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 as well as bispecific antibodies against HER2 and CD3 and their use in the treatment of cancer, in particular cancer overexpressing p95HER2.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and biomedicine, and specifically relates to immune cells expressing specific chimeric antigen receptors targeting p95 fragments of HER2, bispecific antibodies targeting HER2 and CD3, and uses thereof in cancer treatment. Background Art

[0002] Immune cell redirection against tumor-associated or tumor-specific antigens is a successful treatment strategy for some hematological malignancies. In contrast, it has failed so far in the treatment of solid tumors. T cells can be redirected using bispecific antibodies, which are often called bispecific T cell engagers (BiTEs) or T cell bispecific antibodies (TCBs), which simultaneously bind to tumor antigens and the invariant subunits of the T cell receptor (TCR), usually CD3. Alternatively, T cells can be manipulated to express chimeric antigen receptors (CARs), which are engineered proteins comprising the antigen binding domains of antibodies and the signaling domains of TCRs and co-activation receptors.

[0003] In contrast to the success in hematological malignancies, T cell redirection against solid tumors has so far failed. Reasons for this failure include downregulation of the antigens to which the T cells are redirected, heterogeneity in the expression of the antigens, and the immunosuppressive environment established by the tumor.

[0004] Although improving the design of bispecific T cell engagers to overcome such escape mechanisms has inherent limitations, CART cells can be used as a platform for delivering additional anti-tumor factors (such as cytokines or antibodies). The 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). Armored CAR constitutes an expandable platform to offset the above-mentioned drug resistance mechanisms.

[0005] Given the scarcity of tumor-specific antigens, the vast majority of CARs developed to date are directed against tumor-associated antigens. As a result, frequent and severe side effects caused by off-target tumor activity limit their use. Although in principle they should be free of side effects, CARs directed against the few available tumor-specific antigens have not been effective. This failure is at least in part due to the low levels and / or heterogeneous expression of these tumor-specific antigens. Tumor cells ultimately escape by downregulating them, or tumor cells that do not express them are rapidly selected. In order 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 for use as proof-of-concept glioblastoma.

[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 ~15%. About one-third of these tumors also express a truncated form of HER2, called p95HER2. Although HER2 is also expressed in normal epithelium, although the level is much lower than that of HER2-amplified tumors, p95HER2 is a true tumor-specific antigen. Consistently, we have previously demonstrated that specific TCBs effectively redirect lymphocytes to p95HER2-positive breast tumors, but do not target cells expressing normal levels of HER2.

[0007] Several second-generation CARs targeting p95HER2 have been generated. These CARs have been shown to be highly effective in vitro and in vivo against cell lines but have partial effects against patient-derived tumor xenografts, suggesting that they have limited efficacy in the clinic. Therefore, given the limitations of current cell therapies, there is a need for CAR or bispecific T-cell engager alternatives that exploit the specificity of p95HER2 targeting and the broader effects of HER2 targeting. Summary of the invention

[0008] The present invention discloses armored CAR immune cells targeting p95HER2, which have been further modified to secrete bispecific T cell engagers (BiTEs) that recruit immune cells to target HER2 in order to enhance their anti-tumor therapeutic effects. It is expected that this p95HER2 CAR that secretes HER2 bispecific T cell engagers will combine the specificity (and therefore safety) of targeting p95HER2 with the efficacy of locally targeting HER2.

[0009] In other words, the present invention utilizes a tumor-specific antigen (p95HER2) that is heterogeneously expressed in tumors to safely trigger local targeting of a more abundant antigen, but which is also expressed in normal tissues (HER2). The present invention significantly improves the anti-tumor effects of previously described second-generation p95HER2 CAR immune cells and will likely avoid the typical extra-tumor side effects of HER2-targeted therapies.

[0010] Therefore, a first aspect of the present invention relates to immune cells expressing

[0011] (i) a chimeric antigen receptor, the chimeric antigen receptor comprising:

[0012] οp95HER2-specific antigen binding domain,

[0013] o transmembrane domain and

[0014] o At least one intracellular signaling domain and / or co-stimulatory domain

[0015] as well as

[0016] (ii) a bispecific antibody, the bispecific antibody comprising:

[0017] o a first antigen binding region that specifically binds to HER2 and

[0018] o A second antigen binding region that specifically binds to CD3.

[0019] Another aspect of the present invention relates to a nucleic acid construct comprising

[0020] (i) encoding a first region of a chimeric antigen receptor, the chimeric antigen receptor comprising:

[0021] οp95HER2-specific antigen binding domain,

[0022] o transmembrane domain and

[0023] o At least one intracellular signaling domain and / or co-stimulatory domain

[0024] as well as

[0025] (ii) a second region encoding a bispecific antibody, the bispecific antibody comprising:

[0026] o a first antigen binding region that specifically binds to HER2 and

[0027] o a second antigen binding region that specifically binds to CD3;

[0028] Or a nucleic acid composition, the nucleic acid composition comprising

[0029] (i) a first polynucleotide encoding a chimeric antigen receptor, the chimeric antigen receptor comprising:

[0030] οp95HER2-specific antigen binding domain,

[0031] o transmembrane domain and

[0032] o At least one intracellular signaling domain and / or co-stimulatory domain

[0033] as well as

[0034] (ii) a second polynucleotide encoding a bispecific antibody, the bispecific antibody comprising:

[0035] o a first antigen binding region that specifically binds to HER2 and

[0036] o a second antigen binding region that specifically binds to CD3;

[0037] Another aspect of the invention relates to a vector or a vector composition comprising a nucleic acid construct according to the invention, wherein the first and second nucleic acids of the composition are present in different vectors.

[0038] Yet another aspect of the present invention relates to a polypeptide encoded by the nucleic acid construct according to the present invention.

[0039] Yet another aspect relates to a method for obtaining a cell expressing

[0040] (i) a chimeric antigen receptor, the chimeric antigen receptor comprising:

[0041] οp95HER2-specific antigen binding domain,

[0042] o transmembrane domain and

[0043] o At least one intracellular signaling domain and / or co-stimulatory domain

[0044] as well as

[0045] (ii) a bispecific antibody, the bispecific antibody comprising:

[0046] o a first antigen binding region that specifically binds to HER2 and

[0047] o a second antigen binding region that specifically binds to CD3;

[0048] The method comprises inserting a nucleic acid construct or a nucleic acid composition according to the invention or a vector or a vector composition according to the invention into a cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 - Effect of p95HER2 CAR T on PDX.

[0050] Two patient-derived tumor xenografts (PDTX), p95HER2 negative (A) and positive (B), were orthotopically injected into NSG mice. At the time points indicated by arrows, 3 × 10 H1-14p95HER2 CAR expressing cells were injected intravenously. 6 T cells. Tumor volume is expressed as mean ± SD (standard deviation) (n = 6 per arm). UTD, control non-targeted CAR.

[0051] Figure 2 . Schematic representation of the three constructs described in this document.

[0052] (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 figures: scFv = single chain fragment variable, VL = light chain, VH = heavy chain, LS = leader sequence, TM = transmembrane domain, 6xHis = peptide tag consisting of 6 histidine repeats, T2A = self-cleaving peptide.

[0053] Figure 3 Design and in vitro characterization of HER2-CD3 T cell engagers (TECH2) with different binding affinities for HER2

[0054] (A) Simplified scheme of the retroviral vectors shown. (B) Binding affinity of scFvs to HER2 described in (Liu et al., 2015). (C) Comparison of TECH2-formatted scFvs with different affinities to HER2 and CD3 in HEK 293T-producing cell supernatants by flow cytometry. (D) Wild-type MCF7 cells expressing normal levels of HER2 (left) and PDX-derived cell lines overexpressing both HER2 and p95HER2 (right) were co-cultured with T cells transduced to secrete TECH2 with different affinities for 48 h. Then, live target cells were quantified by flow cytometry using EpCAM as a marker. (E) The same target cells were co-cultured with p95HER2 CAR T cells secreting high or medium affinity TECH2 for 48 h and analyzed as in D.

[0055] Figure 4 In vivo characterization of CAR-TECH2 Me and Hi

[0056] (A)(C) MCF7 wild-type cells expressing normal levels of HER2 or (B)(D) two different PDTX fragments with heterogeneous p95HER2 / HER2 expression were orthotopically injected into NSG mice. At the time points indicated by arrows, 3×10 PDTX expressing the indicated CAR, TECH2, or CAR-TECH2 constructs were injected intravenously. 6 T cells. Tumor volume is expressed as mean ± SD (standard deviation) (n = 3-8 per arm). p values ​​represent the last time point before control mice were sacrificed. UTD, control non-targeted T cells.

[0057] Figure 5 - In vitro characterization of p95HER2.CAR-TECH2 NK cells (CAR-TECH2 NK).

[0058] (A) Figure 3 Transduction efficiency of NK cells at day 4 after transduction of the retroviral vectors shown in A; the percentage of positive CAR among total NK cells is indicated. (B) MCF7 cells expressing empty vector or the same vector expressing p95HER2 (Parra-Palau et al., 2014) were co-cultured with different ratios of p95HER2CAR–TECH2-secreting NK cells or untransduced NK cells for 24 h. Then, live target cells were quantified by flow cytometry using EpCAM as an epithelial cell marker. Results are expressed as the mean of three technical replicates. DETAILED DESCRIPTION

[0059] In view of the limitations of current cell therapy for tumor treatment, the present invention relates to a fourth generation CAR immune cell expressing a bispecific T cell engager 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 p95HER2-specific chimeric antigen receptor and a bispecific antibody targeting HER2 and CD3.

[0060] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] All embodiments and definitions disclosed in the context of one aspect of the invention also apply to the other aspects of the invention.

[0062] Immune cells of the present invention

[0063] Therefore, a first aspect of the present invention relates to an immune cell. From this point on, the immune cell of the present invention expresses

[0064] (i) a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising:

[0065] οp95HER2-specific antigen binding domain,

[0066] o transmembrane domain and

[0067] o At least one intracellular signaling domain and / or co-stimulatory domain

[0068] as well as

[0069] (ii) a bispecific antibody, comprising:

[0070] o a first antigen binding region that specifically binds to HER2 and

[0071] o a second antigen binding region that specifically binds to CD3;

[0072] As used herein, "immune cell" refers to a cell that plays a role in an immune response. Immune cells are derived from hematopoiesis 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 cell is a T cell; NK cell; NKT cell; lymphocytes, such as B cells and T cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0073] In certain embodiments of the methods of the invention, the immune cell is a T cell or a Natural Killer (NK) cell.

[0074] As used herein, the term "T cell" refers to a 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 also be separated or obtained from commercially available sources. T cells are any type of CD3 expression, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Treg) and gamma-delta T cells." Cytotoxic cells" include CD8+T cells, natural killer (NK) cells and neutrophils that can mediate cytotoxic responses. The terms "T cell" and "T lymphocyte" are interchangeable and are used interchangeably herein.

[0075] 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. Additional examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells and NKL cells. NK cells can be detected by specific surface markers such as CD16, CD56 and CD8 in humans. NK cells do not express T cell antigen receptors, pan-T markers CD3 or surface immunoglobulin B cell receptors.

[0076] Natural killer T (NKT) cells are a group of heterogeneous T cells that have common properties of both T cells and natural killer cells. Thus, NKT cells are a subpopulation of T cells that co-express the αβ T cell receptor, but also express a variety of molecular markers commonly associated with NK cells (such as NK1). Many of these cells recognize the non-polymorphic CD1d molecule, which is an antigen presenting molecule that binds self and foreign lipids and glycolipids. They only account for about 0.1% of all peripheral blood T cells. Natural killer T cells should not be confused with natural killer cells.

[0077] Immune cell of the present invention-chimeric antigen receptor

[0078] As used herein, "chimeric antigen receptor" or "CAR", also referred to as chimeric T cell receptor, T body, artificial T cell receptor and chimeric immune receptor (CIR), is an engineered receptor that transplants any specificity to immune effector cells. In classical CAR, the specificity of monoclonal antibodies is transplanted to T cells. Therefore, CAR is a fusion protein that includes at least an extracellular domain or antigen binding domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide derived from an extracellular domain, and at least one intracellular costimulatory domain.

[0079] 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 that can bind to an antigen. In the present invention, the antigen can be p95HER2, HER2 or CD3, i.e., the antigen binding domain or antigen binding region is specific for p95HER2, HER2 or CD3.

[0080] The antigen binding domain may include an antibody fragment, which refers to at least a portion of a complete antibody or a recombinant variant thereof, such as an antigen variable region of a complete antibody, which is sufficient to allow the antibody fragment to recognize and specifically bind to the target. The antigen binding domain of the present invention includes 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. In the context of the present invention, the antigen binding domain or antibody fragment includes at least one VH region and one VL region, but it may include two VL regions and two VH regions. Therefore, for example, in a specific embodiment, the p95HER2-specific antigen binding domain of the CAR expressed by the immune cells of the present invention is ScFv, and therefore, it will only include one VL region and one VH region. In another embodiment, the p95HER2-specific antigen binding domain of the CAR expressed by the immune cells of the present invention is a Fab fragment, in which case it will include a VL region and a VH region (Fab or Fab') or two VH regions and two VL regions (Fab2 or F(ab')2).

[0081] In certain embodiments, the antigen binding domain is humanized.

[0082] As used herein, "humanized" forms of non-human (e.g., mouse) antibodies or antigen-binding domains are chimeric antibodies or antigen-binding domains that contain minimal or no sequence from non-human immunoglobulins. In most cases, humanized antibodies or antigen-binding domains are human immunoglobulins (receptor antibodies), wherein the residues in the hypervariable regions of receptors are replaced by residues in the hypervariable regions of non-human species (donor antibodies) (such as mice, rats, rabbits, or non-human primates) with desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies or antigen-binding domains may include residues not found in receptor antibodies or donor antibodies. These modifications are generally in order to further improve the performance of antibodies or antigen-binding domains. Typically, humanized antibodies or antigen-binding domains will include substantially all of at least one and generally two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR residues are residues of human immunoglobulin sequences. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0083] The terms "HER2" and "HER2 receptor" are used interchangeably herein and refer to the ErbB2 protein (also referred to in the literature as HER2 / neu). As used herein, these terms are intended to include variants (e.g., splice variants), isomers, and homologs (orthologs and paralogs) of HER2. In some aspects, the binding of the anti-HER2 binding molecules disclosed herein to HER2 inhibits the growth of cells expressing HER2 (i.e., typically tumor cells, and particularly cancer cells expressing high levels of HER2) by inhibiting the formation of heterologous complexes between HER2 and other ErbB family members (e.g., inhibiting heterodimerization with EGFR or HER3).

