genetically sufficiently different to interact antigenically.

[0224] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0225] The term "syngeneic" refers to close genetic similarity or identity, especially with respect to antigens or immune responses. Syngeneic systems include, for example, models in which organs and cells (e.g., cancer cells and their non-cancerous counterparts) are from the same individual, and / or models in which organs and cells are from different individual animals that are of the same inbred line.

[0226] In certain embodiments of the methods of the invention, the cell is a cell from the immune system.

[0227] The immune cells can be obtained from a subject who has or has been diagnosed with cancer, a plasma cell disorder, or an autoimmune disease or disorder. For example, the immune cells can be obtained from a subject who has cancer, such as multiple myeloma, smoldering myeloma, or Waldenstrom's macroglobulenemia. In some embodiments, the immune cells are obtained from a subject who is resistant to anti-BCMA therapy. The immune cells can also be obtained from an allogeneic donor, which is a non-genetically identical individual of the same species as the intended recipient of the cells.

[0228] Immune cells (e.g., human immune cells) that can be used in the present invention include autologous cells obtained from a subject to which the cells are subsequently administered after ex vivo modification and expansion. For example, immune cells can be obtained from an individual who has or has been diagnosed with cancer, plasma cell disorder, or an autoimmune disease or disorder. Immune cells can also be obtained from an allogeneic donor, which is a non-genetically identical individual of the same species as the intended recipient of the cells. Immune cells useful for the present invention include T cells and NK cells.

[0229] In another particular embodiment of the method of the invention, the immune cell is a T cell, a natural killer (NK) cell or a macrophage.

[0230] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines or microbial products, macrophages nonspecifically phagocytose and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack that results in the degradation of the pathogen. Peptides from degraded proteins are presented on the macrophage cell surface, where they can be recognized by T cells and they can directly interact with antibodies on the B cell surface, resulting in activation of T and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0231] In some embodiments, immune cells (e.g., human immune cells) that can be used in the present invention include autologous cells obtained from a subject to which the cells are subsequently administered after ex vivo modification and expansion. For example, immune cells can be obtained from an individual who has or has been diagnosed with cancer. Immune cells can also be obtained from an allogeneic donor, which is a non-genetically identical individual of the same species as the intended recipient of the cells. Immune cells useful for the present invention include T cells, NK cells, and NKT cells.

[0232] Methods for obtaining T cells, NK cells, and NKT cells are known in the art and may be useful for the engineered immune cells described herein. T cells, NK cells, and NKT cells are typically obtained from peripheral blood collected from a subject, for example, by venipuncture or withdrawal through an implanted port or catheter. Optionally, blood can be obtained by a process including leukoreduction, in which leukocytes are obtained from the subject's blood and other blood components are returned to the subject. The blood or leukoreduction product (fresh or frozen) can be processed to enrich for T cells, NK cells, or NKT cells, using methods known in the art. For example, density gradient centrifugation (e.g., using Ficoll) and / or counterflow centrifugal elutriation can be performed to enrich for mononuclear cells (including T cells, NK cells, or NKT cells). In one example, for T cells, a T cell stimulation step can be further performed using, for example, CD3 / CD28 antibodies coated on magnetic beads, or artificial antigen presenting cells (aAPCs) expressing, for example, cell surface-bound anti-CD3 and anti-CD28 antibody fragments (see below), to stimulate the T cells and deplete other cells, for example, B cells. The T cells of the enriched T cell preparation can then be subjected to genetic modification.

[0233] As an alternative to peripheral blood, tissues including bone marrow, lymph nodes, spleen and tumors can be used as sources for T cells and NK cells. The T cells and NK cells can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although any other mammalian cells may be used. In certain embodiments of any aspect, the T cells or NK cells are human.

[0234] Immune cells, such as T cells, NK cells, or NKT cells, can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Any of the many cell lines available in the art (e.g., immune cell lines, such as T cell lines) can also be used.

