CD4-specific chimeric antigen receptor and methods and uses thereof

Abstract

The present invention relates to a CD4-specific chimeric antigen receptor (CAR), a modified immune cell expressing the CD4-specific CAR as well as related polynucleotides, vectors, and methods and uses. The invention also relates to methods and uses for the treatment of a CD4-mediated and / or CD4-associated disease of a subject using the modified immune cell or CAR.

Description

[0001] Fraunhofer-Gesellschaft zur Fbrderung August 5, 2025 der angewandten Forschung e.V. F74640PC PIN / FLZ / PIN

[0002] CD4-specific chimeric antigen receptor and methods and uses thereof

[0003] The present invention relates to a CD4-specific chimeric antigen receptor (CAR), a modified immune cell expressing the CD4-specific CAR as well as related polynucleotides, vectors, and methods and uses. The invention also relates to methods and uses for the treatment of a CD4-mediated and / or CD4-associated disease of a subject using the modified immune cell or CAR.

[0004] Background

[0005] The standard medication for acute myeloid leukemia (AML) and T cell tumors is chemotherapy, antibody-based therapies and the use of targeted inhibitors. Despite good results, for many patients, and especially for younger patients, hematopoietic stem cell transplantation (SCT) is the superior treatment option for early relapse as well as for non-responders. However, due to the progression of the disease, the poor health status does not always permit a required transplantation. In addition, patients transplanted in “non remission” do not have a benefit from SCT. For this reason, both aggressive variants of AML and T cell-derived tumors are still among the tumor types with very high mortality rates. Therefore, there is a high medical need for these kinds of tumors.

[0006] The standard treatment of CD4-mediated autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis or chronic inflammatory bowel disease (ulcerative colitis), is the systemic administration of immunosuppressants or antibody-based therapies. Both approaches provide patients with symptomatic relief, but offer no curative prospects. For HIV infections, there are very effective anti-retroviral therapies, but there is no clinically established cure.

[0007] Cell therapies based on chimeric antigen receptors (CAR) are promising and potentially curative alternatives to current treatment methods for CD4-associated tumors, infectious and autoimmune diseases. To date, there are no CAR cell therapies with marketing authorization available for any of the aforementioned CD4- positive cell-mediated indications. There exist preclinical studies that show an effect against malignant CD4 positive T cell populations using T cells or an NK-92 cell line transduced with CD4-directed CARs (3-5). Only a few anti-CD4 CAR T cell products are already being tested in early clinical trials for the treatment of PTCL (6-9). In particular, a phase I clinical trial in China using anti-CD4-CAR-T cells has shown results in all three patients treated (6). This CAR construct is disclosed in WO201 6138491 A1 . The nucleotide sequence of the single-chain variable fragment (scFv) of this CAR was derived from the humanized monoclonal antibody Ibalizumab (also known as Hu5A8 or TNX-355). The corresponding construct was also transduced in a NK cell line. However, transduction efficiency in the tested NK cell line was low. A further CD4-targeted CAR T cell is disclosed in WO2020 / 228824A1 . To date, no clinical studies with effector cells other than T cells have been described and no preclinical studies have been published or reported with anti-CD4-CAR- modified primary or iPSC-derived NK cells or macrophages.

[0008] Furthermore, no alternative modification methods other than viral gene transfer for anti-CD4 CARs have been described in the literature.

[0009] Despite the good results achieved with CAR-T cells in the treatment of tumor diseases of the B-cell lineage, the establishment of safe and effective cell therapies for other cancers is proving difficult. Even for other tumors of hematopoietic origin, there are currently no approved CAR-T cell products available, even though for both, acute myeloid leukemia (CD123, CD33, CD44v6, NKG2D, CLEC12A and others) and peripheral T cell lymphoma (PTCL) (CD3, CD5, CD7, CD30, CD37, CCR4, TRBC 1 and others) various antigens are available as potential CAR target antigens in preclinical and clinical trials. The latter markers (CD3, CD5, CD7, CD30, CD37, CCR4, TRBC 1 ) are T cell markers that play a role in T cell-mediated autoimmune diseases in which T cells are activated, proliferate and are therefore a potential target for CAR-modified immune cells. In clinical studies, the treatment of autoreactive B cells using B cell-directed CAR T cells is currently being tested.

[0010] The CD4 co-receptor is expressed in the majority of peripheral T cell lymphomas (PTCL) (1 ) as well as in some acute lymphoblastic T cell leukemias. In these CD4- positive malignancies, the receptor is usually expressed uniformly by the tumor cells. In addition, CD4 is also detectable on cells of some AML diseases (2). In T cell-mediated autoimmune diseases, the CD4-expressing subpopulation plays the key role in the development of the disease. Also, in the case of HIV infections, a transient depletion of CD4-positive cells, which the virus can infect latently or lytically, is a possible treatment approach. An efficient depletion could be achieved by CD4-directed CAR immune cells. The use of a CD4-targeted CAR cell therapy is therefore suitable for all indications and diseases in which the diseased cells exhibit CD4 cell surface expression.

[0011] Anti-CD4 antibody MAX.16H5 is disclosed in WO2012 / 072268.

[0012] The starting material for all currently approved CAR products is leukapheresis from which autologous T cells are obtained. However, patients who are eligible for CAR T cell therapy are usually heavily pre-treated with various chemotherapy regimens and therefore have a significantly reduced pool of suitable T cells (10). Obtaining suitable autologous starting material for the production of CAR T cell represents a particular challenge in case of patients suffering from T cell malignancies. In addition, T cells, especially in the context of an anti-CD4 CAR product, are committing "fratricide" by killing normal and therapeutic, CAR-carrying CD4 positive T cells and only the CD8 positive T cells can be used for CAR T cell production.