[0084] HER2 is a receptor tyrosine kinase and consists of an extracellular domain (ECD) consisting 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 accession number P04626 of Uniprot, version 29 September 2021, consisting of 1,255 amino acids.

[0085] The antigen binding domain of the CAR expressed by the immune cells of the present invention specifically recognizes the amino terminal fragment of p95HER2 located outside the cell. In particular, the antigen binding domain is targeted to the p95HER2 amino acid sequence PIWKFPDE (SEQ ID NO: 5) of human p95HER2, which is located at 34 amino acids of the transmembrane domain that anchors p95HER2 to the membrane.

[0086] As used herein, the term "p95HER2" refers to a polypeptide resulting from a deletion of the N-terminal region of the HER2 receptor protein. The polypeptide is the carboxyl terminal fragment (CTF) of the HER2 receptor protein, also referred to as "611-CTF" or "100-115kDap95HER2". p95HER2 is characterized by an amino acid sequence corresponding to amino acids 611-1,255 of HER2, according to SEQ ID NO: 11.

[0087] SEQ ID NO:11

[0088] MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV

[0089] In certain embodiments, the p95HER2 - specific antigen - binding domain of the CAR expressed by the immune cells of the present invention is a ScFv.

[0090] As used herein, "single-chain variable fragment (ScFv)" means 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 technology, and wherein the variable (Fv) regions of immunoglobulin heavy chains (VH chains) and light chain (VL chains) fragments are connected via spacer sequences. A variety of methods for preparing ScFv are known, and include methods described in U.S. Patent Nos. 4,694,778, Nature, vol.334, p.54454 (1989) and Science, vol.242, pp.1038-1041 (1988). In another specific embodiment, the p95HER2-specific antigen binding domain of the CAR expressed by the immune cells of the present invention is a ScFv with a VL region at the N-terminus and a VH region at the C-terminus. In a specific embodiment, the p95HER2-specific antigen binding domain of the CAR expressed by the immune cells of the present invention is a ScFv with a VH region at the N-terminus and a VL region at the C-terminus. In another specific embodiment, the p95HER2-specific antigen binding domain of the CAR expressed by the immune cells of the present invention is a ScFv having a VL region at the N-terminus and a VH region at the C-terminus.

[0091] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) or complementarity determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.

[0092] The terms "CDR", "hypervariable region", "HVR", "complementarity determining region" or as used herein, refer to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody includes six CDRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). Therefore, the CDRs determine the affinity (roughly, binding strength) and specificity of the protein for a specific antigen. The CDRs of the two chains of each pair of chains are aligned by the framework region to obtain the function of binding to a specific epitope. Therefore, both the variable heavy chain and the variable light chain are characterized by three CDRs, namely VH-CDR1, VH-CDR2, VH-CDR3 and VL-CDR1, VL-CDR2, VL-CDR3.

[0093] As used herein, the term "functionally equivalent variant of a CDR sequence" refers to a sequence variant of a specific CDR sequence that has substantially similar sequence identity thereto and substantially retains its ability to bind to its cognate antigen when used as part of an antibody, antibody fragment, or antigen-binding domain of a ScFv as described herein. For example, a functionally equivalent variant of a CDR sequence may be a polypeptide sequence derivative of the sequence, including 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.

[0094] In a specific embodiment, the CDR1, CDR2 and CDR3 of the VH region of the ScFv of the CAR expressed by the immune cells of the present invention respectively include the sequences of SEQ ID NOs: 39, 40 and 41 or their functionally equivalent variants, and / or the CDR1, CDR2 and CDR3 of the VL region of the ScFv of the CAR expressed by the immune cells of the present invention respectively include the sequences of SEQ ID NOs: 42, 43 and 44 or their functionally equivalent variants.

[0095] district sequence SEQ ID NO: VH-CDR1 TYGMA SEQ ID NO:39 VH-CDR2 TINSNGGKTYHPDSVKG SEQ ID NO:40 VH-CDR3 EGFDY SEQ ID NO:41 VL-CDR1 KASQNVGTAVA SEQ ID NO:42 VL-CDR2 SASNRYT SEQ ID NO:43 VL-CDR3 QQYSTYPLT SEQ ID NO:44

[0096] As used herein, the term "conservative substitution" refers to the replacement of one amino acid by another amino acid having 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 conservatively substituted for each other:

[0097] 1) Alanine (A), serine (S), threonine (T);

[0098] 2) Aspartic acid (D), glutamic acid (E);

[0099] 3) Asparagine (N), glutamine (Q);

[0100] 4) Arginine (R), Lysine (K);

[0101] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0102] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0103] Functionally equivalent variants of the CDR sequences 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 envisioned that functionally equivalent variants of the CDR sequences include additions consisting 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 at the N-terminus, or 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. Likewise, variants are also envisioned to include deletions consisting 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 at the N-terminus, or the C-terminus, or both the N-terminus and the C-terminus of the corresponding amino acid sequence shown in one of the above sequences.

[0104] Functionally equivalent variants of the CDR sequences according to the present invention, when used as part of an antibody fragment or antigen-binding domain (such as the ScFv of the CAR expressed by the immune cells of the present invention), will preferably maintain SEQ ID NO: At least 70% of the ability of the corresponding amino acid sequence shown in one of 39-44 to bind to its cognate antigen, 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. This ability to bind to its cognate antigen can be determined by the affinity, avidity, specificity and / or selectivity of the antibody or antibody fragment to its cognate antigen.

[0105] CDR sequences can be determined according to conventional standards, for example, by IgBLAST: http: / / www.ncbi.nlm.nih.gov / igblast / (Ye et al., 2013, Nucleic Acids Res 41 (Web Server issue: W34-40), 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), or according to the numbering provided by Chothia et al. (1989, Nature 342: 877-83). This specific region has been described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and Chothia et al. al., J.Mol.Biol.196:901-917 (1987), wherein these definitions include overlaps or subsets of amino acid residues when compared to each other. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR. The CDR sequences given herein are generally based on the Kabat definition.

[0106] In the context of two or more amino acid or nucleotide sequences, the term "identity", "same" or "percent identity" refers to two or more sequences or subsequences that are identical or have a specific percentage of nucleotides or amino acid residues that are identical, and when compared and aligned (if necessary, introducing gaps) to achieve maximum correspondence, any conservative substitutions are not considered to be 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 amino acid or nucleotide sequence alignments are known in the art. Sequences can be aligned using publicly available software programs. Those skilled in the art can determine the appropriate parameters for maximum alignment of specific alignment software. In some embodiments, the default parameters of the alignment software are used. In some embodiments, the percent identity "X" of the first nucleotide sequence and the second nucleotide sequence is calculated as 100×(Y / Z), wherein Y is the number of nucleotide residues that are scored as identical matches in the alignment of the first and second sequences (aligned by visual inspection or 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 that considers the entirety of the two sequences is used, so all letters and gaps in each sequence must be aligned. In this case, the same formula as above can be used, but the length of the region in which the first and second sequences overlap is used as the Z value, the region having substantially the same length as the length of the first sequence.

[0107] For example, according to the present invention, 95% identity to a reference sequence is achieved, the parameters are set such that the percent identity is calculated over the entire length of the reference nucleotide sequence, and gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0108] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences in VH (or VL) usually appear in the following order: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0109] As used herein, the term "functionally equivalent variant of a FR sequence" refers to a sequence variant of a specific FR sequence that has substantially similar sequence identity thereto and substantially retains its ability to bind to its cognate antigen when used as part of an antibody or antibody binding domain as described herein. For example, a functionally equivalent variant of a FR sequence may be a polypeptide sequence derivative of the sequence, including additions, deletions or substitutions of one or more amino acids.

[0110] In a specific embodiment, FR1, FR2, FR3 and FR4 of the VH region of the CAR expressed by the immune cells of the present invention respectively include sequences of SEQ ID NOs: 45, 46, 47 and 48 or functionally equivalent variants thereof, and / or FR1, FR2, FR3 and FR4 of the VL region of the CAR expressed by the immune cells of the present invention respectively include sequences of SEQ ID NOs: 49, 50, 51 and 52 or functionally equivalent variants thereof.

[0111]

[0112]

[0113] Functionally equivalent variants of the FR sequences 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 sequences include additions consisting 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 at the N-terminus, or C-terminus, or at the N-terminus and C-terminus of the corresponding amino acid sequence shown in one of the above reference sequences. Likewise, variants are also envisioned to include deletions consisting 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 at the N-terminus, or the C-terminus, or both the N-terminus and the C-terminus of the corresponding amino acid sequence shown in one of the above sequences.

[0114] Functionally equivalent variants of the FR sequences according to the present invention, when used as part of the antigen-binding domain of the present invention, will preferably retain 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 by the affinity, avidity, specificity and / or selectivity values ​​of the antibody or antibody fragment for its cognate antigen.

[0115] In a specific embodiment, the VL of the ScFv of the CAR expressed by the immune cell of 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 with the sequence of SEQ ID NO: 54. In a specific embodiment, the VL of the ScFv of the CAR ScFv expressed by the immune cell of the present invention comprises the sequence of SEQ ID NO: 54. In a specific embodiment, the VL of the ScFv of the CAR ScFv of the present invention consists of the sequence of SEQ ID NO: 54 or consists essentially of the sequence of SEQ ID NO: 54.

[0116] SEQ ID NO:54

[0117] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTAVAWFQQKPGKAPKILIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPLTFGQGTKLEIK

[0118] In a specific embodiment, the VH of the ScFv of the CAR expressed by the immune cell of 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 with the sequence of SEQ ID NO: 55. In a specific embodiment, the VH of the ScFv of the CAR expressed by the immune cell of the present invention comprises the sequence of SEQ ID NO: 55. In a specific embodiment, the VH of the ScFv of the CAR expressed by the immune cell of the present invention consists of the sequence of SEQ ID NO: 55 or consists essentially of the sequence of SEQ ID NO: 55.

[0119] SEQ ID NO:55

[0120] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMAWVRQTPDRRLELVATINSNGGKTYHPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREGFDYWGQGTLVTVSS

[0121] In a specific embodiment, the VL of the ScFv of the CAR expressed by the immune cells of 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:54, and the VH of the ScFv of the CAR expressed by the immune cells of 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:55.

[0122] In a specific embodiment, the VL of the ScFv of the CAR expressed by the immune cell of the present invention comprises the sequence of SEQ ID NO:54, and the VH of the ScFv of the CAR expressed by the immune cell of the present invention comprises the sequence of SEQ ID NO:55.

[0123] In a specific embodiment, the VL of the ScFv of the CAR expressed by the immune cells of the present invention consists of or essentially consists of the sequence of SEQ ID NO:54, and the VH of the ScFv of the CAR expressed by the immune cells of the present invention consists of or essentially consists of the sequence of SEQ ID NO:55.

[0124] In another specific embodiment of the CAR expressed by the immune cells of the present invention, the VH and VL regions of the ScFv of the CAR expressed by the immune cells of the present invention are connected by a linker region.

[0125] The term "flexible polypeptide linker" or "linker" refers to a peptide linker composed of amino acids (such as glycine and / or serine residues) that is used alone or in combination to connect the variable heavy chain and variable light chain regions together; or to connect any or multiple regions of the CAR expressed by the immune cells of the present invention.

[0126] The linker peptide can have any of a variety of amino acid sequences. Proteins can be connected by spacer peptides, which are usually flexible, but other chemical connections are not excluded. The linker can be a peptide of about 6 to 40 amino acids in length, or a peptide of about 6 to 25 amino acids in length. These linkers can be produced with coupled proteins using synthetic linker encoding oligonucleotides. Peptide linkers with a certain degree of flexibility can be used. The connecting peptide can have almost any amino acid sequence, but it should be remembered that a suitable linker will have a sequence that results in a common flexible peptide. The small amino acids used (such as glycine and alanine) can be used to produce flexible peptides. The generation of such sequences is conventional for those skilled in the art.

[0127] Suitable linkers can be readily selected and can be of any suitable different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0128] Exemplary flexible linkers include linkers having the sequence TGSTSGSGKPGSGEGS (SEQ ID NO: 53). Suitable linkers include, for example, glycine polymers (G)n, glycine-serine polymers (including, for example, (GS) n 、(GSGGS) n (SEQ ID NO:67) and (GGGS) n(SEQ ID NO:68), wherein n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In a particular embodiment, the linker comprises a glycine polymer of formula (G4S)3. Glycine and glycine-serine polymers are of interest because these amino acids are relatively unstructured and therefore can serve as neutral tethers between components. Glycine polymers are of particular interest because glycine can enter 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 ordinary skill will recognize that the design of a peptide conjugated to any of the above elements can include a fully or partially flexible linker such that the linker can include a flexible linker and one or more portions of a structure that imparts less flexibility.

[0129] In a specific embodiment, the joint is located between the VH and VL regions of the ScFv of the CAR expressed by the immune cells of the present invention. In another specific embodiment, the VH and VL regions of the ScFv of the CAR expressed by the immune cells of the present invention are connected by a joint region including SEQ ID NO:53. In an embodiment, the ScFv of the CAR expressed by the immune cells of the present invention includes the structure VL-joint-VH. In another embodiment, the ScFv of the CAR expressed by the immune cells of the present invention may have the structure VH-joint-VL or VL-joint-VH. In a specific embodiment, the joint is located at the C-terminus relative to the VL region and the N-terminus relative to the VH region, i.e., VL-joint-VH.

[0130] In a specific embodiment, the ScFv of the CAR expressed by the immune cells of 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%, or at least 99% sequence identity to the sequence of SEQ ID NO:56.