[0235] In one embodiment, immune cells (e.g., T cells, NK cells, or NKT cells) are obtained from a unit of blood drawn from a subject using any suitable technique known in the art, such as Ficoll™ separation. In another embodiment, cells from the subject's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, NK cells, or NKT cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. It will be appreciated that cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. For example, the cells may be washed with phosphate buffered saline (PBS). Alternatively, the wash solution may lack calcium, lack magnesium, or lack many, but not all, divalent cations. An initial activation step in the absence of calcium may result in extended activation. The washing step may be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffering agents. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0236] In some embodiments, immune cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. Specific subpopulations of immune cells, such as T cells, for example CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques known in the art. For example, T cells may be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28) conjugated beads, for example, DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. Additionally or alternatively, a population of T cells may be enriched by negative selection, for example by a combination of antibodies against surface markers specific to the cells to be negatively selected. Cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry may be used.

[0237] It will be understood that cells derived from a subject modified to express the CAR and bispecific antibody expressed by immune cells according to the invention can be stored for a period of time prior to their use (see, for example, the therapeutic methods below). For example, the cells can be frozen, optionally after they have been washed, or they can be incubated under conditions suitable for them (e.g., at 2-10° C. or at room temperature on a rotator) to maintain viability until needed. In this manner, the cells can be stored until such time as they may be needed. They can be stored in an unmodified state (i.e., they do not express the CAR and / or bispecific antibody) or in a modified state (i.e., they have been modified to express the CAR and / or bispecific antibody). Prior to use in therapeutic applications as further described below, the cells can generally be activated and expanded using methods known in the art. For example, T cells can be expanded by contacting the surface to which they are attached with an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cell. In particular, the T cell population may be stimulated as described herein, for example by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjugation with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used, as can other methods commonly known in the art.

[0238] T cells exposed to different stimulation times may exhibit different characteristics. For example, a typical blood or apheresed peripheral blood mononuclear cell product has a higher helper T cell population (TH, CD4+) than a cytotoxic or suppressive T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that, before about day 8-9, is composed predominantly of TH cells, but after about day 8-9, the population of T cells contains an increasingly larger population of TC cells. Thus, depending on the goal of the treatment, infusion of a patient with a T cell population that contains preferentially TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells is isolated, it may be beneficial to expand this subset to a higher degree.

[0239] In a particular embodiment, the immune cell is a T cell or a NK cell. In a more particular embodiment, the T cell is a CD8+ T cell.

[0240] In particular, the host cells of the invention may be expanded prior to transduction with a polynucleotide or vector of the invention.

[0241] It is worth noting that after some cancer treatments, especially those with drugs that damage the immune system, the quality of the immune cells obtained may be optimized or improved in terms of their ability to regenerate ex vivo immediately after the treatment during the period when the patient would normally recover from the treatment. Also, after ex vivo manipulation using the methods described herein, these cells may be in favorable conditions for enhanced engraftment and in vivo propagation. Therefore, in conjunction with the present invention, during this recovery stage, the production of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, is provided. In addition, in some embodiments, mobilization methods (e.g., mobilization using GM-CSF) and the establishment of specific conditions may be used to create conditions in the subject where repopulation, recirculation, regeneration, and / or regeneration of specific cell types is favorable, especially at certain time frames after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0242] The engineered cells of the present disclosure may also include a suicide system. A suicide system provides a mechanism by which the engineered cells can be inactivated or destroyed, as described above. Such a feature allows for precise therapeutic control of any therapy in which the engineered cells are used. As used in the present disclosure, a suicide system provides a mechanism by which the cells that have a suicide system can be inactivated or destroyed. Suicide systems are well known in the art.

[0243] The expression "inserting into a cell a nucleic acid construct according to the invention or a nucleic acid composition according to the invention or a vector of the invention or a vector composition of the invention" as used in the present disclosure refers to the introduction of a nucleic acid or vector into a cell of interest in such a way that said nucleic acid or vector produces or expresses the nucleic acid of interest.

[0244] Methods for introducing and expressing genes into cells are known in the art. In the context of one or more expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0245] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0246] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc.