[0013] T cells from healthy allogeneic sources can only be used after appropriate genetic manipulation, as they trigger a life-threatening graft-versus-host disease (GvHD) in patients via their naturally expressed T cell receptors.

[0014] In view of all these obstacles faced when using autologous or allogeneic T cells, other immune cell types, in particular natural killer (NK) cells and macrophages, are of interest, as these cells possess natural cytotoxic properties and can kill target cells, such as malignant cells, in antigen-independent manner. Both NK cells and macrophages are present in the blood circulation in sufficient amounts and can be derived from peripheral blood, umbilical cord blood, induced pluripotent stem cells, human embryonic stem cells and even cell lines (11 , 12).

[0015] The selection of a suitable CAR construct and expression system has a major impact on the efficiency of genetic modification, viability and functionality of the final product (14).

[0016] Currently there are no reports of CAR constructs that are equally expressible and functional in primary T cells, NK cells and macrophages.

[0017] Summary of the invention

[0018] The inventors have generated CAR constructs that target CD4 and allow expression of the same construct in different immune cell types, including NK cells, macrophages and T cells. In particular, the CAR construct exhibits high transduction efficiency also in primary NK cells and primary macrophages, which are cells known in the art to be difficult to transduce.

[0019] The CARs provided herein having the specific composition and arrangement of domains surprisingly allow for efficient transduction and high functionality when expressed in primary NK cells, macrophages and T cells. The CARs provided herein are therefore suitable for providing modified CAR immune cells for the treatment of CD4-mediated and / or CD4-associated diseases in a subject.

[0020] In an aspect, the invention relates to a CD4-specific chimeric antigen receptor (CAR) comprising:

[0021] (i) an extracellular CD4 binding-domain comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) CDRH1 , CDRH2 and CDRH3 and a light chain variable region (VL) comprising complementarity determining regions CDRL1 , CDRL2 and CDRL3, wherein:

[0022] (a) CDRH1 comprises the amino acid sequence of NYWMH (SEQ ID NO: 1 );

[0023] (b) CDRH2 comprises the amino acid sequence of ALYPGNVDTTYNQKFKD (SEQ ID NO: 2);

[0024] (c) CDRH3 comprises the amino acid sequence of MGTTLEAPLDY (SEQ ID NO: 3);

[0025] (d) CDRL1 comprises the amino acid sequence of SARSSVSYLY (SEQ ID NO: 4);

[0026] (e) CDRL2 comprises the amino acid sequence of DTSNLAS (SEQ ID NO: 5); and / or

[0027] (f) CDRL3 comprises the amino acid sequence of QQWSDYPLT (SEQ ID NO: 6);

[0028] (ii) a hinge domain (hinge),

[0029] (iii) a transmembrane domain (TM); and

[0030] (iv) a cytoplasmic domain comprising at least one signalling domain.

[0031] In embodiments, the CD4 binding-domain: comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and / or comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0032] In embodiments, the sequence of the VH is as set forth in SEQ ID NO: 7, and the sequence of the VL is as set forth in SEQ ID NO: 8.

[0033] In embodiments, the CD4 binding-domain is a single-chain variable fragment (scFv), optionally wherein the amino acid sequence of the scFv is as set forth in SEQ ID NO: 18.

[0034] In embodiments, the VH and VL are joined by flexible peptide linker, optionally wherein the peptide linker is a glycine-linker (Gly)n, a glycine-serine linker (Glyi- 5Sen-5)n, such as (Gly4Ser)3 (SEQ ID NO: 9), a glycine-alanine linker, or a linker with the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10), wherein n is an integer of 1 , 2, 3, 4 or 5.

[0035] In embodiments, the hinge domain is selected from a lgG1 hinge domain, lgG4 hinge domain and a CD28 hinge domain, optionally wherein the hinge domain is a native human domain, codon-optimized domain or a domain comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations as compared to a native domain.

[0036] In embodiments, the hinge domain is a mutated lgG1 hinge domain, wherein

[0037] (i) the mutated IgG 1 hinge domain is a human IgG 1 domain wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions are mutated in the CH2 region, and / or

[0038] (ii) the mutated lgG1 hinge domain exhibits substantially abolished binding to the FcyRs and / or substantially abolished effector function, optionally wherein the sequence of the mutated IgG 1 hinge domain is as set for the in SEQ ID NO: 13.

[0039] In embodiments, the transmembrane domain is (i) from the transmembrane region of CD8, optionally wherein the transmembrane domain exhibits at least 90% sequence identity with SEQ ID No: 14, or (i) from the transmembrane region of CD28, optionally wherein the transmembrane domain exhibits at least 90% sequence identity with SEQ ID No: 15.

[0040] In embodiments, the CAR comprises cytoplasmic domain comprising at least one co-stimulatory signalling domain and at least one stimulatory signalling domain, optionally wherein the at least one stimulatory signalling domain is selected from a CD3 , DAP10 and DAP12 stimulatory signalling domain, and / or wherein the at least one co-stimulatory signalling domain is selected from a 4-1 BB, a CD28, DNAM1 and 2B4 co-stimulatory signalling domain.

[0041] In embodiments, the CD4-specific CAR has the following structure, from N- to C- terminus:

[0042] Anti-CD4 binding scFv - mutated lgG1 hinge - CD8 TM - CD28 Co-stimulatory domain - CD3 stimulatory domain.