[0131] SEQ ID NO:56

[0132] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTAVAWFQQKPGKAPKILIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPLTFGQGTKLEIKTGSTSGSGKPG SGEGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMAWVRQTPDRRLELVATINSNGGKTYHPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREGFDYWGQGTLVTVSS

[0133] In another specific embodiment, the ScFv of the CAR expressed by the immune cell of the present invention comprises the sequence of SEQ ID NO: 56. In yet another specific embodiment, the ScFv of the CAR expressed by the immune cell of the present invention consists of the sequence SEQ ID NO: 56 or consists essentially of the sequence SEQ ID NO: 56.

[0134] The second element of the CAR expressed by the immune cells of the present invention is a transmembrane domain attached to the extracellular domain of the CAR.

[0135] As used herein, "transmembrane domain" (TMD) refers to the region where CAR passes through the cell membrane. The transmembrane domain of the CAR expressed by the immune cells of the present invention is a transmembrane domain, an artificial hydrophobic sequence or a combination thereof of a transmembrane protein (e.g., type I transmembrane protein). A transmembrane domain may include one or more additional amino acids adjacent to a transmembrane region, for example, one or more amino acids associated with the extracellular region of a protein derived from a transmembrane protein (e.g., 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the extracellular region) and / or one or more additional amino acids associated with the intracellular region of a protein derived from a transmembrane protein (e.g., 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the intracellular region). On the one hand, a transmembrane domain is a domain associated with one of the other domains of the CAR used. In some cases, a transmembrane domain may be selected or modified by amino acid replacement to avoid such domains from being combined with the transmembrane domain of the same or different surface membrane proteins, for example, to minimize the interaction with other members of the receptor complex. In a specific embodiment, the transmembrane domain of the CAR expressed by the immune cells of the present invention is capable of homodimerization with another CAR on the surface of the CAR T cell. In different specific embodiments of the CAR expressed by the immune cells of the present invention, the amino acid sequence of the transmembrane domain can be modified or replaced to minimize the interaction with the binding domain of the natural binding partner present in the same CAR T.

[0136] The transmembrane domain can be derived from a natural source or a recombinant source. When the source is a natural source, the domain can be derived from any membrane-bound or transmembrane protein. On the one hand, whenever CAR is bound to a target, the transmembrane domain can conduct signals to one or more intracellular domains. Non-limiting examples or specific uses of transmembrane domains particularly used in the present invention may include at least one or more transmembrane regions of the following: for example, α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD3ζ, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD 278), 4-1BB (CD137), GITR, CD40, CTLA4, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4 CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDGA, CDGA, CD103, ITGAL, CDLa, LFA-1, ITGAM, CDllb, ITGAX, CDlc, ITGB1, CD2 9. ITGB2, CD18, LFA-1, LGAITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT 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, including NKG2D, and / or a transmembrane domain selected from the transmembrane domain of NKG2C.

[0137] In a specific embodiment of the CAR expressed by the immune cells of the present 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 CD3ζ transmembrane domain.

[0138] In a specific embodiment, the transmembrane domain is a CD28 transmembrane domain. In a specific embodiment, the CD28 transmembrane domain comprises the sequence SEQ ID NO:57.

[0139] SEQ ID NO:57

[0140] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0141] In another specific embodiment of the CAR expressed by the immune cells of the present 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%, or at least 99% sequence identity to the sequence of SEQ ID NO:57.

[0142] In another specific embodiment of the CAR expressed by the immune cells of the present invention, the transmembrane domain includes the sequence of SEQ ID NO:57.

[0143] In another specific embodiment of the CAR expressed by the immune cell of the present invention, the transmembrane domain consists of or essentially consists of the sequence SEQ ID NO:57.

[0144] The CAR according to the present invention comprises at least one intracellular signaling domain and / or a co-stimulatory domain.

[0145] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule, and more specifically refers to any oligopeptide or polypeptide known to be used as a domain for transmitting signals to cause activation or inhibition of biological processes in cells. The intracellular signaling domain generates signals that stimulate the immune effector functions of CAR-containing cells (e.g., CAR-T cells). For example, the effector function of a T cell can be a cytolytic function or an auxiliary activity, including the secretion of cytokines. Therefore, the intracellular signaling domain can be a part of a protein that transmits effector function signals and instructs cells (e.g., T cells) to perform specialized functions.

[0146] Typically, the entire intracellular signaling domain can be used; however, it is understood that the entire domain does not have to be used, as long as any portion of the signaling domain used is still capable of transmitting the effector function signal. It is also understood that variants of such intracellular signaling domains with substantially the same or greater functional capacity can also be used. By this we include the meaning that the variant should have substantially the same or greater effector function signal transduction. Typically, substantially the same or greater signal transduction includes at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120%, or more of the signal transduction of the unmodified intracellular signaling domain, wherein the signal transduction of the unmodified intracellular signaling domain corresponds to 100%. Methods for evaluating effector function signal transduction are well known to those skilled in the art and include, for example, evaluating the amount and / or activity of molecules (e.g., proteins, such as cytokines) that indicate the transduction signal. Thus, when the signal is the cytolytic function of T cells, the method may involve measuring one or more cytokines secreted by T cells that are known to have cytolytic activity (e.g., IFNγ). Another method of assessing cytolytic function is by CFSE staining and counting positive cells by flow cytometry or chromium release assay, as is well known in the art.

[0147] Examples of intracellular signaling domains for CARs expressed by immune cells of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capability.

[0148] It is known that the signal generated by a single TCR is usually insufficient to fully activate T cells, and secondary and / or co-stimulatory signals may also be required. Therefore, it can be said that T cell activation is mediated by two different types of intracellular signal sequences: sequences that initiate antigen-dependent primary activation through TCR (primary intracellular signaling domains) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary intracellular signaling domains, such as co-stimulatory domains). Co-stimulatory domains promote the activation of effector functions, and can also promote the persistence of effector functions and / or the survival of cells.

[0149] In a specific embodiment of the CAR expressed by the immune cell of the present invention, at least one intracellular signaling domain of the CAR includes a co-stimulatory domain, a primary signaling domain, or any combination thereof.

[0150] The primary intracellular signaling domain regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary intracellular signaling domain that acts in a stimulatory manner can include a signaling motif, which is referred to as an activation motif or ITAM (e.g., 2, 3, 4, 5 or more ITAMs) based on immunoreceptor tyrosine. Therefore, the intracellular signaling domain can include one or more ITAMs. It should be understood that one or more ITAMs of the intracellular signaling domain can be modified, for example, by mutation. Compared with the native ITAM domain, modification can be used to increase or decrease the signaling function of ITAM.

[0151] Examples of ITAMs that are particularly useful in the present invention that contain primary intracellular signaling domains include those of CD3ζ, Fc receptor γ, Fc receptor β, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In a specific embodiment, at least one intracellular signaling domain of the CAR expressed by the immune cells of the present invention is selected from the group consisting of CD3ζ, Fc receptor γ, Fc receptor β, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In a more specific embodiment, at least one intracellular signaling domain of the CAR expressed by the immune cells of the present invention is a CD3-ζ intracellular domain.

[0152] The term "CD3" refers to the human CD3 protein complex, which consists of six different chains (CD3γ chain (SwissProt P09693), CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766) and a CD3ζ chain homodimer (SwissProt P20963) (εγ:εδ:ζζ), and associates with the T cell receptor α and β chains. The term includes any CD3 variants, isoforms and species homologs that are naturally expressed by cells (including T cells) or expressed on cells transfected with genes or cDNAs encoding the above chains.

[0153] The term "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCR-ζ" is defined as the protein represented by GenBank entry number BAG36664.1, or equivalent residues from non-human species (such as mouse, rodent, monkey, primate, etc.), and "ζ stimulatory domain" or alternatively "CD3 ζ stimulatory domain" or "TCR ζ stimulatory domain" is defined as the amino acid residues of the cytoplasmic domain of the ζ chain that are sufficient to functionally transmit the primary signal required for activation of T cells, etc. In one aspect, the ζ cytoplasmic domain includes residues 52 to 164 included in the protein of GenBank entry BAG36664.1, or equivalent residues from non-human species (e.g., mouse, rodent, monkey, primate, etc.), which are their functional orthologs.

[0154] As described above, the intracellular signaling domain can include a primary intracellular signaling domain alone, or it can include a combination of a primary intracellular signaling domain and one or more secondary intracellular signaling domains (such as one or more costimulatory signaling domains). Therefore, the intracellular signaling domain of the CAR can include 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).

[0155] The costimulatory signaling domain refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule.

[0156] The term "costimulatory molecule" refers to an identifiable T cell binding partner, which specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response produced by T cells, such as but not limited to proliferation. Co-stimulatory molecules are cell surface molecules other than antigen-specific receptors or their ligands, which are essential for an effective immune response. Co-stimulatory molecules can be cell surface molecules other than antigen receptors or their ligands, which are essential for an effective response of immune cells (such as lymphocytes) to antigens. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins) and NK cell activation 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 a ligand that specifically binds to CD83, etc. For example, CD27 co-stimulation has been shown to enhance in vitro expansion, effector function, and survival of human CAR T cells, and enhance human T cell persistence and in vivo anti-tumor activity (Song et al. Blood. 2012; 1 19(3): 696-706).

[0157] In another embodiment, the at least one intracellular signaling domain comprises an intracellular domain of a co-stimulatory molecule selected from OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP 12 and 4-1BB (CD137), or any combination thereof.

[0158] In a specific embodiment, the CAR expressed by the immune cell of the present invention includes the intracellular domain of the costimulatory molecule CD28. In a more specific embodiment, the intracellular domain of the costimulatory molecule includes the sequence of SEQ ID NO:58.

[0159] SEQ ID NO:58

[0160] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0161] In a specific embodiment, at least one intracellular signaling domain of the CAR expressed by the immune cells of the present invention further includes a CD3-ζ intracellular domain. In a more specific embodiment, at least one intracellular signaling domain of the CAR expressed by the immune cells of the present invention further includes a sequence of SEQ ID NO: 59.

[0162] SEQ ID NO:59

[0163] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0164] In a specific embodiment, at least one intracellular signaling domain of the CAR expressed by the immune cell of the present invention is arranged at the N-terminal side relative to the CD3-zeta intracellular domain.

[0165] The intracellular signaling sequences in the intracellular part of the CAR expressed by the immune cells of the present invention can be connected to each other in a random or specified order. Optionally, short oligonucleotides or polypeptide linkers, for example, a length of between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) can form a connection between intracellular signaling sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In another embodiment, a single amino acid (such as alanine or glycine) can be used as a suitable linker.

[0166] In one embodiment, the intracellular signaling domain is designed to include two or more, e.g., 3, 4, 5 or more costimulatory signaling domains. In an embodiment, two or more, e.g., 2, 3, 4, 5 or more costimulatory signaling domains are separated by a linker molecule (such as a linker molecule 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.

[0167] In a preferred embodiment, the intracellular portion of the CAR expressed by the immune cell of the present invention comprises:

[0168] - the signaling domain of CD3ζ and the signaling domain of CD28,

[0169] - the signaling domain of CD3-ζ and the signaling domain of 4-1BB,

[0170] - the signaling domain of CD3-ζ and the signaling domain of OX40,

[0171] - the signaling domain of CD3-ζ and the signaling domain of ICOS,

[0172] -The signaling domain of CD3-ζ and the signaling domain of DAP10

[0173] -The signaling domain of CD3-ζ, the signaling domain of 4-1BB, and the signaling domain of OX40.

[0174] -The signaling domain of 4-1BB and the signaling domain of CD28.

[0175] The intracellular signaling domain may include the entire intracellular portion, or the entire native intracellular signaling domain, its source molecule, or a functional fragment thereof.

[0176] In another embodiment, the CAR expressed by the immune cell of the present invention further includes a hinge domain between the antigen binding domain and the transmembrane domain.

[0177] As used herein, "hinge domain", "hinge region" or "spacer" refers to an amino acid region that allows separation and flexibility of the binding moiety and T cell membrane. The length of the flexible hinge also allows for better binding to relatively inaccessible epitopes, for example, allowing for a longer hinge domain for optimal binding. Those skilled in the art will be able to determine the appropriate hinge for a given CAR target.

[0178] In some cases, the first polypeptide of the CAR expressed by the immune cell of the present invention includes 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 hinge domain derived from CD8).

[0179] The length of the hinge domain may be between about 10 amino acids and about 200 amino acids, preferably between 50 and 150 amino acids, and more preferably between 75 and 125 amino acids.

[0180] Exemplary hinge domains include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:67), and (GGGS)n (SEQ ID NO:68), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine can enter significantly more phi-psi space than alanine and is much less constrained 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), etc.

[0181] In some cases, the hinge domain in the first polypeptide of the CAR expressed by the immune cells of the present invention includes at least one cysteine. For example, in some cases, the hinge domain may include the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 75). If present, the cysteine ​​in the hinge domain of the first CAR can be used to form a disulfide bond with the hinge domain in the second CAR.

[0182] 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 a non-limiting example, an immunoglobulin hinge domain can include 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); ELKTPLGDTTHT (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); etc.

[0183] The hinge domain may include the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 hinge domain. The hinge domain may include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge domain. For example, His 229 of a human IgG1 hinge may be replaced by Tyr so that the hinge domain includes the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 86); see, e.g., Yan et al. (2012) J. Biol. Chem. 287: 5891).

[0184] The hinge domain may include an amino acid sequence derived from human CD8 or a variant thereof.

[0185] In specific embodiments, the hinge domain comprises, consists of, or consists essentially of a CD8 hinge domain.

[0186] In another specific embodiment, the hinge domain comprises the sequence of SEQ ID NO:60.

[0187] SEQ ID NO:60

[0188] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0189] 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 co-stimulatory domain.

[0190] In a specific embodiment, the CAR expressed by the immune cells of the present invention includes a CD8 hinge domain, a CD28 transmembrane domain, a CD3ζ intracellular signaling domain, and a CD28 co-stimulatory domain.

[0191] In another embodiment, the CAR expressed by the immune cells of the present invention includes, 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 co-stimulatory signaling domain, followed by a CD3ζ intracellular signaling domain.