[0247] Chemical means for introducing polynucleotide into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).In the case where a non-viral delivery system is utilized, exemplary delivery vehicles are liposomes.The use of lipid formulations is contemplated for the introduction (in vitro, ex vivo, or in vivo) of nucleic acid into host cells.In another embodiment, nucleic acid can be associated with lipids. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist as micelles, in bilayer structures, or in "disintegrating" structures. They may also simply be interspersed in the solution, potentially forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring lipids or synthetic lipids. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as the class of compounds that contain long chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0248] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl / phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol.

[0249] "Liposome" is a general term that encompasses a variety of mono- and multi-lamellar lipid vesicles formed by the creation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions with structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0250] In certain embodiments, the methods of the invention further comprise the step of selecting cells that express the CAR on their surface and secrete the bispecific antibody.

[0251] Regardless of the method used to introduce an exogenous polynucleotide into a host cell or otherwise expose the cell to a polynucleotide of the present disclosure, various assays may be performed to confirm the presence of the recombinant DNA sequence in the host cell, which allows the selection of those cells that express CAR on their surface and secrete bispecific antibodies. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR and PCR, to identify agents within the scope of the present disclosure; "biochemical" assays, such as detection of the presence or absence of a particular peptide, for example, by immunological means (EL1SA and Western blot) or by the assays described herein. It will be clear to those skilled in the art that, to select cells that express both CAR and bispecific antibodies, two or more of the techniques exemplified above may be used to obtain confirmation of the expression of both polypeptides of interest. EXAMPLES

[0252] The present invention is illustrated by the following examples, which are merely illustrative and should not be construed as limiting the scope of the invention.

[0253] methodology Vector design and retrovirus production Vector plasmids encoding h1-214 p95HER2 CAR (H1-14 CAR), HER2-CD3 bispecific T cell engager high affinity (TECH2 Hi), HER2-CD3 bispecific T cell engager medium affinity (TECH2e), HER2-CD3 bispecific T cell engager low affinity (TECH2 Lo), p95HER2.CAR HER2-TECH2 high affinity (CAR-TECH2 Hi) or p95HER2.CAR HER2-TECH2 medium affinity (TECH2e) were synthesized and cloned into pMSGV-1 retroviral vector (Genscript, Netherlands). A scheme of the different constructs is shown in Figure 2. Retroviral stocks of the aforementioned constructs and empty (UTD) retroviruses were then produced. Briefly, 0.7ug of envelope plasmid (RD-114) and 1.5ug of transfer plasmid (H1-14 CAR, TECH2 Hi, TECH2e, TECH2 Lo, CAR-TECH2 Hi, CAR-TECH2e, UTD in pMSGV-1) were co-transfected in GP2-293 packaging cell line (No. 631458, Takara). After 3 days, cell supernatant containing retroviral particles was collected and stored at -80°C for future transduction.

[0254] Transduction and expansion of CAR and TECH2-secreting T cells PBMCs were stimulated with 10 ng / ul anti-CD3 (OKT3) (no. 16-0037-85, Thermo-Fisher) and 300 U / ml IL-2 (no. 703892-4, Novartis) for 48 hours prior to transduction. Retroviral supernatants were then thawed and centrifuged at 2000 g for 2 hours in Retronectin (no. T100A, Takara) coated 6-well plates. 2 × 10 6 Stimulated PBMCs were added on top and centrifuged at 400g for 10 min. Five days later, CAR expression and cytotoxicity assays were performed. Untransduced T cells (UTD) were transduced with empty CAR retrovirus.

[0255] CAR expression analysis 0.2×106 CAR T were double-diluted with 1x PBS and resuspended in 1x PBS, 2.5mM EDTA, 1% BSA, and 5% horse serum for 20 minutes. Cells were then stained with 1 / 20 biotin goat anti-mouse IgG (no. 115-065-072, Jackson ImmunoResearch) for 30 minutes and washed twice with 1x PBS. APC-streptavidin antibody (no. 405207, Biolegend) at 1 / 150 and 1 / 300 anti-CD3-PE (no. 300408, Biolegend) was added for 30 minutes. Zombie Aqua (no. 423101, Biolegend) was used as a viability marker at 1:1000 dilution. CAR expression was measured on a FACS Celesta (BD Bioscience) and analyzed using FlowJo software.