[0043] In a further aspect, the invention relates to a polynucleotide encoding a CD4-specific CAR provided herein, optionally wherein the polynucleotide further encodes a CD8 leader sequence at the N-terminus of the CAR.

[0044] In a further aspect, the invention relates to an expression vector comprising the polynucleotide provided herein, optionally wherein the expression vector:

[0045] (i) is a viral vector, optionally wherein the viral vector is selected from an SV40 vector, adenovirus vector, adeno-associated virus (AAV), lentiviral vector, and retroviral vector; or

[0046] (ii) the expression vector is a non-viral vector, optionally wherein the non-viral vector is selected from a transposon-based expression vector, or an expression vector for providing mRNA encoding the CD4-specific CAR.

[0047] In a further aspect, the invention relates to a modified immune cell

[0048] (i) comprising a polynucleotide provided herein, or an expression vector provided herein, and / or

[0049] (ii) expressing a CD4-specific CAR provided herein.

[0050] In embodiments, the immune cell is selected from a T cell, NK cell and macrophage, optionally wherein the immune cell is a primary cell and / or autologous cell or an allogeneic cell.

[0051] In a further aspect, the invention relates to a method for preparing the modified immune cell of the invention, comprising the steps of (A) or (B):

[0052] (A) (i) providing mRNA encoding the CD4-specific CAR provided herein, optionally wherein the mRNA further encodes a CD8 leader sequence at the N-terminus of the CAR,

[0053] (ii) preparing lipid nanoparticles comprising the mRNA of (ii), and

[0054] (iii) transfecting immune cells with lipid nanoparticles comprising the mRNA obtained in (ii), thereby obtaining the modified immune cell;

[0055] (B)

[0056] (i) providing an expression vector comprising a polynucleotide encoding the CD4-specific CAR provided herein, optionally wherein the polynucleotide further encodes a CD8 leader sequence at the N-terminus of the CAR encoding the CD4-specific CAR,

[0057] (ii) introducing the expression vector of (i) into an immune cell, thereby obtaining the modified immune cell.

[0058] In a further aspect, the invention relates to a modified immune cell provided herein, for use in the treatment of a CD4-mediated and / or CD4-associated disease in a subject.

[0059] In embodiments, the CD4-mediated and / or CD4-associated disease is selected from:

[0060] (i) a CD4-positive cancer, optionally wherein the CD4-positive cancer is a CD4- positive T cell lymphoma or CD4 positive T cell leukemia or a CD4-positive malignancy, or wherein the CD4 positive cancer is selected from anaplastic large cell lymphoma, peripheral T cell lymphoma (PTL), or acute myeloid leukemia (AML), cutaneous T cell lymphoma, and Sezary Syndrome; optionally wherein the subject treated exhibits a blast crisis, a minimal residual disease, has undergone lymphodepletion or depletion of CD4 positive cells and / or has undergone stem cell transplantation and / or will undergo stem cell transplantation;

[0061] (ii) a CD4 negative cancer, in which non-malignant CD4 positive T cells represent a population within or surrounding the tumour tissue;

[0062] (iii) a T cell-mediated autoimmune disease, optionally wherein the T cell-mediated autoimmune disease is rheumatoid arthritis or ulcerative colitis; and

[0063] (iv) an HIV, HTLV-1 or HLTV-2 infection, optionally wherein the subject has undergone lymphodepletion or depletion of CD4 positive cells. Figure Legend

[0064] Figure 1 : Exemplary design of a CD4-specific chimeric antigen receptor suitable for the expression in primary T cells, NK cells and macrophages, as gene fragment (upper panel) and protein structure (lower panel). SP: signal (or leader) peptide; hinge: hinge domain; TM: transmembrane domain; Co- Stim: co-stimulatory domain; SD: stimulatory domain

[0065] Figure 2: Sequence of the MAX.16H5-derived scFv in light chain - heavy chain (VL- VH) orientation including a CD8 signal peptide

[0066] Figure 3: Comparison of the impact of different hinge domains on the CD4-specific CAR expression in NK cells. (A) Comparison of expression of the different hinge regions derived from lgG1 (mutated; “IgGl mut”), CD28 (“CD28”) or a short variant of lgG4 (“lgG4short”) transduced in primary NK cells relative to the expression of a CD4-specific CAR with a unmutated lgG1 hinge region (“CD4norm”). Ratio of expression was determined by assessing % CAR positive cells in flow cytometry. (B) Assessment of functionality of CAR NK cells transduced with CD4- specific CARs carrying the different hinge regions and an unmutated hinge region from lgG1 (“aCD4norm”) in a flow cytometry based PBMC cocultivation assay. Relative decrease of CD4+ T cells are determined when cocultured with CAR NK cells of two independent healthy donors (black). Absolute CAR expression for these two donors is presented in grey.

[0067] Figure 4: Expression data of the MAX.16H5-derived CD4-specific CAR containing an lgG1 hinge domain on (A) primary T cells, either stably transduced (left) or transiently transfected with mRNA (right), (B) primary NK cells in a G-rex cell culture system, and (C) primary macrophages transiently modified with CD4-CAR mRNA 24 hours post infection.

[0068] Figure 5: Growth kinetics and phenotyping of CD4-specific CAR NK cells in a G- rex 24-well-plate, which can be scaled up to GMP-compliant bioreactors. (A) Growth kinetics of CD4-CAR NK cells and unmodified NK cells. Transduction was carried out on day 3 post isolation. (B) Expression of NK cell markers on day 14 post-isolation on CAR- and unmodified NK cells show normal NK cell receptor expression and cellular activation.