[0192] In a specific embodiment, the CAR expressed by the immune cell of the present invention includes SEQ ID NO:61.

[0193] SEQ ID NO:61

[0194] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTAVAWFQQKPGKAPKILIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPLTFGQGTKLEIKTGSTSGSG KPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMAWVRQTPDRRLELVATINSNGGKTYHPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREGFDYWGQGT LVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0195] Immune cells of the present invention-bispecific antibodies

[0196] In addition to CAR, the immune cells of the present invention further express bispecific antibodies that specifically bind to HER2 and CD3.

[0197] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of them, which has the ability to specifically bind to an antigen under typical physiological conditions. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region (Ab) of the antibody can mediate the binding of the immunoglobulin to host tissues or factors (including various cells of the immune system). As mentioned above, unless otherwise stated or clearly contradictory to the context, the term antibody herein includes fragments of antibodies, which are previously defined antigen-binding fragments. It should also be understood that, unless otherwise stated, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAb), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen provided by any known technology.

[0198] In the context of the present invention, the term "bispecific antibody" refers to an antibody that has two different antigen-binding regions defined by different antibody sequences.

[0199] In a specific embodiment of the bispecific antibody expressed by an immune cell of the present invention, the first and second antigen binding regions of the bispecific antibody are ScFv.

[0200] In another specific embodiment, the bispecific antibody expressed by the immune cell of the present invention is characterized by:

[0201] - the VH region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or functionally equivalent sequences thereof;

[0202] - the VL region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 4, SEQ ID NO: 100 and SEQ ID NO: 6 or functionally equivalent sequences thereof;

[0203] - the VH region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or functionally equivalent sequences thereof; and / or

[0204] - the VL region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12 or functionally equivalent sequences thereof,

[0205] The antibody is characterized in that it exhibits high affinity for HER2 and will be referred to as a "high affinity" antibody hereinafter. In a specific embodiment of the bispecific antibody, the dissociation constant (K) of the ScFv of the high affinity antibody is D ) is about 0.1 to about 1.0 nM, preferably about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM or about 0.9 nM.

[0206] In another specific embodiment, the bispecific antibody expressed by the immune cell of the present invention is characterized by:

[0207] - the VH region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 102 or functionally equivalent sequences thereof;

[0208] - the VL region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 4, SEQ ID NO: 103 and SEQ ID NO: 6 or functionally equivalent sequences thereof;

[0209] - the VH region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or functionally equivalent sequences thereof; and / or

[0210] - The VL region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12 or functionally equivalent sequences thereof.

[0211] The antibody is characterized in that it exhibits a medium affinity for HER2 and will be referred to as a "medium affinity (Med Aff.")" antibody hereinafter. In a specific embodiment of the bispecific antibody, the dissociation constant (K) of the ScFv of the medium affinity antibody is D) is 500 to 2000nM, preferably about 600nM, about 700nM, about 800nM, about 900nM, about 1000nM, about 1100nM, about 1200nM, about 1300nM, about 1400nM, about 1500nM, about 1600nM, about 1700nM, about 1800nM, about 1900nM, about 2000nM, more preferably about 1020nM, about 1040nM, about 1060nM, about 1080nM, about 1120nM, about 1140nM, about 1160nM, about 1180nM.

[0212]

[0213] As used herein, the term "binding" in the context of binding of an antibody / ScFv / FV / CDR to a predetermined antigen or epitope generally refers to binding with an affinity or "binding affinity" corresponding to about 10 when measured by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as ligand and the antibody as analyte. -7 M or lower KD, such as about 10 -8 M or lower, such as about 10 -9 M or lower, about 10 -10 M or lower, or about 10 -11 M or even lower, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten times lower, such as at least 100 times lower, for example at least 1,000 times lower, such as at least 10,000 times lower, for example at least 100,000 times lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount of affinity reduction depends on the KD of the antibody, so that when the KD of the antibody is extremely low (i.e., the antibody is highly specific), then the affinity for the antigen is at least 10,000 times lower than the amount of affinity for the non-specific antigen.

[0214] The above method for determining antibody / ScFv / FV / CDR binding affinity can also be used to compare the binding affinity between antibodies. In another specific embodiment of the bispecific antibody expressed by the immune cell of the present invention, the bispecific antibody Med Aff of the immune cell of the present invention has a binding affinity that is at least 2 times lower, at least 3 times lower, at least 4 times lower, or at least 5 times lower than the bispecific antibody High Aff of the immune cell of the present invention.

[0215] In another specific embodiment of the immune cell-expressed bispecific antibody of the present invention:

[0216] - the VH region of the ScFv of the first antigen-binding region has FR1, FR2, FR3 and FR4, wherein FR1, FR2, FR3 and FR4 respectively include the sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 or functionally equivalent sequences thereof;

[0217] - the VL region of the ScFv of the first antigen-binding region has FR1, FR2, FR3 and FR4, wherein FR1, FR2, FR3 and FR4 respectively include the sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 or a functionally equivalent sequence thereof;

[0218] - the VH region of the ScFv of the second antigen-binding region has FR1, FR2, FR3 and FR4, wherein FR1, FR2, FR3 and FR4 respectively include the sequences of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 or functionally equivalent sequences thereof; and / or

[0219] - The VL region of the ScFv of the second antigen-binding region has FR1, FR2, FR3 and FR4, wherein FR1, FR2, FR3 and FR4 respectively include the sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28 or functionally equivalent sequences thereof.

[0220]

[0221]

[0222] In a specific embodiment of the bispecific antibody expressed by the immune cell of the present invention:

[0223] - 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,

[0224] - 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,

[0225] - 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 SEQ ID NO: 31, and / or,

[0226] -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%, or at least 99% sequence identity to the sequence of SEQ ID NO:32.

[0227] SEQ ID NO:29

[0228] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS

[0229] SEQ ID NO:104

[0230] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFVAMDVWGQGTLVTVSS

[0231] SEQ ID NO:30

[0232] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0233] SEQ ID NO:105

[0234] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0235] SEQ ID NO:31

[0236] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE

[0237] SEQ ID NO:32

[0238] DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0239] In a specific embodiment of the bispecific antibody expressed by the immune cell of the present invention:

[0240] - 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,

[0241] - 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,

[0242] - the VH region of the ScFv of the second antigen-binding region comprises the sequence of SEQ ID NO: 31 and / or,

[0243] - The VL region of the ScFv of the second antigen-binding region comprises the sequence of SEQ ID NO:32.

[0244] In a specific embodiment of the bispecific antibody expressed by the immune cell of the present invention:

[0245] - the VH region of the ScFv of the first antigen-binding region consists of or essentially consists of the sequence of SEQ ID NO: 29 or SEQ ID NO: 104,

[0246] - the VL region of the ScFv of the first antigen-binding region consists of or essentially consists of the sequence of SEQ ID NO: 30 or SEQ ID NO: 105,

[0247] - the VH region of the ScFv of the second antigen-binding region consists of or essentially consists of the sequence of SEQ ID NO: 31, and / or,

[0248] - the VL region of the ScFv of the second antigen-binding region consists of or essentially consists of the sequence of SEQ ID NO:32.

[0249] It will be clear to experts in the field that the organization of the ScFv domains of the first and second antigen binding regions can be varied without affecting their function. Therefore, in a specific embodiment, the bispecific antibody expressed by the immune cell of the present invention has the following region arrangement:

[0250] -VL(HER2)-VH(HER2)-VH(CD3)-VL(CD3);

[0251] -VL(HER2)-VH(HER2)-VL(CD3)-VH(CD3);

[0252] -VH(HER2)-VL(HER2)-VH(CD3)-VL(CD3);

[0253] -VH(HER2)-VL(HER2)-VL(CD3)-VH(CD3);

[0254] -VL(CD3)-VH(CD3)-VH(HER2)-VL(HER2);

[0255] -VL(CD3)-VH(CD3)-VL(HER2)-VH(HER2);

[0256] -VH(CD3)-VL(CD3)-VH(HER2)-VL(HER2); or

[0257] -VH(CD3)-VL(CD3)-VL(HER2)-VH(HER2).

[0258] Among them, VH(HER2) is the VH region of the ScFv of the anti-HER2 antibody, VL(HER2) is the VL region of the ScFv of the anti-HER2 antibody, VH(CD3) is the VH region of the ScFv of the anti-CD3 antibody, and VL(CD3) is the VL region of the ScFv of the anti-CD3 antibody.

[0259] In a specific embodiment of the bispecific antibody expressed by immune cells of the present invention, 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.

[0260] In another specific embodiment of the immune cell-expressed bispecific antibody of the present invention, the linker is selected from SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35.

[0261] SEQ ID NO:33GGGGSGGGGSGGGGS

[0262] SEQ ID NO:34GGGGS

[0263] SEQ ID NO:35GGSGGSGGSGGSGGVD

[0264] In a more specific embodiment of the bispecific antibody expressed by the immune cell of the present invention

[0265] - 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,

[0266] -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.

[0267] SEQ ID NO:36

[0268] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS

[0269] SEQ ID NO:37

[0270] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0271] SEQ ID NO:106

[0272] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFVAMDVWGQGTLVTVSS

[0273] In another specific embodiment of the bispecific antibody expressed by immune cells of the present invention,

[0274] - the ScFv of the first antigen-binding region comprises the sequence of SEQ ID NO: 36 or SEQ ID NO: 106,

[0275] - The ScFv of the second antigen binding region comprises the sequence of SEQ ID NO:37.

[0276] In a more specific embodiment of the bispecific antibody expressed by the immune cell of the present invention

[0277] - the ScFv of the first antigen-binding region consists of or essentially consists of the sequence of SEQ ID NO: 36 or SEQ ID NO: 106,

[0278] - The ScFv of the second antigen binding region consists of or essentially consists of the sequence of SEQ ID NO:37.

[0279] In yet another specific embodiment, the bispecific antibody expressed by the immune cells of the present invention has the sequence of SEQ ID NO:38 or SEQ ID NO:107.

[0280] SEQ ID NO:38

[0281] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSGG GGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSV EGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0282] SEQ ID NO:107

[0283] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGSGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFVAMDVWGQGTLVTVSSGG GGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSV EGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0284] Uses of immune cells of the present invention

[0285] The immune cells of the invention find use in medicine, in particular in cancer treatment. Thus, one aspect of the invention is the immune cells of the invention for use in medicine.

[0286] Another aspect of the present invention is the use of the immune cell of the present invention in a method for preventing or treating cancer.

[0287] As used herein, the terms "treat", "treatment", "treatment" or "amelioration" are used. The term refers to therapeutic treatment, the purpose of which is to reverse, reduce, inhibit, delay or stop the progression or severity of a condition associated with a disease or illness. The term "treatment" includes reducing or alleviating at least one adverse effect or condition of a condition (such as cancer), a disease or an illness. Treatment is usually "effective" when one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if disease progression is delayed or stopped. That is, "treatment" includes not only the improvement of symptoms or markers, but also interrupts at least one condition that indicates the expected symptom progression or deterioration in the absence of treatment. Whether detectable or not, a beneficial or desired clinical outcome is a reduction in one or more symptoms, a reduction in the extent of the disease, a stabilization of the disease condition (i.e., no aggravation), a delay or slowing of the progression of the disease (these include but are not limited to), an improvement or alleviation of the disease state, and relief (partial or complete). The term "treatment" of a disease also includes alleviating the symptoms or side effects of the disease (including symptomatic treatment). In some embodiments, treating cancer includes reducing tumor size, reducing the number of cancer cells, inhibiting cancer metastasis, prolonging life, reducing cancer cell growth, reducing cell survival, or reducing the state of cancer. This involves improving various physiological symptoms involved.

[0288] In certain embodiments of the present disclosure, immune cells are delivered to individuals in need thereof, such as individuals with cancer. These cells then enhance the immune system of the individual to attack the corresponding cancer cells. In some cases, one or more doses of immune cells are provided to the individual. If two or more doses of immune cells are provided to the individual, the interval between the two administrations should be sufficient to allow the immune cells to proliferate in the individual, and in a specific embodiment, the interval between the two doses is 1, 2, 3, 4, 5, 6, 7 days or more days.

[0289] In certain embodiments, the growth factor that promotes immune cell growth and activation is administered to the subject simultaneously with the immune cell or after the immune cell. The immune cell growth factor can be any suitable growth factor that promotes immune cell growth and activation. The example of suitable immune cell growth factor includes 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.

[0290] A therapeutically effective amount of immune cells can be administered by a variety of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection or infusion.

[0291] The immune cell population can be administered according to a treatment regimen consistent with the disease, such as a single dose or several doses within one to several days to improve the disease state or regular doses over an extended period of time to inhibit disease progression and prevent disease recurrence. The exact dose used in the preparation will also depend on the route of administration and the severity of the disease or illness, and should be determined according to the doctor's judgment and the circumstances of each patient. The therapeutically effective number of immune cells will depend on the subject being treated, the severity and type of the ailment, and the mode of administration. In some embodiments, the therapeutically effective number of immune cells can vary from about 5×106 cells per kg body weight to about 7.5×108 cells per kg body weight, such as about 2×107 cells per kg body weight to about 5×108 cells, or about 5×107 cells per kg body weight to about 2×108 cells. Those skilled in the art can easily determine the exact number of immune cells based on the age, weight, sex, and physiological condition of the subject. The effective dose can be inferred from a dose-response curve derived from an in vitro or animal model test system.

[0292] In certain embodiments, the compositions and methods of the present embodiments relate to a combination of an immune cell population and at least one additional therapy. The additional therapy may be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virotherapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy.

[0293] In some embodiments, additional therapy is administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, additional therapy is administration of side effect limiting agents (e.g., agents intended to reduce the occurrence and / or severity of treatment side effects, such as antiemetics, etc.). In some embodiments, additional therapy is radiotherapy. In some embodiments, additional therapy is surgery. In some embodiments, additional therapy is a combination of radiotherapy and surgery. In some embodiments, additional therapy is gamma irradiation. In some embodiments, additional therapy is therapy targeting PBK / AKT / mTOR pathways, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors and / or chemopreventive agents. Additional therapy can be one or more chemotherapeutic agents known in the art.