[0256] CAR T cytotoxicity assay MCF7 p95HER2 / empty cells were co-cultured with CAR or TECH2-secreting T cells at the indicated ratios in 96-well flat-bottom plates. After 48 hours of incubation, the cell mixture was detached, washed with 1×PBS, and resuspended in 1×PBS, 2.5 mM EDTA, 1% BSA, and 5% horse serum for 20 minutes. The cell suspension was then stained with anti-EpCAM-AF647 (no. 324212, Biolegend) at a dilution of 1:300 as a target cell marker, and zombie Aqua (no. 423101, Biolegend) at a dilution of 1:1000 as a viability marker. EpCAM-positive viable target cells were counted in an LSR Fortessa (BD Bioscience) and analyzed using FlowJo software.

[0257] In vivo patient-derived tumor (PDTX) and cell line-derived (CDX) models Patient-derived tumor xenografts (PDTXs) ​​from human breast tumors used in this study were obtained from biopsies or surgical resections at the Vall d'Hebron University Hospital, Barcelona, ​​in accordance with institutional guidelines. The Institutional Review Board (IRB) of the Vall d'Hebron Hospital provided approval for this study in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients who provided samples.

[0258] Breast cancer fragments from patient samples or wild type MCF7 cells were orthotopically implanted into NOD.Cg-Prkdcscid Il2rgtm1WjI / SzJ (NSG) (number 005557, Charles River Laboratories) mice. Tumor xenografts were measured twice weekly using calipers and tumor volumes were calculated using the formula: (length × width) 2 )x(π / 6).

[0259] Tumor volume is 200-300mm 3 When this is reached, the animals are removed from the 6 were treated intravenously (iv) with CAR+ or TECH2-secreting+ T cells and a second dose was administered 10 days later.

[0260] Generation and characterization of NK cells expressing CAR-TECH2 Hi NK cells (CD56+CD3-) were isolated from PBMCs using an NK cell isolation kit (no. 130-092-657, Miltenyi). NK cells were expanded and activated for 48 hours prior to transduction using NK MACS medium with 5% AB serum and human cytokines IL-2 (no. 703892-4, Novartis, 500 U / mL) and IL-15 (no. 130-095-765, Miltenyi, 140 U / mL). Cells were then transduced as previously described for T cells. After 5 days, CAR expression was determined similarly to T cells. Cytotoxicity assays were performed in MCF7 p95HER2 / empty cells co-cultured with CAR-TECH2 Hi NK cells at the indicated ratios in 96-well flat-bottom plates. After 24 hours of incubation, the cell mixture was scraped off, washed with 1x PBS, and resuspended in 1x PBS, 2.5 mM EDTA, 1% BSA, and 5% horse serum for 20 minutes. The cell suspension was then stained with anti-EpCAM-AF647 (No. 324212, Biolegend) at a dilution of 1:300 as a target cell marker, and zombie Aqua (No. 423101, Biolegend) at a dilution of 1:1000 as a viability marker. EpCAM-positive viable target cells were counted in an LSR Fortessa (BD Bioscience) and analyzed using FlowJo software.

[0261] result Second-generation p95HER2 CAR T has modest efficacy against patient-derived tumor xenografts (PDTX) The antitumor activity of h1-214 anti-p95HER2 CAR against cell line derived tumors has been previously described in an international patent application published as WO2021239965. Here, we tested the efficacy of p95HER2 CAR against breast cancer patient derived tumor xenografts. p95HER2 CAR had no effect against HER2 positive, p95HER negative PDTX (Figure 1A), highlighting the specificity of the CAR. In contrast, p95HER2 CAR showed antitumor efficacy against p95HER2 positive PDTX (Figure 1B). The efficacy of second generation p95HER2 CAR against PDTX was overtly lower than that against cell lines expressing p95HER2, indicating limited efficacy in the clinic.

[0262] One of the mechanisms behind this resistance in PDTX is the heterogeneity and low level expression of tumor antigens, as seen by immunohistochemical analysis of PDTX samples. In p95HER2-positive tumors, HER2 expression is more abundant and uniform than that of p95HER2, but the second generation is tumor-specific and therefore a safer target. This suggests that p95HER2 may be a safe target for the specific redirection of T cells to tumors, but targeting HER2 may greatly increase tumor eradication due to its abundance.