[0069] The CAR has the structure as shown in the upper panel of Figure 1 .

[0070] Figure 6: CD4-CAR-NK cells are highly functional and specific. (A) Expression of CD4 on diverse AML (left) and TCL (right) cell lines. (B) CAR-mediated cytotoxicity of CD4-CAR-NK cells towards diverse cell lines assessed by a calcein-based cytotoxicity assay in an E:T ratio of 2.5:1. To assess CAR-mediated cytoxicity, natural cytotoxicity (% killing executed by unmodified NK cells) was subtracted. (C) Cytotoxicity in E:T ratios 2.5:1 and 1 :1 towards selected cell lines comparing CD4-CARs and irrelevant CD19-CARs. The CAR has the structure as shown in the upper panel of Figure 1 .

[0071] Figure ?: Cytotoxicity of CD4-specific CAR NK cells towards patient-derived, primary samples of (A) T cell malignancies and (B) acute myeloid leukemia, assessed in a flow cytometry-based cytotoxicity assay. The CAR has the structure as shown in the upper panel of Figure 1 .

[0072] Figure 8: Cytotoxicity of CD4-specific CAR NK cells towards activated, primary T cells from unmatched donors in a flow-cytometry based cytotoxicity assay. The CAR has the structure as shown in the upper panel of Figure 1.

[0073] Figure 9: Cytotoxicity of (A) stably transduced and mRNA-transfected CD4-specific CAR T cells towards primary activated T cells and (B) CD4-specific virally transduced CAR T cells towards AML and TCL cell lines. The CAR has the structure as shown in the upper panel of Figure 1 .

[0074] Figure 10: Phagocytosis of mRNA-transfected CD4-specific CAR macrophages towards the CD4-positive cell line Sup-M2 in an E:T ratio of 1 :1 for 4 hours. M<t>: macrophage. The CAR has the structure as shown in the upper panel of Figure 1 .

[0075] Detailed Description

[0076] The inventors have generated Chimeric Antigen Receptors (CARs) that target the extracellular portion of CD4 and allow expression of the same construct in different immune cell types, including NK cells, macrophages and T cells. In particular, the CAR construct surprisingly exhibits high transduction efficiency also in primary NK cells and primary macrophages. Primary NK cells and primary macrophages cells are known to be difficult to transduce.

[0077] Thereby, platform CD4-specific CAR constructs are provided which may be used off-the-shelf for transducing an immune cell selected from NK cells, macrophages and T cells, or a mixture thereof.

[0078] The CD4-specific CARs provided herewith can be expressed either constitutively (e.g., by viral or via non-viral methods) or transiently (e.g., as mRNA) in the selected immune cells and show a high expression rate and functionality.

[0079] In particular, the modified CAR NK cells, CAR macrophages and CAR T cells provided herein were found to exhibit excellent recognition and killing of CD4 positive cells, which make them particularly suitable for the treatment of CD4- mediated and / or CD4-associated diseases in a subject.

[0080] The advantageous CAR constructs provided herein comprise an antigen-binding domain which comprises the CDR regions of antibody MAX.16H5. Antibody MAX.16H5, which is for example described in WO2012 / 072268, differs both in epitope bound on CD4 and in affinity from other CD4-specific antibodies.

[0081] The CARs provided herein having the specific composition and arrangement of domains surprisingly allow for efficient transduction and high functionality when expressed in NK cells, macrophages and T cells. The CARs provided herein are therefore suitable for providing modified CAR immune cells for the treatment of CD4- mediated and / or CD4-associated diseases in a subject.

[0082] In an aspect, the invention relates to a CD4-specific chimeric antigen receptor (CAR) comprising:

[0083] (i) an extracellular CD4 binding-domain comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) CDRH1 , CDRH2 and CDRH3 and a light chain variable region (VL) comprising complementarity determining regions CDRL1 , CDRL2 and CDRL3, wherein: (a) CDRH1 comprises the amino acid sequence of NYWMH (SEQ ID NO: 1 ); (b) CDRH2 comprises the amino acid sequence of ALYPGNVDTTYNQKFKD (SEQ ID NO: 2);

[0084] (c) CDRH3 comprises the amino acid sequence of MGTTLEAPLDY (SEQ ID NO: 3);

[0085] (d) CDRL1 comprises the amino acid sequence of SARSSVSYLY (SEQ ID NO: 4);

[0086] (e) CDRL2 comprises the amino acid sequence of DTSNLAS (SEQ ID NO: 5); and / or

[0087] (f) CDRL3 comprises the amino acid sequence of QQWSDYPLT (SEQ ID NO: 6);

[0088] (ii) a hinge domain (hinge),

[0089] (iii) a transmembrane domain (TM); and

[0090] (iv) a cytoplasmic domain comprising at least one signalling domain.

[0091] The invention relates, in an aspect, to an CD4-specific chimeric antigen receptor (CAR).

[0092] The term “chimeric antigen receptor” or “CAR” is known in the art and is understood as engineered, membrane bound receptor comprising at least one antigen-binding domain, a hinge domain, a transmembrane domain, and an endodomain comprising at least one stimulatory domain. In various embodiments, CARs are genetically engineered receptors that redirect immune effector cells toward a target cell expressing an epitope on a target protein.

[0093] Chimeric antigen receptors (CARs) are therefore molecules that combine antibodybased specificity for a desired antigen or epitope with an effector immune cell receptor-activating intracellular domain (i.e. at least one stimulatory domain) to generate a chimeric protein that exhibits a specific cellular immune activity.