[0294] Various combinations can 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":

[0295] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0296] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0297] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0298] Administration of any compound or therapy of the present embodiments to a patient will follow the general regimen 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 toxicity due to the combination therapy.

[0299] A 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 refer to a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their intracellular activity mode, for example, whether and at which stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, insert DNA, or induce chromosome and mitotic aberrations by affecting nucleic acid synthesis.

[0300] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylmelamine; polyacetogenins (particularly blatacin) and acetogenins. camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including its synthetic analogue KW-218 9 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednisone prednimustine, trofosfamide and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II); dynemicins, including dynemicin A;Bisphosphonates such as clodronate; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycins, authrarnycins, azaserines, bleomycins, cactinomycins, carabicins, carminomycins, carzin ophilin), chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolites, such as methotrexate and 5-fluorouracil (5-FU); folate analogs, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, fluorouracil deoxynucleoside, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents, such as mitotane and trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anguidine); urethanes; vindesine; dacarbazine; mannomustine; dibromomannitol;dibromodulan (mitolactol); pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine; 6-mercaptoguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate (edatrexate) exate); daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin; procarbazine, plicomycin, gemcitabien, navelbine, farnesol protein transferase inhibitors, transplatinum, and any pharmaceutically acceptable salts, acids or derivatives thereof. ;

[0301] The definition of "chemotherapeutic agent" also includes: (i) anti-hormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapristone, and (toremifine citrate); (ii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, (Megestrol acetate), (exemestane; Pfizer), formestanie, fadrozole, Rl (vorozole), (letrozole; Novartis) and (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors, such as ME inhibitors (WO 2007 / 044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly antisense oligonucleotides that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, for example, PKC-α, Raf and H-Ras, such as oblimersen ( Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g., ) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, for example, and VAX1 rlL-2; topoisomerase 1 inhibitors, such as rmRH; (ix) anti-angiogenic agents, such as bevacizumab ( Genentech); and any pharmaceutically acceptable salts, acids and derivatives thereof.

[0302] Protein kinase inhibitors include tyrosine kinase inhibitors, which inhibit the tyrosine kinase activity of tyrosine kinases (such as ErbB receptors) to a certain extent. Examples of tyrosine kinase inhibitors include drugs targeting EGFR-10, such as: (i) antibodies that bind to EGFR, including MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US 4943533, Mendelsohn et al.) and variants thereof, such as chimeric 225 (C225 or cetuximab; Imclone) and remodeled human 225 (H225) (WO 96 / 40210, Imclone Systems Inc.); antibodies that bind to type II mutant EGFR (US5212290); humanized and chimeric antibodies that bind to EGFR (US5891996); and human antibodies that bind to EGFR, such as ABX-EGF (WO 98 / 50433); (ii) anti-EGFR antibodies conjugated to cytotoxic agents (EP 659439A2); and small molecules that bind to EGFR, including ZD1839 or gefitinib (Gefitinib) QRESSA TM ; Astra Zeneca), Erlotinib HC1 (CP-358774, TARCEVA TM ; Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen), quinazolines (such as PD 153035, 4-(3-chloroanilino)quinazoline), pyridopyrimidines, pyrirnidopyrirnidines, 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)phthalimide), tyrosine kinase inhibitors containing a nitrothiophene moiety (tyrphostine); PD-0183805 (Warner-Lambert) and antisense molecules (e.g., molecules that bind to ErbB encoding nucleic acids).

[0303] In yet another embodiment, the ADC cytotoxic agent or chemotherapeutic agent of the present invention is an anti-tubulin agent. In a more specific embodiment, the cytotoxic agent is selected from the group consisting of vinca alkaloids, podophyllotoxin, taxanes, baccatin derivatives, scutellarin, maytansinoids, combretastatin and dolastatin. 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, auristatin or other dolastatin derivatives such as auristatin E or auristatin F, AEB, AEVB, AEFP, MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), eruterobin or netropsin.

[0304] In addition, potent chemotherapeutic agents such as CC-1065 analogs, calikiamycin, maytansine, dolastatin 10 analogs, lysoxins, and palytoxins can be attached to ADCs using conditionally stable linkers to form potent immunoconjugates.

[0305] In a specific embodiment, the chemotherapeutic agent is selected from lapatinib, tucatinib, and neratinib.

[0306] As used herein, the term "cancer" or "tumor" or "tumor disease" refers to a large class of diseases involving unregulated cell growth, and it is also known as a malignant neoplasm. The term is generally applied to diseases characterized by uncontrolled cell division (or increased survival or apoptosis resistance), and the cells are able to invade other adjacent tissues (invasion) and spread through lymphatic vessels and blood vessels to other parts of the body where cells are usually not present (metastasis), circulate through the bloodstream, and then invade normal tissues in other parts of the body. Tumors are classified as benign and malignant according to whether they can spread by invasion and metastasis: benign tumors are tumors that cannot spread by invasion and metastasis, that is, they only grow locally; and malignant tumors are tumors that can spread by invasion and metastasis. Known biological processes associated with cancer include angiogenesis, immune cell infiltration, cell migration and metastasis. Cancers generally share some of the following characteristics: maintaining proliferation signaling, escaping growth inhibitors, resisting cell death, achieving replicative immortality, inducing angiogenesis, and activating invasion and ultimately metastasis. Cancer attacks nearby parts of the body and may also spread to more distant parts of the body through the lymphatic system or bloodstream. Cancers are classified by the similar cell types of the tumor cells, so it is speculated where the tumor originated.

[0307] Examples of cancers or tumors include, but are not limited to, 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, and liver tumors. In particular, the tumor / cancer may be selected from the group consisting of adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell tumor, glioblastoma, glioma, hemangioendothelioma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary carcinoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, teratoma, acral lentigo melanoma, actinic keratinizing adenocarcinoma, adenoid cystic carcinoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, Bartholin's gland carcinoma, basal cell carcinoma, bronchial adenocarcinoma, carcinosarcoma, bile duct carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, swing sarcoma, local focal nodular hyperplasia, germ cell tumor, glucagonoma, hemangioblastoma, hemangioma, hemangioma, hepatic adenoma, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, epithelial-mesenchymal squamous cell tumor, 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 cancer, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm's tumor.

[0308] In certain embodiments, the cancer is a p95HER2-positive cancer.

[0309] The terms "p95HER2" and "HER2" have been defined in the context of the CAR of the present invention, and said definitions apply equally to the present diagnostic method.

[0310] "p95HER2 positive cancer" refers to a cancer in which at least a portion of the cancer cells contain p95HER2, as determined by immunohistochemistry (IHC), Western blot, Determination (Monogram Biosciences) or flow cytometry. In some embodiments, cancer is determined to be p95HER2 positive by IHC. In some such embodiments, the method described in Sperinde et al., Clin.Canc.Res., 2010, 16 (16): 4226-4235, such as the method using anti-p95 antibody clone D9 in the VeraTag assay, is used to determine that cancer is p95HER2 positive. In some embodiments, the method described in U.S. Patent No. 8,389,227B2, such as the method using antibodies produced by hybridoma cell lines with the deposit number of DSMACC2904 or DSMACC2980 deposited in the German Microbiological Collection (Deutschland Sammlung von Mikroorganismen and Zellen), is used to determine that cancer is p95HER2 positive. In some embodiments, cancer is determined to be p95HER2 positive according to the guidelines of the assay manufacturer or assay laboratory. p95HER2 refers to a collection of carboxyl-terminal HER2 fragments, which in some embodiments can be divided into 95 to 100 kDa fragments and 100 to 115 kDa fragments. See, for example, Arribas et al., Cancer Res., 2011, 71: 1515-1519. In some embodiments, p95HER2-positive cancers contain 100 to 115 kDa HER2 fragments.

[0311] Nucleic acids and vectors of the present invention

[0312] The immune cells of the present invention can be obtained by using a nucleic acid construct and / or composition encoding a CAR and a bispecific antibody to be expressed by the T cells of the present invention.

[0313] Therefore, another aspect of the present invention relates to a nucleic acid construct, starting from here on, a nucleic acid construct of the present invention, which comprises

[0314] (i) encoding a first region of a chimeric antigen receptor, the chimeric antigen receptor comprising:

[0315] ○p95HER2-specific antigen binding domain,

[0316] ○ Transmembrane domain and

[0317] ○ At least one intracellular signaling domain and / or co-stimulatory domain

[0318] as well as

[0319] (ii) a second region encoding a bispecific antibody, the bispecific antibody comprising:

[0320] ○The first antigen binding region that specifically binds to HER2 and

[0321] ○ A second antigen binding region that specifically binds to CD3;

[0322] or a nucleic acid composition, hereinafter referred to as a nucleic acid composition of the invention, comprising

[0323] (i) a first polynucleotide encoding a chimeric antigen receptor, the chimeric antigen receptor comprising:

[0324] ○p95HER2-specific antigen binding domain,

[0325] ○ Transmembrane domain and

[0326] ○ At least one intracellular signaling domain and / or co-stimulatory domain

[0327] as well as

[0328] (ii) a second polynucleotide encoding a bispecific antibody, the bispecific antibody comprising:

[0329] ○The first antigen binding region that specifically binds to HER2 and

[0330] ○ A second antigen binding region that specifically binds to CD3;

[0331] In a specific embodiment of the nucleic acid construct of the present invention or the nucleic acid composition of the present invention, the p95HER2-specific CAR antigen binding domain, the CAR transmembrane domain, the at least one CAR intracellular signaling domain and / or CAR co-stimulatory domain, the bispecific antibody anti-HER2 antigen binding region and the bispecific antibody anti-CD3 antigen binding region are as defined in any one of the aforementioned aspects and / or embodiments.

[0332] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single-stranded or double-stranded form. Unless otherwise limited, the term covers nucleic acids containing known natural nucleotide analogs, the binding abilities of these analogs are similar to reference nucleic acids, and their metabolic patterns are similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also means conservatively modified variants (e.g., replaced with degenerate codons), alleles, orthologs, SNPs and complementary sequences, and sequences indicated in direct form. In particular, replacement with degenerate codons can be obtained by creating a sequence, wherein the third position of one or more selected (or all) codons is replaced by residues with mixed bases and / or deoxyinosine residues.

[0333] As used herein, the term "nucleic acid construct" refers to a single nucleotide sequence comprising all elements (i.e., the first and second regions of a polynucleotide) required to encode a chimeric antigen receptor and a bispecific antibody. As used herein, the term "nucleic acid composition" refers to a composition comprising at least two nucleotide sequences or polynucleotides, one encoding a CAR expressed by an immune cell of the present invention, and the other encoding a bispecific antibody expressed by an immune cell of the present invention.

[0334] In a specific embodiment of the nucleic acid construct of the present invention or the nucleic acid composition of the present invention, the chimeric antigen receptor encoded by the first region of the nucleic acid construct or the first polynucleotide of the nucleic acid composition and the bispecific antibody encoded by the second region of the nucleic acid construct or the second polynucleotide of the nucleic acid composition further include a signal sequence.

[0335] The term "signal sequence", also referred to as a "leader peptide", is used herein according to its usual meaning in the art and refers to a peptide of about 5-30 amino acids in length. The leader peptide is present at the N-terminus of a newly synthesized protein that forms part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to, proteins that reside in certain organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), are secreted from cells, or are inserted into cell membranes. In some embodiments, the leader peptide forms part of the transmembrane domain of a protein.

[0336] In some embodiments, the isolated nucleic acid encodes a protein from N-terminus to C-terminus: a leader peptide is present at the N-terminus of newly synthesized proteins that form part of the secretory pathway.

[0337] In a specific embodiment of the nucleic acid construct of the invention or the nucleic acid composition of 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.

[0338] In a specific embodiment of the nucleic acid construct of the invention or the nucleic acid composition of 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.

[0339] SEQ ID NO:62MALPVTALLLPLALLLHAARP

[0340] SEQ ID NO:63MDFQVQIFSFLLISASVIMSRG

[0341] It is obvious to experts in the field that the nucleic acid construct of the present invention includes two regions, one region encoding the CAR expressed by the immune cells of the present invention, and the other region encoding the bispecific antibody expressed by the immune cells of the present invention, so as to obtain two separate polypeptides, i.e., CAR and bispecific antibodies. There are several mechanisms that can be used to produce two or more polypeptides from the same nucleic acid construct. One such way is that there are different transcription promoting elements in the nucleic acid, for example, promoters and enhancers, for each different coding region (see aspects and embodiments below). Another way is that there is a single promoter element, which produces a unique messenger RNA (mRNA) encoding both CAR and bispecific antibodies. In this case, mRNA can be monocistronic, i.e., both regions are included in the same open reading frame of mRNA, or bicistronic, wherein, mRNA includes two different open reading frames, one encoding corresponds to the first district of CAR expressed by the immune cells of the present invention, and the other encoding corresponds to the second district of the bispecific antibody expressed by the immune cells of the present invention.

[0342] In a specific embodiment, the nucleic acid construct of the invention is a monocistronic construct, wherein the chimeric antigen receptor encoded by the first region and the bispecific antibody encoded by the second region are located in the same open reading frame and are separated by a self-cleaving peptide region.

[0343] As used herein, the term "self-cleaving peptide region" refers to a peptide sequence having a cleavage activity that occurs 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 or 2A-like peptides, cleavage occurs between the glycine and proline residues on these peptides. This is caused by the "ribosome skipping mechanism", which prevents the formation of normal peptide bonds between glycine residues 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 mRNA molecule. The self-cleaving peptide used in the present invention can be obtained from a viral 2A peptide or a 2A-like peptide with equivalent functions. Examples of 2A self-cleaving peptides include 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV) (VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 87)), 2A peptide (E2A) derived from equine rhinitis virus type A (ERAV) (QCTNYALLKLAGDVESNPGP (SEQ ID NO: 88)), 2A peptide (P2A) derived from porcine teschovirus (PTV-1) (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 89)), and 2A peptide (T2A) derived from thosea signa virus (TaV) (EGRGSLLTCGDVEENPGP (SEQID NO: 64)). Mutations can be appropriately introduced into the self-cleaving peptide domain as long as its activity is not significantly impaired.