[0263] Generation of a bispecific T cell engager (TECH2) targeting HER2 and CD3, and a p95HER2 CAR T secreting bispecific CD3-HER2 T cell engager (p95HER2.CAR-TECH2) construct The first step in creating an armed CAR T was to design a construct for expression and secretion of TECH2 by T cells using publicly available sequences.

[0264] We call the bispecific T cell engager secreted by the transduced T cells "TECH2", which is formed by two scFvs that recognize and bind to two antigens: HER2 on tumor cells and CD3 on T cells, respectively. Secreted TECH2 as a bispecific engager redirects T cells toward tumor cells expressing HER2 through CD3 binding. Unless specified, TECH2 refers to the bispecific T cell engager with high affinity for HER2, or "TECH2 Hi".

[0265] We included TECH2 co-expressed with the p95HER2 CAR as described (Choi et al., 2019, Nature Publishing Group 37, 1049-1058). A simplified scheme of the three constructs is shown in Figure 2.

[0266] Generation and binding characterization of TECH2 with reduced binding affinity to HER2 We altered the amino acid sequence of the HER2 scFv of TECH2 Hi to generate two additional molecules with different binding affinities for HER2, as described by Carter et al. (1992, PNAS 89, 4285-4289): TECH2 with intermediate affinity (TECH2e) and TECH2 with low affinity (TECH2 Lo) (Figure 3A). The binding affinities of the scFvs for immobilized HER2 were reported in Liu et al., 2015, Cancer Res 75(17), 3596-607 (Figure 3B).

[0267] To compare the binding affinity of HER2scFv tuned to different affinities in the TECH2 format, we overexpressed the constructs in 293T cells, collected media containing TECH2, and measured binding to both CD3 and HER2 targets by flow cytometry. HER2 binding affinity was measured to HER2 expressed in PDX433, and CD3 binding was measured to PBMC (Figure 3C). TECH2 Hi had 230-fold higher binding to HER2 than TECH2e, and they both bound equally to CD3, as expected, since the CD3 binding domain was not modified.

[0268] In vitro characterization of TECH2 and p95HER2.CAR-TECH2 with reduced binding affinity to HER2 In vitro cytotoxicity assays showed that TECH2e and TECH2 Lo secreting T cells did not kill targeted MCF7 cells expressing normal levels of HER2, but TECH2 Hi had cytotoxicity against this cell line, as previously shown (Figure 3D). The results also show that TECH2e is effective against PDX (PDX433) overexpressing both p95HER2 and full-length HER2.

[0269] We observed the same results when we tested constructs expressing p95HER2.CAR and TECH2 simultaneously (Figure 3E). Altogether, these results suggest that TECH2e is less effective than TECH2 Hi, but has a better safety / efficacy profile.

[0270] Effect of p95HER2.CAR-TECH2 high affinity in vivo NSG mice were orthotopically implanted with MCF7 wild-type cells expressing normal levels of HER2 (Figure 4A), or fragments of two different breast cancer patient-derived tumor xenografts (PDTX) overexpressing both p95HER2 and full-length HER2 (Figure 4B). Mice were cultured until tumors reached 300 mm 3 When it reaches 3×10 6Patients were treated with T cells secreting H1 214 p95HER2 CAR+ T cells (p95HER2 CAR), TECH2 high affinity (TECH2 Hi) and p95HER2.CAR+TECH2 high affinity (p95HER2.CAR-TECH2 Hi). Untransduced T cells (UTD) were used as a control.

[0271] Armed CAR-TECH2 was clearly superior to the second generation p95HER2 CAR, which showed very limited antitumor efficacy in contrast to the complete eradication of tumors mediated by CAR-TECH2 (Figure 1B). TECH2 alone also showed limited efficacy (Figure 4B), reinforcing the idea that the combination of the two components is more effective than each of them alone.

[0272] This shows that CAR-TECH2 high affinity is highly effective against two different PDTXs expressing heterogeneous levels of p95HER2, yet it kills MCF7-derived tumors. Further studies in real "healthy tissue" models need to be performed to accurately evaluate the safety of this construct, but alternatively, modulation of the affinity of the HER2 binding arm would increase the safety profile.