[0094] The VH and VL regions of the CD4-binding domain herein comprise the CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 regions with the SEQ ID NOs as set forth in SEQ ID NOs: 1 , 2, 3, 4, 5, and 6. This set of CDR regions corresponds to the CDR regions of antibody MAX.16H5.

[0095] MAX.16H5 binds to an epitope in the extracellular portion of human CD4. Therefore, the CARs herein bind to the extracellular portion of CD4. Thus, in embodiments, the CD4-binding domain binds to human CD4. In embodiments, the CD4-binding domain binds to an extracellular portion of human CD4.

[0096] The term “chimeric” describes being composed of parts of different proteins or DNAs from different origins.

[0097] As used herein, the term “CDR” or “complementarity determining region” means the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991 ), and by Chothia et al., J. Mol. Biol. 196:901 -917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996)) where the definitions include overlapping or subsets of amino acid residues when compared against each other.

[0098] Preferably, the term “CDR” is a CDR as defined by Kabat et al.

[0099] CDRs usually are numbered CDR1 , CDR2 and CDR3 for the heavy chain variable region and the light chain variable region, respectively. As a result, an arm of an antibody usually has 6 CDRs, which together form an antigen-binding site. In general, CDRs usually each are 1 to 25 amino acids in length, preferably 3 to 20 amino acids in length, such as 3 to 16 amino acids in length. An antibody may comprise one, two or more arms, i.e. one, two or three antigen-binding sites.

[0100] An “antigen-binding domain” is a fragment of an antibody which exhibits essentially the same specificity as the complete antibody of which the fragment is derived from. An antigen-binding domain comprises a VH variable region and a VL variable region, which together form a minimal antigen-binding domain.

[0101] In a VH, CDRs are surrounded by framework regions (FR). A VH typically has the format FR1 , CDRH1 , FR2, CDRH2, FR3, CDRH3, and FR4. In a VL, CDRs are surrounded by framework regions. A VL typically has the format FR1 , CDRL1 , FR2, CDRL2, FR3, CDRL3, and FR4.

[0102] In the present CAR, the antigen-binding domain is an extracellular CD4-binding domain. An “extracellular CD4-binding domain” is understood as that the CD4- binding domain of the CAR is located extracellularly when expressed in a host cell. “CD4” or “cluster of differentiation 4” is a glycoprotein that serves as a co-receptor for the T cell receptor (TCR). CD4 is found on the surface of immune cells such as helper T cells, monocytes, macrophages, and dendritic cells. In humans, the CD4 protein is encoded by the CD4 gene.

[0103] “Specific binding” is understood that the binding of the binding molecule to human CD4 is at least 50-fold, preferably at least 100-fold stronger than the binding to a control protein such as albumin, as determined e.g. by methods known to the person skilled in the art, such as surface plasmon resonance-based kinetic binding analyses. Alternatively, also methods such as Western Blot analysis, Enzyme-linked Immunosorbent Assay (ELISA) or determining shifts in the fluorescent signal in cytometer-based assays may be used. Such specific binding may be based on any interaction between an antibody and its antigen known to the person skilled in the art, such as non-covalent bonds (e.g. van der Waals contacts, hydrogen-bond formation or hydrophobic interactions).

[0104] The CD4 binding domain comprises a heavy chain variable region (VH) comprising CDRs CDRH1 , CDRH2 and CDRH3 and a light chain variable region (VL) comprising complementarity determining regions CDRL1 , CDRL2 and CDRL.

[0105] The CD4 binding domain is therefore, in embodiments, an antibody or an antigenbinding fragment of an antibody. In embodiments, the CD4 binding domain is an scFv, Fab or diabody. In embodiments, the CD4 binding domain is an scFv or a Fab.

[0106] The term "antibody" is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (example IgG, IgM, IgA, IgD or IgE) and includes monoclonal recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; dAb, sdAb, antigen binding antibody fragments, Fab, F(ab’)2, Fv, disulfide linked Fv, scFv, disulphide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing.

[0107] The Fab fragment is composed of the variable domain at the amino terminus, variable heavy (VH) or variable light (VL), and the constant domain at the carboxyl terminus, CH1 (heavy) and CL (light). The terms full, whole or intact antibody, used interchangeably herein, refer to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 Daltons. An intact antibody is composed of two identical heavy chains (HCs) and two identical light chains (LCs) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains comprising two antigen-binding fragments, known as Fab fragments, and a Fc crystallizable fragment. The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc may elicit effector functions by binding to receptors on immune cells or by binding C1q, the first component of the classical complement pathway. The five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences, which are called p, a, y, s and 5 respectively, each heavy chain can pair with either a K or A light chain. The majority of antibodies in the serum belong to the IgG class, and there are four isotypes of human IgG, lgG1 , lgG2, lgG3 and lgG4.

[0108] In one embodiment, the CD4-specific CAR comprises a humanized CD4 binding domain.

[0109] A “humanized binding domain” refers to a type of engineered antigen-binding domain having its CDRs derived from a non-human donor antibody, the remaining framework regions being derived from an acceptor human antibody. In addition, framework support residues may be altered to preserve binding affinity (see e.g., Queen, et al., Proc. Natl Acad Sci USA. 1989; 86(24): 10029-10032 and Hodgson, et al., Biotechnology, 1991 ; 9(5): 421 -5). In addition, one or more positions in the CDR regions may be humanized.

[0110] It is preferred that the CD4 binding domain is an scFv.

[0111] An “scFv” or “single chain Fv” is an antibody wherein a VH and VL are present on a single protein chain, optionally linked by a flexible peptide linker.