[0344] In another specific embodiment of the nucleic acid construct of the present invention, the self-cleaving peptide region comprises the sequence of SEQ ID NO:64.

[0345] In another specific embodiment, the nucleic acid construct of the present invention is a monocistronic construct, wherein the chimeric antigen receptor encoded by the first region and the bispecific antibody encoded by the second region are located in the same open reading frame and are separated by a cis-hydrolase element.

[0346] "Cis-hydrolase element" or "CHYSEL" refers to a peptide sequence that causes the ribosome to release the growing polypeptide chain being synthesized without dissociating from the mRNA. In this regard, the ribosome continues to translate and thus produces a second polypeptide. Peptides such as FMDV 2A sequence (GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP, SEQ ID NO:90), sponge (Amphimedon queenslandica) 2A sequence (LLCFLLLLLSGDVELNPGP, SEQ ID NO:91; or HHFMFLLLLLAGDIELNPGP, SEQ ID NO:92); acorn worm (Saccoglossuskowalevskii) (WFLVLLSFILSGDIEVNPGP, SEQ ID NO:93) 2A sequence; amphioxus (Branchiostoma floridae) (KNCAMYMLLLSGDVETNPGP, SEQ ID NO:94; or MVISQLMLKLAGDVEENPGP, SEQ ID NO:95) 2A sequence, porcine teschovirus-1 (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO:96). ID NO: 96) 2A sequence; Thoseaa signa virus (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 97) 2A sequence; and Equine rhinitis virus type A (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO: 98) 2A sequence is CHYSEL used in the present invention. In some embodiments, the 2A sequence is a naturally occurring or synthetic sequence, which includes the 2A consensus sequence DXEX-NPGP (SEQ ID NO: 99), wherein X is any amino acid residue.

[0347] In a specific embodiment, the nucleic acid construct of the invention is a multicistronic construct, wherein the chimeric antigen receptor encoded by the first region and the bispecific antibody encoded by the second region are present in different open reading frames and are separated by an internal ribosome entry site or a cis-hydrolase element.

[0348] The term "internal ribosome entry site" or "IRES" defines a sequence motif that facilitates the attachment of ribosomes to this motif on an internal mRNA sequence. Thus, an mRNA containing an IRES sequence motif produces two translation products, one starting from the 5'-end of the mRNA and the other starting through the IRES-mediated internal translation mechanism. Many IRESs have been described and can be used in the nucleic acid constructs of the present invention. See, e.g., US 8,192,984; WO 2010 / 119257; and US 2005 / 0112095.

[0349] In addition, the nucleic acid construct of the present invention or the nucleic acid composition of the present invention may further include a "marker", "label" or "tag", which can improve or promote certain properties of the protein, i.e., protein stability or resistance to degradation, or provide technical advantages in the production process, i.e., promote expression and / or purification. Examples of such tags are biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, PA tags, fluorescent protein tags, etc. The tag can be fused or conjugated to the amino terminus or carboxyl terminus of a polypeptide, such as a CAR expressed by an immune cell of the present invention or a bispecific antibody expressed by an immune cell of the present invention. The "polyhistidine" sequence of the His tag or histidine amino acid is bound by a peptide bond, which is fused or conjugated to the amino terminus of the protein of the present invention and / or to the carboxyl terminus of the protein of the present invention. In a preferred embodiment, the polyhistidine region comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 histidine residues.

[0350] In a specific embodiment of the nucleic acid construct of the present invention or the nucleic acid composition of the present invention, the chimeric antigen receptor encoded by the first region of the nucleic acid construct or the first polynucleotide of the nucleic acid composition and / or the bispecific antibody encoded by the second region of the nucleic acid construct or the second polynucleotide of the nucleic acid composition further comprises a tag.

[0351] In another specific embodiment of the nucleic acid construct of the present invention or the nucleic acid composition of the present invention, the tag is located at the C-terminus of the bispecific antibody.

[0352] In a more specific embodiment of the nucleic acid construct according to the present invention or the nucleic acid composition according to the present invention, the tag is a hexahistidine tag.

[0353] Another aspect of the present invention relates to a polypeptide encoded by the nucleic acid construct of the present invention.

[0354] The terms "polypeptide" or "peptide" or "protein (if single chain)" are used interchangeably herein and generally refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, or it may be interrupted by non-amino acids. The term also includes 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 may be isolated from natural sources, may be produced by recombinant technology from eukaryotic or prokaryotic hosts, or may be products of artificial synthesis, as long as they can be expressed.

[0355] In a specific embodiment, the polypeptide encoded by the nucleic acid construct of the present invention includes the sequence of SEQ ID NO:65, or SEQ ID NO:66, or SEQ ID NO:108 or SEQ ID NO:109.

[0356] SEQ ID NO:65

[0357]

[0358] SEQ ID NO:66

[0359]

[0360] SEQ ID NO:108

[0361]

[0362] SEQ ID NO:109

[0363]

[0364] Another aspect of the invention relates to a vector comprising the nucleic acid construct of the invention, from here on a vector of a composition, or a vector composition comprising a vector, wherein the first and second nucleic acids of the composition are present in different vectors, from here on a vector composition of the invention.

[0365] As used herein, "vector", "cloning vector" and "expression vector" are vehicles that transform hosts and can achieve expression (e.g., transcription and translation) of introduced sequences. The term "vector" is also intended to include plasmids, phages, viruses, etc.

[0366] The nucleotide sequence encoding any CAR expressed by the immune cells of the present invention and / or the bispecific antibody expressed by the immune cells of the present invention may be present in an expression vector and / or a cloning vector. The expression vector may include selectable markers, replication origins, and other features for vector replication and / or maintenance. Suitable expression vectors include, for example, plasmids, viral vectors, etc.

[0367] A large number of suitable vectors and promoters are known to those skilled in the art; many vectors and promoters are commercially available for use in generating the subject recombinant constructs. The following vectors are provided as examples. Bacteria: 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 organisms: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0368] Expression vectors usually have convenient restriction sites near the promoter sequence to insert the nucleic acid sequence encoding the heterologous protein. There may be selectable markers operable in the expression host. Suitable expression vectors include, but are not limited to, viral vectors, particularly lentiviruses (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses (such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc.

[0369] In a specific embodiment of the vector of the present invention or the vector composition of the present invention, the vector is a lentiviral vector.

[0370] As used herein, the term "promoter" or "regulatory sequence" refers to a DNA sequence recognized by the transcriptional machinery of a cell / host that is required to initiate specific transcription of a polynucleotide sequence and / or to express a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also include an enhancer sequence and other regulatory elements required to express a gene product. The promoter / regulatory sequence may, for example, be a sequence that expresses a gene product in a tissue-specific manner.

[0371] Examples of promoters / regulatory sequences suitable for use in 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-1α (CHEF-1) (Ebadat, et al, PLoS One, 2017, 12(10): e018596), human elongation factor-1α (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 (WO 2018085690), murine stem cell virus (MSCV) long terminal repeat (LTR) (Hughes, M. et al., Hum Gene Ther. 2005Apr; 16(4): 457–472) and NFAT-minimal IL2 promoter (Hoojberg et al., Blood, 2000Jul; 96(2): 459-466).

[0372] In a specific embodiment of the vector of the present invention or the vector composition of the present invention, wherein the nucleic acid construct, the first polynucleotide and / or the second polynucleotide are under the operational control of one or more promoters, wherein the promoters may be the same or different. In a specific embodiment of the vector of the present invention or the vector composition of the present invention, wherein the nucleic acid construct, the first polynucleotide are under the operational control of the LTR promoter, and the second polynucleotide are under the operational control of the NFAT-minimal IL2 promoter.

[0373] In another specific embodiment of the vector of the present invention or the vector composition of the present invention, the nucleic acid construct, the first polynucleotide and / or the second polynucleotide are under the operational control of the murine stem cell virus (MSCV) long terminal repeat sequence.

[0374] The term "operably linked" or alternatively "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence has a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be adjacent to each other and, for example, in the case where it is desired to link two protein coding regions, be in the same reading frame.

[0375] As described above, in some embodiments, the nucleic acid comprising any CAR of the present invention will be RNA in some embodiments, for example, RNA synthesized in vitro. Methods for synthesizing RNA in vitro are known in the art; RNA comprising nucleotide sequences encoding the first and / or second polypeptides of heterodimerization, conditionally active CAR disclosed herein can be synthesized using any known method. Methods for introducing RNA into host cells are known in the art. The introduction of RNA comprising nucleotide sequences encoding the first and / or second polypeptides of heterodimerization, conditionally active CAR disclosed herein into host cells can be performed in vitro or in vitro or in vivo. For example, RNA comprising nucleotide sequences encoding the first and / or second polypeptides of heterodimerization, conditionally active CAR disclosed herein can be used to electroporate host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) in vitro or in vitro.

[0376] In order to evaluate the expression of the CAR polypeptide or part thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both, so as to identify and select expressing cells from a cell population attempted to be transfected or infected by a viral vector; in other aspects, the selectable marker can be carried on a separate DNA fragment and used for a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable them to be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neomycin. Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present or expressed in a recipient organism or tissue, and the expression of the polypeptide encoded by it is expressed as some easily detectable properties, such as enzymatic activity. After the DNA is introduced into the recipient cell, the expression of the reporter gene is determined at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared or commercially available using known techniques. Typically, constructs with minimal 5' flanking regions that show the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate the ability of agents to modulate promoter-driven transcription.

[0377] Use of the nucleic acid construct, nucleic acid composition, vector and / or vector composition of the present invention

[0378] Another aspect of the present invention relates to the use of the nucleic acid construct of the present invention, the nucleic acid composition of the present invention, the vector of the present invention, or the vector composition of the present invention for medicine.

[0379] Another aspect of the present invention relates to use of the nucleic acid construct of the present invention, or the nucleic acid composition of the present invention, or the vector of the present invention, or the vector composition of the present invention in a method for preventing or treating cancer.

[0380] In a specific embodiment, the nucleic acid construct of the present invention, the nucleic acid composition of the present invention, the vector of the present invention or the vector composition of the present invention is used in a method for preventing or treating cancer, wherein the cancer is p95HER2 positive.

[0381] All previously described definitions and embodiments are equally valid for this aspect and its embodiments.

[0382] Method for obtaining immune cells of the present invention

[0383] The present invention further comprises a method allowing to obtain cells of the present invention expressing CAR and bispecific antibodies. Therefore, another aspect of the present invention relates to a method, from here on the method of the present invention, for obtaining cells expressing:

[0384] (i) a chimeric antigen receptor, the chimeric antigen receptor comprising:

[0385] οp95HER2-specific antigen binding domain,

[0386] o transmembrane domain and

[0387] o At least one intracellular signaling domain and / or co-stimulatory domain

[0388] as well as

[0389] (ii) a bispecific antibody, comprising:

[0390] o a first antigen binding region that specifically binds to HER2 and

[0391] o a second antigen binding region that specifically binds to CD3,

[0392] The method comprises inserting a nucleic acid construct of the invention or a nucleic acid composition of the invention or a vector of the invention or a vector composition of the invention into a cell.

[0393] The term "cell" or "engineered cell" means any cell of any organism modified, transformed or manipulated by adding or modifying a gene, DNA or RNA sequence, or protein or polypeptide. It also refers to the offspring of such cells. The cells or genetically engineered cells of the present invention include isolated immune cells, such as T, NK or NKT cells, which contain DNA or RNA sequences encoding p95HER2-specific CAR or CAR complex and HER2-specific bispecific antibodies, and express the CAR on the cell surface and secrete the bispecific antibody. Isolated cells and engineered cells can be used, for example, to enhance NK or NKT cell activity or T lymphocyte activity, treat cancer, and treat infectious diseases.

[0394] In a particular embodiment of the method of the invention, the cell is a mammalian cell.

[0395] 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 No. 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.

[0396] In some cases, the cell is not an immortalized cell line, but is a cell obtained from an individual (e.g., a primary cell). For example, in some cases, the cell is an immune cell obtained from an individual.

[0397] Engineered cells can be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor infiltrating lymphocytes, lymph node tissue or thymus tissue. Host cells can include placental cells, embryonic stem cells, induced pluripotent stem cells or hematopoietic stem cells. Cells can be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats and transgenic species thereof. Cells can be obtained from established cell lines.

[0398] The above cells can be obtained by any known method. For recipients of engineered cells, the cells can be autologous, homologous, allogeneic or xenogeneic. The term "autologous" refers to any material derived from the same individual that will be reintroduced into the individual later.

[0399] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be genetically different enough to allow antigenic interaction to occur.

[0400] The term "xenogeneic" refers to a transplant that originates from an animal of a different species.

[0401] The term "homologous" refers to close genetic similarity or identity, especially with respect to antigens or immune responses. Homologous systems include, for example, models in which organs and cells (e.g., cancer cells and their noncancerous counterparts) are derived from the same individual, and / or models in which organs and cells are derived from different animal individuals of the same inbred strain.

[0402] In a particular embodiment of the method of the invention, the cell is a cell from the immune system.

[0403] Immune cells can be obtained from subjects suffering from or diagnosed with cancer, plasma cell disorders, or autoimmune diseases or disorders. For example, immune cells can be obtained from subjects suffering from cancer (e.g., multiple myeloma, smoldering myeloma, or Waldenstrom's macroglobulinemia). In some embodiments, immune cells are obtained from subjects resistant to anti-BCMA therapy. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipient of the cell.

[0404] Immune cells (e.g., human immune cells) that can be used for the present invention include autologous cells, which are obtained from a subject to which cells will be administered later after ex vivo modification and amplification. For example, immune cells can be obtained from individuals suffering from or diagnosed as suffering from cancer, plasma cell disorders, or autoimmune diseases or disorders. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipient of the cell. Immune cells used for the present invention include T cells and NK cells.

[0405] In another specific embodiment of the method of the present invention, the immune cell is a T cell, a natural killer (NK) cell or a macrophage.