[0273] Effect of p95HER2.CAR-TECH2 intermediate affinity in vivo NSG mice were orthotopically implanted with MCF7 wild-type cells (Figure 4C) or PDX overexpressing both p95HER2 and full-length HER2 (PDX67) (Figure 4D). Mice were transplanted with 3x10 cells / mL or 1x10 cells / mL when tumors reached 300 mm3, as shown in the graph. 6The subjects were treated with T cells secreting H1 214 p95HER2 CAR T cells (p95HER2 CAR), HER2 CAR T cells (HER2.CAR), TECH2 intermediate affinity (TECH2e) and p95HER2.CAR+TECH2 intermediate affinity (p95HER2.CAR-TECH2e). Untransduced T cells (UTD) were used as controls. The in vivo results correlate with previous in vitro findings: TECH2e alone or in combination with p95HER2 CAR has no cytotoxic effect on cells expressing normal levels of HER2 (Figure 4C). Moreover, p95HER2.CAR-TECH2e showed increased efficacy over its two components: TECH2e (Figure 4D) and p95HER2 CAR alone (Figure 1B). In addition, two of four animals showed complete response (CR) for at least 80 days of follow-up after complete tumor regression (Figure 4D, left). These results demonstrate the advantage of dual, yet safe, targeting of p95HER2 and HER2 to achieve significant antitumor responses.

[0274] Generation and in vitro characterization of p95HER2.CAR-TECH2 in NK cells Our constructs could also be used in other immune cells, as an example of which the CAR-TECH2 vector could efficiently retrovirally transduce NK cells (Figure 5A).

[0275] In vitro cytotoxicity analysis showed that the p95HER2 CAR portion of the construct did not target HER2-expressing MCF7 cells (Figure 5B). Given the purification of NK cells prior to these experiments, no CD3+ cells were present and therefore no effect of the TECH2 portion would be expected. The results also showed that p95HER2.CAR-TECH2 was effective in killing cells expressing p95HER2, demonstrating the specificity of the product (Figure 5B).

Claims

1. An immune cell, (i) - an antigen-binding domain specific for p95HER2, a transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A chimeric antigen receptor comprising and (ii) a first antigen-binding region that specifically binds to HER2, and a second antigen-binding region that specifically binds to CD3 A bispecific antibody comprising expressing, immune cells.

2. The immune cell of claim 1 , wherein the first and second antigen-binding regions of the bispecific antibody are ScFvs.

3. the VH region of said ScFv of said first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the VL region of said ScFv of said first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 4, SEQ ID NO: 100 and SEQ ID NO: 6, respectively; - the VH region of said ScFv of said second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; and / or the VL region of said ScFv of said second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12, respectively; or the VH region of said ScFv of said first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:102, respectively; the VL region of said ScFv of said first antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 4, SEQ ID NO: 103 and SEQ ID NO: 6, respectively; - the VH region of said ScFv of said second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; and / or - the VL region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3 comprising the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12, respectively.

4. - the VH region of the ScFv of the first antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; - the VL region of the ScFv of the first antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, respectively; - the VH region of the ScFv of the second antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively; and / or - the VL region of the ScFv of the second antigen-binding region has FR1, FR2, FR3 and FR4 comprising the sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28, respectively.

5. - the VH region of the ScFv of the first antigen-binding region comprises the sequence of SEQ ID NO: 29 or SEQ ID NO: 104, - the VL region of the ScFv of the first antigen-binding region comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 105, - the VH region of the ScFv of the second antigen-binding region comprises the sequence of SEQ ID NO: 31, and / or The immune cell of claim 4, wherein the VL region of the ScFv of the second antigen-binding region comprises the sequence of SEQ ID NO:

32.

6. The immune cell of any one of the preceding claims, wherein the VH and VL regions of the ScFv of the first antigen binding region, the VH and VL regions of the ScFv of the second antigen binding region, and / or the ScFvs of the first and second antigen binding regions are connected by a peptide linker.