[0112] In embodiments, the scFv may have the structure, from N- to C-terminus, VH-linker- VL, VL-linker-VH, VH-VL, or VL-VH, wherein “linker” is a flexible peptide linker. In embodiments, the scFv has the structure VH-linker-VL or VL-linker-VH.

[0113] In yet further embodiments, the scFv has the following structure, from N- to C- terminus: VL-linker-VH. As shown in the Examples, CD4-specific CARs which comprise the CDR regions of MAX.16H5 in the scFv format exhibit the advantageous and surprising efficient transduction and high functionality in various effector immune cells, including primary NK cells and primary macrophages, in addition to primary T cells.

[0114] As shown in Figure 4, advantageous expression was found for a CAR construct of the invention in primary T cells, primary NK cells and primary macrophages. For primary T cells, this was found in Figure 4(A) both for stably transduced cells (left) and transiently transfected cells with mRNA (right). For primary NK cells, advantageous expression was found (Figure 4(B)). Primary NK cells are known to be difficult to transduce. For macrophages, this was found in primary macrophages transiently modified with CD4-CAR mRNA 24 hours post infection (Figure 4(C)).

[0115] Further, CAR immune cells provided herein exhibit high functionality and specificity. Specifically, Figure 6 demonstrates that CD4-CAR-NK cells are highly functional and specific. In the experiment underlying this Figure, functionality was shown for cytotoxicity towards various AML and TCL cell lines. Figure 7 demonstrates cytotoxicity of CD4-specific CAR NK cells provided herein towards patient-derived, primary samples of T cell malignancies and acute myeloid leukemia.

[0116] Figure 8 shows the cytotoxicity of CD4-specific CAR NK cells towards activated, primary T cells from unmatched donors in a flow-cytometry based cytotoxicity assay. Figure 9 shows the cytotoxicity of (A) stably transduced and mRNA-transfected CD4-specific CAR T cells towards primary activated T cells and (B) CD4-specific virally transduced CAR T cells towards AML and TCL cell lines. Figure 10 shows phagocytosis of mRNA-transfected CD4-specific CAR macrophages towards the CD4-positive cell line Sup-M2.

[0117] It is further disclosed herein for all embodiments, that the VH region sequences, VL region sequences, and CDRs, may “comprise” or “consist” of the respective indicated sequences.

[0118] In the present invention, the CAR binds to human CD4 with an affinity (KD) of 100 nM or less, 50 nM, or less 20 nM or less, 10 nM or less, or 1 nM or less, such as down to 0.1 nM.

[0119] Methods for determining the binding affinity of an antibody or antigen-binding domain thereof are well known to the person skilled in the art and exemplarily also described above in the context of specific binding of an antibody. For example, surface plasmon resonance using a Biacore® device may be used, at 20°C.

[0120] The term “epitope” describes the part of an antigen that is recognized by antibodies or related binding molecules. For example, the epitope is the specific piece of the antigen that an antibody binds to. Epitopes may be conformational epitopes or linear epitopes. A conformational epitope is usually composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope (binding site on antibody) based on the 3D surface features and shape or tertiary structure of the antigen. Linear epitopes are epitopes that are usually recognized by an antibody via their amino acid sequence or primary structure.

[0121] In embodiments, the CD4 binding-domain: comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and / or comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0122] In embodiments, the CDRs are specified as in the aspect herein above and the percent identity variation is due to the remaining portion of the variable region(s) VH and / or VL, i.e., in one or more of the framework sequences.

[0123] For example, the VH and VL regions of the CD4-binding domain comprise the CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 regions with the SEQ ID NOs as set forth in SEQ ID NOs: 1 , 2, 3, 4, 5, and 6, and the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 7. In such embodiments, the VH exhibits one or more non-identical positions in one or more of the heavy chain framework sequences FR1 , FR2, FR2, and FR4 as compared to SEQ ID NO: 7.

[0124] For example, the VH and VL regions of the CD4-binding domain comprise the CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 regions with the SEQ ID NOs as set forth in SEQ ID NOs: 1 , 2, 3, 4, 5, and 6, and the VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 8. In such embodiments, the VL exhibits one or more non-identical positions in one or more of the light chain framework sequences FR1 , FR2, FR2, and FR4 as compared to SEQ ID NO: 8.

[0125] The sequences of various CD4-specific CARs of the invention and domains, regions and sub-sequences thereof as well as of polynucleotide sequences of the invention encoding CARs or domains, regions and sub-sequences thereof are provided in Table 1.

[0126] In embodiments, the sequence of the VH is as set forth in SEQ ID NO: 7, or the sequence of the VL is as set forth in SEQ ID NO: 8. In embodiments, the sequence of the VH is as set forth in SEQ ID NO: 7, and / or the sequence of the VL is as set forth in SEQ ID NO: 8. In embodiments, the sequence of the VH is as set forth in SEQ ID NO: 7, and the sequence of the VL is as set forth in SEQ ID NO: 8.

[0127] Such variable regions correspond to the variable regions of antibody MAX.16H5. An scFv wherein the sequence of the VH is as set forth in SEQ ID NO: 7, and the sequence of the VL is as set forth in SEQ ID NO: 8 was successfully used in CD4- specific CARs and CAR constructs herein.

[0128] In embodiments, the CD4-binding domain comprises the variable regions of humanized antibody MAX.16H5.