[0406] The term "macrophage" refers to a subpopulation of phagocytes differentiated from monocytes. Macrophages activated by inflammation, immune cytokines or microbial products non-specifically engulf and kill foreign pathogens in macrophages by hydrolysis and oxidative attacks, thereby causing degradation of pathogens. The peptides of degraded proteins are displayed on the macrophage cell surface, where they can be recognized by T cells, and they can directly interact with antibodies on the surface of B cells, thereby causing T cells and B cells to activate and further stimulate an immune response. Macrophages belong to the class of antigen presenting cells. In one embodiment, the macrophage is a spleen macrophage.

[0407] In embodiments, immune cells (e.g., human immune cells) that can be used in the present invention include autologous cells, which are obtained from a subject to which cells will be administered later after ex vivo modification and amplification. For example, immune cells can be obtained from an individual suffering from or 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 cell. Immune cells that can be used in the present invention include T, NK, and NKT cells.

[0408] Methods for obtaining T, NK and NKT cells are known in the art and can be used for engineered immune cells as described herein. T, NK and NKT cells are generally obtained from peripheral blood collected from a subject by, for example, venipuncture or by implantation of a port or catheter. Optionally, blood can be obtained by a process including leukocyte apheresis, wherein leukocytes are obtained from the blood of the subject, and other blood components are returned to the subject. Blood or leukocyte apheresis products (fresh or cryopreserved) are processed using methods known in the art to enrich T, NK or NKT cells. For example, density gradient centrifugation (using, for example, Ficoll) and / or countercurrent centrifugation can be performed to enrich monocytes (including T, NK or NKT cells). In an example, for T cells, a T cell stimulation step can be further performed to stimulate T cells and deplete other cells, such as B cells, and the T cell stimulation step uses, for example, CD3 / CD28 antibodies coated on magnetic beads or expresses, for example, artificial antigen presenting cells (aAPC) of anti-CD3 and anti-CD28 antibody fragments bound to cell surfaces (see below). The T cells of the enriched T cell preparation can then be genetically modified.

[0409] As a substitute for peripheral blood, tissues including bone marrow, lymph nodes, spleen and tumors can be used as the source of T cells and NK cells. T cells and NK cells can be T cells and NK cells of human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cells can be used. In some embodiments of any aspect, T cells or NK cells are human.

[0410] Immune cells (such as T, NK or NKT cells) can be obtained from many sources (peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue and tumor). Any number of cell lines available in the art (e.g., immune cell lines, such as T cell lines) can also be used.

[0411] In embodiments, any suitable technique known in the art (such as Ficoll TMSeparation) is to obtain immune cells (such as T, NK or NKT cells) from a unit of blood collected from a subject. In another embodiment, cells in the circulating blood of a subject are obtained by a single blood collection method. The single blood collection product generally contains lymphocytes, including T, NK or NKT cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. It is understood that the cells collected by the single blood collection method can be washed to remove the plasma fraction and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. For example, cells can be washed with phosphate buffered saline (PBS). Alternatively, the washing solution lacks calcium, and may lack magnesium, or may lack many (if not all) divalent cations. In the absence of calcium, the initial activation step can lead to amplified activation. The washing step can be completed by methods known to those skilled in the art, such as using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, BaxterCytoMate or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, undesirable components of the aliquot can be removed and the cells resuspended directly in culture medium.

[0412] In an embodiment, immune cells are separated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL™ gradient centrifugation or countercurrent centrifugal separation. Specific subsets of immune cells, such as T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further separated by positive or negative selection techniques known in the art. For example, the immune cells can be isolated by conjugating beads (such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells) with anti-CD3 / anti-CD28 (e.g., 3x28). T cells can be isolated by incubating with M-450CD3 / CD28T) for a period of time sufficient to positively select the desired T cells. Additionally or alternatively, T cell populations can be enriched by negative selection, such as by a combination of antibodies for surface markers specific to negatively selected cells. Cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry can be used.

[0413] It should be understood that cells derived from the subject will be modified to express CAR and bispecific antibodies expressed by the immune cells of the present invention, and can be stored for a period of time before use (see, for example, the treatment methods below). For example, the cells can be frozen, optionally frozen after washing the cells, or the cells can be incubated under appropriate conditions to keep them alive until needed (e.g., on a rotator at 2-10 ° C or room temperature). In this way, the cells can be stored until they may be needed. They can be stored in an unmodified state (i.e., where they do not express CAR and / or bispecific antibodies) or a modified state (i.e., where they have been modified to express CAR and / or bispecific antibodies). Before being used for therapeutic applications further described below, cells can generally be activated and amplified using methods known in the art. For example, T cells can be amplified by contacting the surface of a reagent attached to a signal associated with the CD3 / TCR complex and a ligand stimulating a co-stimulatory molecule on the surface of the T cell. In particular, a T cell population can be stimulated as described herein, such as by contacting an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody fixed on the surface, or by contacting a protein kinase C activator (e.g., bryostatin) conjugated to a calcium ionophore. To costimulate the accessory molecules on the surface of T cells, a ligand that binds to the accessory molecules is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and other methods generally known in the art can also be used.

[0414] T cells exposed to different stimulation times can show different characteristics. For example, typical blood or a single peripheral blood mononuclear cell product has a helper T cell group (TH, CD4+) greater than a cytotoxic or inhibitory T cell group (TC, CD8+). The in vitro expansion of T cells by stimulating CD3 and CD28 receptors produces a T cell group, which is mainly composed of TH cells before about 8-9 days, and after about 8-9 days, the T cell group includes more and more TC cell groups. Therefore, according to the purpose of treatment, it may be advantageous to infuse a T cell group mainly including TH cells to a subject. Similarly, if an antigen-specific TC cell subset has been isolated, it may be beneficial to expand the subset to a greater extent.

[0415] In a specific embodiment, the immune cell is a T cell or a NK cell. In a more specific embodiment, the T cell is a CD8+ T cell.

[0416] In particular, the host cells of the invention may be expanded prior to transduction with a polynucleotide or vector of the invention.

[0417] It is noteworthy that after some cancer treatments, especially after treatment with drugs that damage the immune system, the quality of the immune cells obtained shortly after treatment may be optimized or improved in terms of ex vivo propagation capacity during the time period when the patient should usually recover from treatment. In addition, after ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced implantation and in vivo proliferation. Therefore, relevant to the present invention is that at this stage of recovery, the generation of blood cells (including T cells, dendritic cells or other cells of the hematopoietic system) is provided. In addition, in some aspects, the establishment of mobilization modes (e.g., mobilization using GM-CSF) and specific conditions can be used to create conditions for re-proliferation, recirculation, regeneration and / or propagation of specific cell types in subjects, particularly within a certain time window after therapy. Exemplary cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0418] The engineered cells of the present disclosure may also include a suicide system. The suicide system provides a mechanism by which the engineered cells as described above can be inactivated or destroyed. This feature allows for precise therapeutic control of any treatment using engineered cells. As used herein, the suicide system provides a mechanism by which cells having the suicide system can be inactivated or destroyed. Suicide systems are well known in the art.

[0419] As used herein, the expression "inserting a nucleic acid construct of the present invention or a nucleic acid composition of the present invention or a vector of the present invention or a vector composition of the present invention into a cell" means introducing a nucleic acid or a vector into a target cell such that the nucleic acid or vector will produce or express the target nucleic acid.

[0420] Methods for introducing genes into cells and expressing them are known in the art. In the context of one or more expression vectors, the vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical or biological means.

[0421] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells comprising 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.

[0422] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and in particular retroviral vectors, have become the most widely used methods for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0423] Chemical methods for introducing polynucleotides 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. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is envisioned that lipid formulations are used to introduce nucleic acids into host cells (in vitro, in vitro or in vivo). On the other hand, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, attached to liposomes via connecting molecules associated with both liposomes and oligonucleotides, embedded in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, containing or compounded with micelles, or otherwise associated with lipids. The composition associated with lipids, lipid / DNA or lipid / expression vectors is not limited to any specific structure in solution. For example, they may exist in a bilayer structure, as micelles, or have a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include naturally occurring fat droplets in the cytoplasm and compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0424] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("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; dimyristoylphosphatidylglycerol ("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 sole solvent because it evaporates more easily than methanol.

[0425] "Liposome" is a general term that encompasses a variety of monolayer and multilayer lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are characterized by a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components rearrange themselves before forming a closed structure and trap water and dissolved solutes between the lipid bilayers. However, compositions in solution that differ in structure from the normal vesicle structure are also encompassed. For example, lipids may present a micellar structure or exist only as heterogeneous aggregates of lipid molecules. Liposome transfectamine-nucleic acid complexes are also contemplated.

[0426] In a specific embodiment, the method of the present invention further comprises the step of selecting cells that express CAR on their surface and secrete bispecific antibodies.

[0427] Regardless of the method used to introduce exogenous polynucleotides into host cells or otherwise expose cells to the polynucleotides of the present disclosure, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells, which will allow the selection of cells expressing CAR on its surface and secreting bispecific antibodies. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as, by immunological methods (ELISA and protein blotting) or by assays described herein to detect the presence or absence of specific peptides to identify reagents belonging to the scope of the present disclosure. It is obvious to those skilled in the art that in order to select cells expressing CAR and bispecific antibodies, two or more of the above-mentioned examples can be used to obtain confirmation of the expression of two target polypeptides.

[0428] ***

[0429] The present invention will be described by the following examples, which are merely illustrative and do not limit the scope of the present invention.

[0430] Example

[0431] method

[0432] Vector design and retrovirus production

[0433] 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 intermediate affinity (TECH2 Me), HER2-CD3 bispecific T cell engager low affinity (TECH2Lo) p95HER2.CAR HER2-TECH2 high affinity (CAR-TECH2 Hi) or p95HER2.CAR HER2-TECH2 intermediate affinity (TECH2 Me) were synthesized and cloned into the pMSGV-1 retroviral vector (Genscript, The Netherlands). The schemes of the different constructs are as follows Figure 2 As shown. Then, retroviral stocks of the above constructs and empty (UTD) retrovirus were generated. Briefly, 0.7ug of envelope plasmid (RD-114) and 1.5ug of transfer plasmid (H1-14 CAR, TECH2 Hi, TECH2 Me, TECH2 Lo, CAR-TECH2 Hi, CAR-TECH2 Me, UTD in pMSGV-1) were co-transfected into GP2-293 packaging cell line (#631458, Takara). After 3 days, cell supernatants containing retroviral particles were collected and stored at -80C for future transduction.

[0434] Transduction and expansion of CAR- and TECH2-secreting T cells

[0435] Before transduction, PBMCs were stimulated with 10 ng / ul of anti-CD3 (OKT3) (#16-0037-85, Thermo-Fisher) and 300 U / ml IL-2 (#703892-4, Novartis) for 48 hours. Then, the retroviral supernatant was thawed and centrifuged at 2000 g for 2 hours in a 6-well plate coated with retronectin (#T100A, Takara). Next, 2×10 6 10 stimulated PBMCs were added to the top and centrifuged at 400 g for 10 minutes. After 5 days, CAR expression and cytotoxicity assays were performed. Untransduced T cells (UTD) were transduced with empty CAR retrovirus.

[0436] CAR expression analysis

[0437] 0.2×10 6Each CAR T was washed twice with 1×PBS and resuspended in 1×PBS, 2.5mM EDTA, 1% BSA and 5% horse serum for 20 minutes. Then, the cells were stained with 1 / 20 biotin goat anti-mouse IgG (#115-065-072, Jackson ImmunoResearch) for 30 minutes and washed twice with 1×PBS. 1 / 150 APC-Streptavidin antibody (#405207, Biolegend) and 1 / 300 anti-CD3-PE (#300408, Biolegend) were added for 30 minutes. ZombieAqua (#423101, Biolegend) was used as a viability marker at a dilution of 1:1000. CAR expression was measured on FACS Celesta (BD Bioscience) and analyzed using FlowJo software.

[0438] CAR T cell toxicity assay

[0439] MCF7 p95HER2 / empty cells were co-cultured with CAR or TECH2 secreting T cells in 96-well flat-bottom plates at the indicated ratios. After 48 hours of incubation, the cell mixture was separated and washed with 1×PBS and then resuspended in 1×PBS, 2.5mM EDTA, 1% BSA, and 5% horse serum for 20 minutes. The cell suspension was then stained with anti-EpCAM-AF647 (#324212, Biolegend) at a dilution of 1:300 as a target cell marker and with zombie Aqua (#423101, Biolegend) at a dilution of 1:1000 as a viability marker. EpCAM-positive live target cells were counted on the LSR Fortessa (BD Bioscience) and analyzed with FlowJo software.

[0440] In vivo patient-derived tumor (PDTX) and cell line-derived (CDX) models

[0441] Human breast tumor patient-derived tumor xenografts (PDTX) used in this study were obtained from biopsies or surgical resections at Vall d'Hebron University Hospital, Barcelona, ​​and were obtained in accordance with institutional guidelines. The Institutional Review Board (IRB) of the Vall d'Hebron Hospital approved this study in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients who provided samples.

[0442] Patient breast cancer fragments or wild-type MCF7 cells were orthotopically implanted into NOD.Cg-PrkdcscidIl2rgtm1WjI / SzJ(NSG)(#005557, Charles River Laboratories) mice. Tumor xenografts were measured twice a week with calipers, and tumor volume was determined using the following formula: (length × width 2 )×(π / 6).

[0443] Once the tumor volume reaches 200-300mm 3 , use 3×10 6 Animals were treated intravenously (iv) with either CAR+ or TECH2-secreting+ T cells and a second dose was given 10 days later.

[0444] Generation and characterization of NK cells expressing CAR-TECH2 Hi

[0445] NK cells (CD56+CD3-) were isolated from PBMC using an NK cell isolation kit (#130-092-657, Miltenyi). Before transduction, NK cells were expanded and activated for 48 hours using NK MACS medium containing 5% AB serum and human cytokines IL-2 (#703892-4, Novartis, 500U / mL) and IL-15 (#130-095-765, Miltenyi, 140U / mL). Then, the cells were transduced as previously explained for T cells. After 5 days, the determination of CAR expression was similar to that of T cells. Cytotoxicity assays were performed in MCF7p95HER2 / 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 separated and washed with 1×PBS and then resuspended in 1×PBS, 2.5mM EDTA, 1% BSA, and 5% horse serum for 20 minutes. Then, the cell suspension was stained with anti-EpCAM-AF647 (#324212, Biolegend) at a dilution of 1:300 as a target cell marker and with zombie Aqua (#423101, Biolegend) at a dilution of 1:1000 as a viability marker. EpCAM-positive live target cells were counted on an LSR Fortessa (BD Bioscience) and analyzed using FlowJo software.