7. The immune cell of claim 6, wherein the linker is selected from SEQ ID NO: 33, 34 or 35.

8. - the ScFv of the first antigen-binding region comprises the sequence of SEQ ID NO: 36 or SEQ ID NO: 106, The immune cell of claim 7, wherein the ScFv of the second antigen-binding region comprises the sequence of SEQ ID NO:

37.

9. The immune cell of claim 8, wherein the bispecific antibody has the sequence of SEQ ID NO: 38 or SEQ ID NO:

107.

10. 2. The immune cell of any one of the preceding claims, wherein the p95HER2-specific antigen-binding domain of the CAR is an ScFv.

11. The immune cell of claim 10, wherein the CDR1, CDR2 and CDR3 of the VH region of the CAR ScFv comprise the sequences of SEQ ID NOs: 39, 40 and 41, respectively, and / or the CDR1, CDR2 and CDR3 of the VL region of the CAR ScFv comprise the sequences of SEQ ID NOs: 42, 43 and 44, respectively.

12. The immune cell of claim 11, wherein the FR1, FR2, FR2 and FR4 of the VH region of the CAR comprise the sequences of SEQ ID NOs: 45, 46, 47 and 48, respectively, and / or the FR1, FR2, FR3 and FR4 of the VL region of the CAR comprise the sequences of SEQ ID NOs: 49, 50, 51 and 52, respectively, or functionally equivalent variants thereof.

13. The immune cell of claim 12, wherein the VH and VL regions of the CAR ScFV are connected by a linker region.

14. The immune cell of claim 13 , wherein the linker region comprises SEQ ID NO:

53.

15. The immune cell of claim 13, wherein the VL of the CAR ScFv comprises the sequence of SEQ ID NO: 54 and the VH of the CAR ScFv comprises the sequence of SEQ ID NO:

55.

16. The immune cell of claim 15, wherein the CAR ScFv comprises the sequence of SEQ ID NO:

56.

17. 2. The immune cell of any one of the preceding claims, wherein the transmembrane domain of the CAR is selected from the group consisting of CD4 transmembrane domain, CD8 transmembrane domain, CD28 transmembrane domain, 4-1BB transmembrane domain, CTLA4 transmembrane domain, CD27 transmembrane domain and CD3 zeta transmembrane domain.

18. The immune cell of claim 17, wherein the transmembrane domain comprises the sequence of SEQ ID NO:

57.

19. 2. The immune cell of any one of the preceding claims, wherein the at least one intracellular signaling domain of the CAR comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

20. 20. The immune cell of claim 18, wherein the at least one intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

21. The immune cell of claim 20, wherein the intracellular domain of the costimulatory molecule comprises the sequence of SEQ ID NO:

58.

22. 10. The immune cell of any one of the preceding claims, wherein the at least one intracellular signaling domain of the CAR further comprises the sequence of SEQ ID NO:

59.

23. The immune cell of claim 22, wherein the at least one intracellular signaling domain is located N-terminal to the CD3 zeta intracellular domain.

24. 23. The immune cell of claim 22, wherein the CAR further comprises a hinge domain between the antigen binding domain and the transmembrane domain.

25. The immune cell of claim 24, wherein the hinge domain comprises the sequence of SEQ ID NO: 60 (CD8 hinge domain).

26. The immune cell of claim 25, wherein the CAR comprises the sequence of SEQ ID NO:

61.

27. 20. An immune cell according to any one of the preceding claims for use as a medicament.

28. 20. An immune cell according to any one of the preceding claims for use in a method for preventing or treating cancer.

29. The immune cell of claim 28, wherein the cancer is p95HER2 positive.

30. (i) a first region encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: - an antigen-binding domain specific for p95HER2, a transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A first region including: (ii) a second region encoding a bispecific antibody, the bispecific antibody comprising: a first antigen-binding region that specifically binds to HER2, and a second antigen-binding region that specifically binds to CD3 A second region including A nucleic acid construct comprising: or (i) a first polynucleotide encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: - an antigen-binding domain specific for p95HER2, a transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A first polynucleotide comprising: (ii) a second polynucleotide encoding a bispecific antibody, said bispecific antibody comprising: a first antigen-binding region that specifically binds to HER2, and a second antigen-binding region that specifically binds to CD3 and a second polynucleotide comprising A nucleic acid composition comprising:

31. 31. The nucleic acid construct or composition of claim 30, wherein the p95HER2-specific CAR antigen-binding domain, the CAR transmembrane domain, the at least one CAR intracellular signalling domain and / or CAR costimulatory domain, the anti-HER2 antigen-binding region and the anti-CD3 antigen-binding region of the bispecific antibody are as defined in any one of claims 1 to 26.