[0129] In embodiments, the VH and VL are joined by flexible peptide linker, optionally wherein the peptide linker is a glycine-linker (Gly)n, a glycine-serine linker (Glyi- 5Sen-5)n, such as (Gly4Ser)3 (SEQ ID NO: 9), a glycine-alanine linker, or a linker with the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10), wherein n is an integer of 1 , 2, 3, 4 or 5.

[0130] A “flexible peptide linker” is understood as an amino acid sequence that connects the VH and VL domains and provides a spacer function compatible with interaction of the two variable domains so that the resulting CD4-binding domain retains a specific binding affinity to the target (CD4) as an antibody that comprises the same VH and VL.

[0131] In particular embodiments, further peptide linker(s) may separate CD4-binding domain, hinge domain, transmembrane domain, co-stimulatory domain(s) and / or signalling domain(s). Such peptide linker(s), if present, may be chosen independently from each other. A CAR herein can comprise one, two, three, four, five or more peptide linkers. In particular embodiments, the length of a peptide linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, about 10 to about 20 amino acids or any intervening length of amino acids. In some embodiments, the peptide linker is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more amino acids long. Examples of peptide linkers, in particular flexible peptide linkers, include a glycine-linker (Gly)n, a glycineserine linker (Glyi-5Sen-5)n, such as (Gly4Ser)3 (SEQ ID NO: 9), a glycine-alanine linker, or a linker with the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10), wherein n is an integer of 1 , 2, 3, 4 or 5.

[0132] In embodiments, the flexible peptide linker linking VL and VH in an scFv is selected from a glycine-serine linker (Glyi-5Sen-5)n, such as (Gly4Ser)3 (SEQ ID NO: 9), and a linker with the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10), wherein n is an integer of 1 , 2, 3, 4 or 5. In embodiments, the flexible peptide linker linking VL and VH in an scFv is selected from a (Gly4Ser)3 (SEQ ID NO: 9) linker, and a linker with the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10). In embodiments, the flexible peptide linker linking VL and VH in an scFv is a (Gly4Ser)3 (SEQ ID NO: 9) linker. In other embodiments, the flexible peptide linker linking VL and VH in an scFv is a linker with the sequence as set forth in SEQ ID NO: 10 (“Whitlow linker”; GSTSGSGKPGSGEGSTKG).

[0133] In preferred embodiments, the flexible peptide linker linking VL and VH in an scFv is a linker with the sequence as set forth in SEQ ID NO: 10 (“Whitlow linker”; GSTSGSGKPGSGEGSTKG).

[0134] In embodiments, the scFv of the CAR herein has a structure selected from one of the following: VL- (Gly4Ser)3 linker - VH, VH- (Gly4Ser)3 linker - VL, VL- Whitlow linker - VH, and VH- Whitlow linker - VL. In embodiments, the scFv of the CAR herein has a structure selected from VL- (Gly4Ser)3 linker - VH, and VL- Whitlow linker - VH.

[0135] In further embodiments thereof, the sequence of the VH is as set forth in SEQ ID NO: 7, and / or the sequence of the VL is as set forth in SEQ ID NO: 8.

[0136] In embodiments, the CD4 binding-domain is a single-chain variable fragment (scFv), optionally wherein the amino acid sequence of the scFv is as set forth in SEQ ID In the Examples provided herein underlying Figure 3 to 10, the CD4 binding-domain is a single-chain variable fragment (scFv), wherein the amino acid sequence of the scFv is as set forth in SEQ ID NO: 18.

[0137] The CD4-specific CARs herein further comprise a hinge domain.

[0138] A “hinge domain” or “hinge”, also referred to as a “spacer” in the art, is an extracellular region of the CAR that separates the antigen-binding domain from the transmembrane domain. A hinge domain typically plays a role in positioning the antigen-binding domain away from the effector cell surface to enable proper cell / cel I contact, antigen-binding and activation.

[0139] The hinge domain may be derived either from a natural, synthetic, semi-synthetic or recombinant source. In embodiments, the hinge domain may be an amino acid sequence of a naturally occurring immunoglobulin hinge region or an amino acid sequence of a human immunoglobulin hinge region. In embodiments, the hinge domain is a human IgG hinge domain. In embodiments, the hinge domain is selected from a human lgG1 hinge domain, a human lgG2 hinge domain, a human lgG3 hinge domain or a human lgG4 hinge domain. In embodiments, the hinge domain is selected from a human lgG1 hinge domain, or a human lgG4 hinge domain.

[0140] Suitable hinge regions are for example disclosed in Guedan S et al (Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev. 2018 Dec 31 ;12:145-156).

[0141] In embodiments, a human IgG constant domain which may be used herein as human IgG hinge domain has the structure short hinge - CH2 - CH3.

[0142] In certain embodiments, a native human IgG hinge region of human lgG1 , lgG2, lgG3 or lgG4, in particular a native human IgG hinge region of human IgG 1 , or lgG4, may be used. A native human IgG hinge region of human lgG1 is depicted in SEQ ID No: 11.

[0143] In certain embodiments, it is advantageous that the human IgG hinge domain exhibits substantially abolished binding to the FcyRs and / or substantially abolished effector function. Such human IgG hinge domains which exhibit substantially abolished binding to the FcyRs and / or substantially abolished effector function are known in the art.

[0144] For example, it is known that the binding sites for FcyRs are located in the CH2 domains of IgG constant domains. Therefore, a human IgG hinge domain which exhibits substantially abolished binding to the FcyRs and / or substantially abolished effector function may be selected from a short hinge of human IgG 1 , lgG2, lgG3 or lgG-4, in particular a short hinge of human lgG1 , or lgG4. A short hinge of human lgG4 is depicted in SEQ ID No: 12.