[0446] result

[0447] Second-generation p95HER2 CAR T has a moderate effect on patient-derived tumor xenografts (PDTX)

[0448] The anti-tumor activity of h1-214 anti-p95HER2 CAR against cell line-derived tumors has been previously described in international patent applications such as WO2021239965. We have now tested the efficacy of p95HER2 CAR against breast cancer patient-derived tumor xenografts. p95HER2 CAR has no effect on HER2-positive, p95HER-negative PDTX ( Figure 1 A), emphasizing the specificity of CAR. In contrast, p95HER2 CAR showed antitumor efficacy against p95HER2-positive PDTX ( Figure 1 B). The second-generation p95HER2 CAR showed significantly lower efficacy against PDTX than against p95HER2-expressing cell lines, suggesting limited clinical efficacy.

[0449] One of the mechanisms of this resistance in PDTX is tumor antigen heterogeneity and low-level expression, as shown by immunohistochemical analysis of PDTX samples. In p95HER2-positive tumors, HER2 expression is more abundant and uniform than p95HER2, although the second is tumor-specific and therefore a safer target. This suggests that p95HER2 may be a safe target for T cell-specific redirection to the tumor, but once there, targeting HER2 could significantly increase tumor eradication due to its abundance.

[0450] Generation of bispecific T cell engagers (TECH2) targeting HER2 and CD3 and secreting bispecific CD3-HER2 T cells p95HER2CAR T of cell-engagement (p95HER2.CAR-TECH2) construct

[0451] The first step in generating armored CAR-T is to design a construct for T cell expression and secretion of TECH2 using available public sequences.

[0452] We refer to "TECH2" as a bispecific T cell engager secreted by transduced T cells, which is formed by two scFvs that recognize and bind to two antigens: HER2 on tumor cells and CD3 on T cells. Secreted TECH2 acts as a dual engager, redirecting T cells to HER2-expressing tumor cells through CD3 binding. Unless otherwise specified, TECH2 refers to a bispecific T cell engager with high affinity for HER2 or "TECH2 Hi."

[0453] We co-expressed TECH2 with p95HER2 CAR as described (Choi et al., 2019, Nature Publishing Group 37, 1049–1058). Figure 2 Simplified schemes of the three constructs are shown.

[0454] Generation and binding characterization of TECH2 with reduced binding affinity for HER2

[0455] We altered the amino acid sequence of the HER2 scFv of TECH2 Hi to generate two additional molecules with different binding affinities to HER2 as described by Carter et al. (1992, PNAS 89, 4285-4289): TECH2 with intermediate affinity (TECH2 Me) and TECH2 with low affinity (TECH2 Lo) ( Figure 3 A). Liu et al., 2015, Cancer Res 75(17), 3596-607 reported the binding affinity of scFv to immobilized HER2 ( Figure 3 B).

[0456] To compare the binding affinity of HER2 scFvs with different affinity modulations in the TECH2 format, we overexpressed the constructs in 293T cells, collected the culture medium containing TECH2, and measured binding to both targets, CD3 and HER2, by flow cytometry. HER2 binding affinity was measured against HER2 expressed in PDX433, and CD3 binding was measured against PBMCs ( Figure 3 C) TECH2 Hi binds HER2 230-fold more strongly than TECH2 Me, and as expected, they bind CD3 equally well since the CD3 binding domain has not been modified.

[0457] In vitro characterization of TECH2 and p95HER2.CAR-TECH2 with reduced HER2 binding affinity

[0458] In vitro cytotoxicity analysis showed that TECH2 Me and TECH2 Lo secreting T cells did not kill targeted MCF7 cells expressing normal levels of HER2, whereas TECH2 Hi was cytotoxic against this cell line as shown previously ( Figure 3 D). The results also showed that TECH2 Me was effective against PDX overexpressing both p95HER2 and full-length HER2 (PDX433).

[0459] We observed the same results when we tested constructs expressing both p95HER2.CAR and TECH2 ( Figure 3 E). In summary, these results suggest that TECH2 Me, although less effective than TECH2 Hi, has a better safety / effectiveness profile.

[0460] In vivo effects of high affinity p95HER2.CAR-TECH2

[0461] NSG mice were orthotopically implanted with MCF7 wild-type cells, which express normal levels of HER2 ( Figure 4 A) or orthotopically implanted with two different breast cancer patient-derived tumor xenograft (PDTX) fragments overexpressing p95HER2 and full-length HER2 ( Figure 4 B) When the tumor reaches approximately 300 mm 3 When 3×10 6 Mice were treated with H1 214p95HER2 CAR+T cells (p95HER2 CAR), TECH2 high affinity (TECH2 Hi), and p95HER2.CAR+TECH2 high affinity (p95HER2.CAR-TECH2 Hi) secreting T cells. Untransduced T cells (UTD) were used as a control.

[0462] Armored CAR-TECH2 is significantly superior to the second-generation p95HER2 CAR ( Figure 1 B), the second generation p95HER2 CAR showed very limited anti-tumor efficacy compared to complete tumor eradication mediated by CAR-TECH2. TECH2 alone also showed limited efficacy ( Figure 4 B), which further supports the idea that the combination of the two components is more effective than either one alone.

[0463] Although the high affinity of CAR-TECH2 was highly effective against 2 different PDTXs expressing heterogeneous levels of p95HER2, it exhibited killing effects against MCF7-derived tumors. Further studies in real “healthy tissue” models are needed to accurately assess the safety of this construct, but alternatively, modulation of the HER2 binding arm affinity may also improve safety.

[0464] In vivo effects of p95HER2.CAR-TECH2 intermediate affinity

[0465] NSG mice were orthotopically implanted with MCF7 wild-type cells ( Figure 4 C) or PDX overexpressing both p95HER2 and full-length HER2 (PDX67) ( Figure 4 D) When the tumor reached approximately 300 mm3, 3 × 10 6 Mice were treated with H1 214p95HER2 CAR T cells (p95HER2 CAR), HER2 CAR T cells (HER2.CAR), TECH2 intermediate affinity (TECH2 Me), and p95HER2.CAR+TECH2 intermediate affinity (p95HER2.CAR-TECH2 Me) secreting T cells as indicated. Untransduced T cells (UTD) were used as controls. The in vivo results correlated with previous in vitro findings: TECH2 Me alone or in combination with p95HER2 CAR had no cytotoxic effect on cells expressing normal levels of HER2 ( Figure 4C). In addition, p95HER2.CAR-TECH2 Me showed higher efficacy than its two individual components: TECH2 Me ( Figure 4 D) and p95HER2 CAR ( Figure 1 B). In addition, 2 of 4 animals showed complete remission (CR) at a follow-up of at least 80 days after complete tumor regression ( Figure 4 D left). These results demonstrate the advantage of dual but safe targeting of p95HER2 and HER2, achieving significant anti-tumor responses.

[0466] Generation and in vitro characterization of p95HER2.CAR-TECH2 in NK cells

[0467] Our constructs can also be used in other immune cells. For example, CAR-TECH2 vectors can also be efficiently retrovirally transduced in NK cells ( Figure 5 A).

[0468] In vitro cytotoxicity analysis showed that the p95HER2 CAR portion of the construct did not target HER2-expressing MCF7 cells ( Figure 5 B). Given that NK cells were purified prior to these experiments, there were no CD3+ cells present, and it was therefore expected that the TECH2 portion should not have any effect. The results also showed that p95HER2.CAR-TECH2 was able to effectively kill cells expressing p95HER2, demonstrating the specificity of the product ( Figure 5 B).

Claims

1. An immune cell expressing (i) a chimeric antigen receptor, the chimeric antigen receptor comprising: o p95HER2-specific antigen binding domain, o Transmembrane domain and o At least one intracellular signaling domain and / or co-stimulatory domain as well as (ii) a bispecific antibody, the bispecific antibody comprising: o The first antigen binding region that specifically binds to HER2 and o A second antigen binding region that specifically binds to CD3.

2. The immune cell according to claim 1, wherein The first antigen-binding region and the second antigen-binding region of the bispecific antibody are ScFv.

3. The immune cell according to claim 2, wherein: - the VH region of the ScFv of the first antigen-binding region has CDR1, CDR2 and CDR3, which respectively include the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, - the VL region of the ScFv of the first antigen-binding region comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise the sequences of SEQ ID NO: 4, SEQ ID NO: 100 and SEQ ID NO: 6, respectively, - the VH region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise 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, wherein the CDR1, CDR2 and CDR3 comprise the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12, respectively, or where: - the VH region of the ScFv of the first antigen-binding region comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 102, respectively, - the VL region of the ScFv of the first antigen-binding region comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise the sequences of SEQ ID NO: 4, SEQ ID NO: 103 and SEQ ID NO: 6, respectively, - the VH region of the ScFv of the second antigen-binding region has CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise 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, and the CDR1, CDR2 and CDR3 include the sequences of SEQ ID NO: 10, SEQ ID NO: 101 and SEQ ID NO: 12, respectively.

4. The immune cell according to claim 3, wherein: - the VH region of the ScFv of the first antigen-binding region has FR1, FR2, FR3 and FR4, wherein FR1, FR2, FR3 and FR4 include 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, wherein FR1, FR2, FR3 and FR4 respectively include the sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, - the VH region of the ScFv of the second antigen-binding region has FR1, FR2, FR3 and FR4, wherein FR1, FR2, FR3 and FR4 include 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, and the FR1, FR2, FR3 and FR4 include the sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, respectively.

5. The immune cell according to claim 4, wherein: - 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 VL region of the ScFv of the second antigen-binding region comprises the sequence of SEQ ID NO:

32.

6. The immune cell according to 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 ScFv of the first antigen-binding region and the second antigen-binding region are connected via a peptide linker.

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

8. The immune cell according to claim 7, wherein - 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 of SEQ ID NO:

37.

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

107.

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

11. The immune cell according to claim 10, wherein The CDR1, CDR2 and CDR3 of the VH region of the CAR ScFv include 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 include the sequences of SEQ ID NOs: 42, 43 and 44, respectively.

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

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

14. The immune cell according to claim 13, wherein The linker region includes SEQ ID NO:

53.

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

56.

17. An immune cell according to any one of the preceding claims, wherein The transmembrane domain of the CAR 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 CD3ζ transmembrane domain.

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

57.

19. An immune cell according to 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. The immune cell according to claim 18, wherein The at least one intracellular signaling domain comprises an intracellular domain of a co-stimulatory molecule selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP 12, and 4-1BB (CD137), or any combination thereof.

21. The immune cell according to claim 20, wherein The intracellular domain of the co-stimulatory molecule comprises the sequence of SEQ ID NO:

58.

22. An immune cell according to any one of the preceding claims, wherein At least one intracellular signaling domain of the CAR further comprises the sequence of SEQ ID NO:

59.

23. The immune cell according to claim 22, wherein The at least one intracellular signaling domain is arranged at the N-terminal side relative to the CD3zeta intracellular domain.

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

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

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

61.

27. An immune cell according to any preceding claim for use in medicine.

28. 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 for use according to claim 28, wherein: The cancer is p95HER2 positive.

30. A nucleic acid construct comprising (i) encoding a first region of a chimeric antigen receptor, the chimeric antigen receptor comprising: o p95HER2-specific antigen binding domain, o Transmembrane domain and o At least one intracellular signaling domain and / or co-stimulatory domain as well as (ii) a second region encoding a bispecific antibody, the bispecific antibody comprising: o The first antigen binding region that specifically binds to HER2 and o The second antigen binding region that specifically binds to CD3, or a nucleic acid composition comprising (i) a first polynucleotide encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: o p95HER2-specific antigen binding domain, o Transmembrane domain and o At least one intracellular signaling domain and / or co-stimulatory domain as well as (ii) a second polynucleotide encoding a bispecific antibody, the bispecific antibody comprising: o The first antigen binding region that specifically binds to HER2 and o A second antigen binding region that specifically binds to CD3.

31. The nucleic acid construct or nucleic acid composition according to claim 30, wherein: The p95HER2-specific CAR antigen binding domain, the CAR transmembrane domain, the at least one CAR intracellular signaling domain and / or CAR co-stimulatory 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. The nucleic acid construct or nucleic acid composition according to claim 30 or 31, wherein: The chimeric antigen receptor encoded by the first region of the nucleic acid construct or the first polynucleotide of the nucleic acid composition and the bispecific antibody encoded by the second region of the nucleic acid construct or the second polynucleotide of the nucleic acid composition further include a signal sequence.

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

63.

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

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

64.

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

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

38. The nucleic acid construct or nucleic acid composition according to 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. 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. A vector or a vector composition, wherein the vector comprises a nucleic acid construct according to any one of claims 31 to 38, wherein the vector composition comprises a vector, wherein The first and second nucleic acids of the composition are present in different vectors.

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

43. A carrier or carrier composition according to 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 a vector composition according to any one of claims 41 to 43 for use in medicine.

45. A method for preventing or treating cancer using 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.

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

47. A method for obtaining a cell, the cell expressing (i) a chimeric antigen receptor, the chimeric antigen receptor comprising: o p95HER2-specific antigen binding domain, o Transmembrane domain and o At least one intracellular signaling domain and / or co-stimulatory domain as well as (ii) a bispecific antibody, the bispecific antibody comprising: o The first antigen binding region that specifically binds to HER2 and o A second antigen binding region that specifically binds to CD3; The method comprises inserting a nucleic acid construct or a nucleic acid composition according to any one of claims 31 to 38 or a vector or a vector composition according to any one of claims 41 to 43 into the cell, and selecting those cells that express CAR on their surface and secrete the bispecific antibody.

48. The method of claim 47, wherein: The cells are cells from the immune system.

49. The method of claim 48, wherein: The cells from the immune system are T cells, NK cells or macrophages.

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