32. 32. The nucleic acid construct or nucleic acid composition of claim 30 or 31, wherein the chimeric antigen receptor encoded by the first polynucleotide of the first region of the nucleic acid construct or the nucleic acid composition and the bispecific antibody encoded by the second polynucleotide of the second region of the nucleic acid construct or the nucleic acid composition further comprise a signal sequence.

33. 33. The nucleic acid construct or composition of claim 32, wherein the signal sequence forming part of the chimeric antigen receptor comprises SEQ ID NO: 62 and / or the signal sequence forming part of the bispecific antibody comprises SEQ ID NO:

63.

34. 34. The nucleic acid construct of any one of claims 30 to 33, which is a polycistronic construct, wherein the chimeric antigen receptor encoded by the first region and the bispecific antibody encoded by the second region are found in the same open reading frame and are separated by a self-cleaving peptide region.

35. 35. The nucleic acid construct of claim 34, wherein the self-cleaving peptide region comprises the sequence of SEQ ID NO:

64.

36. 36. The nucleic acid construct or nucleic acid composition of any one of claims 31 to 35, wherein the chimeric antigen receptor encoded by the first polynucleotide of the first region of the nucleic acid construct or of the nucleic acid composition and / or the bispecific antibody encoded by the second polynucleotide of the second region of the nucleic acid construct or of the nucleic acid composition further comprises a tag.

37. 37. The nucleic acid construct or composition of claim 36, wherein the tag is located at the C-terminus of the bispecific antibody.

38. 38. The nucleic acid construct or composition of claim 37, wherein the tag is a hexahistidine tag.

39. A polypeptide encoded by the nucleic acid construct of any one of claims 31 to 38.

40. 37. The polypeptide of claim 36, comprising the sequence of SEQ ID NO:65, or SEQ ID NO:66, or SEQ ID NO:108, or SEQ ID NO:

109.

41. 39. A vector composition comprising a vector containing the nucleic acid construct of any one of claims 31 to 38, or wherein the first and second nucleic acids of said composition are found in different vectors.

42. 42. The vector or vector composition of claim 41 , wherein the nucleic acid construct, the first polynucleotide and / or the second polynucleotide are under the operative control of a murine stem cell virus (MSCV) long terminal repeat sequence.

43. 43. The vector or vector composition of claim 41 or 42, wherein the vector is a lentiviral vector.

44. A nucleic acid construct or a nucleic acid composition according to any one of claims 31 to 38, or a vector or vector composition according to any one of claims 41 to 43, for use in medicine.

45. A nucleic acid construct or nucleic acid composition according to any one of claims 31 to 38, or a vector or vector composition according to any one of claims 41 to 43, for use in a method for preventing or treating cancer.

46. 46. ​​The nucleic acid construct, nucleic acid composition, vector or vector composition for use according to claim 45, wherein the cancer is p95HER2 positive.

47. (i) - an antigen-binding domain specific for p95HER2, a transmembrane domain, and at least one intracellular signaling domain and / or costimulatory domain A chimeric antigen receptor comprising (ii) a first antigen-binding region that specifically binds to HER2, and a second antigen-binding region that specifically binds to CD3 and a bispecific antibody comprising A method for obtaining a cell expressing 46. ​​A method comprising inserting into said cell a nucleic acid construct or nucleic acid composition according to any one of claims 31 to 38, or a vector or vector composition according to any one of claims 41 to 43, and selecting cells which express said CAR on their surface and secrete said bispecific antibody.

48. 48. The method of claim 47, wherein the cell is a cell from the immune system.

49. 49. The method of claim 48, wherein the cell from the immune system is a T cell, a NK cell or a macrophage.

Images