[0145] In other embodiments, the CH2 region may be deleted. Such human IgG hinge domain has the structure short hinge - CH3.

[0146] In yet further embodiments, a mutated IgG 1 hinge domain may be used wherein the mutated lgG1 hinge domain is a human lgG1 hinge domain wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions are mutated, in particular substituted, in the CH2 region. The mutated lgG1 hinge domain exhibits substantially abolished binding to the FcyRs and / or substantially abolished effector function. Such mutated hinge domains are known in the art and include the mutated IgG 1 hinge domain which is as set forth in SEQ ID NO: 13. The mutated lgG1 hinge domain as set for the SEQ ID NO: 13 exhibits 5 substitutions as compared to the native sequence in SEQ ID NO: 11 .

[0147] In following sequence pursuant to SEQ ID NO: 13, the letters in bold and underlined are substituted with a different amino acid as compared to SEQ ID NO: 11 :

[0148] KDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCWVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK

[0149] ( SEQ ID NO : 13 )

[0150] In embodiments, the hinge domain is a mutated lgG1 hinge domain, wherein

[0151] (i) the mutated lgG1 hinge domain is a human lgG1 domain wherein 11 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions are mutated in the CH2 region, and / or

[0152] (ii) the mutated lgG1 hinge domain exhibits substantially abolished binding to the FcyRs and / or substantially abolished effector function, optionally wherein the sequence of the mutated IgG 1 hinge domain is as set for the in SEQ ID NO: 13. An IgG hinge domain, which exhibits substantially abolished binding to the FcyRs is understood as hinge, a domain which exhibits a binding to human FcyRs I, Ila, lib, Illa and 11 lb which is reduced by at least 80%, 85%, 90%, 95% or 96% as compared the binding of a corresponding native IgG constant domain, without mutations, to human FcyRs I, Ila, lib, Illa and lllb.

[0153] As the binding to human FcyRs is typically determining effector function of such IgG hinge domain, by binding to the FcyRs on the cell surface of effector cells, such mutated IgG hinge domain with substantially abolished binding to the FcyRs is also a mutated IgG hinge domain with substantially abolished effector function. Assays for determining FcyR binding are known in the art and include Alpha screen assays.

[0154] An IgG hinge domain which exhibits substantially abolished effector function is understood as hinge a domain which exhibits an effector function which is reduced by at least 80%, 85%, 90%, 95% or 96% as compared the effector function of a corresponding native IgG constant domain, without mutations. “Effector function” is understood as ADCC and / or CDC mediated by the IgG constant domain. Assays for determining effector function are known in the art and include in vitro cytotoxicity assays.

[0155] In other embodiments, the hinge domain is a hinge domain of human CD28.

[0156] In embodiments, the hinge domain is selected from a lgG1 hinge domain, lgG4 hinge domain and a CD28 hinge domain, optionally wherein the hinge domain is a native human domain, codon-optimized domain or a domain comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations as compared to a native domain.

[0157] In embodiments, the hinge domain is a lgG1 hinge domain. In embodiments, the lgG1 hinge domain is a short lgG1 hinge domain. In other embodiments, the lgG1 hinge domain is an lgG1 hinge domain comprising or lacking a CH2 region. In other embodiments, the lgG1 hinge domain is an lgG1 hinge domain comprising a CH2 and a CH3 region.

[0158] In embodiments, the lgG1 hinge domain exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 11. In embodiments, the lgG1 hinge domain has the sequence as set forth in SEQ ID No: 11. In embodiments, the lgG1 hinge domain exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 13. In embodiments, the sequence of the lgG1 hinge domain is as set forth in SEQ ID No: 13.

[0159] The use of lgG1 hinge domains is particularly preferred.

[0160] In other embodiments, the hinge domain is a CD28 hinge domain. In embodiments, the CD28 hinge domain exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 27. In embodiments, the sequence of the CD28 hinge domain is as set forth in SEQ ID No: 27.

[0161] In embodiments, the hinge domain is a lgG4 hinge domain. In preferred embodiments, the lgG4 hinge domain is a short lgG1 hinge domain. In other embodiments, the hinge domain is an lgG4 hinge domain comprising or lacking a CH2 region. In other embodiments, the hinge domain is an lgG4 hinge domain comprising a CH2 and a CH3 region. In embodiments, the lgG4 hinge domain exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 12. In embodiments, the sequence of the lgG4 hinge domain is as set forth in SEQ ID No: 14.

[0162] The CD4-specific CAR comprises a transmembrane domain (TM). A “transmembrane domain” or “TM” is the portion of the CAR that fuses the extracellular portion and the intracellular, cytoplasmic portion comprising signalling domains (including co-stimulatory domain(s) and signalling domain(s)) and anchors the CAR to the plasma membrane of the immune cell by traversing the cell membrane.

[0163] The TM may be derived either from a natural, synthetic, semi-synthetic or recombinant source. The TM domain may comprise the transmembrane region of alpha or beta chain of the T cell receptor, CD35, CD3s, CD3y, CD3 , CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (4-1 BB), CD152, CD154, CD278 (ICOS) or PD1 .

[0164] In embodiments, the transmembrane domain is from the transmembrane region of CD8, or from the transmembrane region of CD28. Such TMs were successfully used in CD4-specific CARs provided in the Examples. The sequence of the transmembrane region of CD8 is as set forth in SEQ ID No: 14. The sequence of the transmembrane region of CD28 is as set forth in SEQ ID No: 15.

[0165] In embodiments, the transmembrane domain is (i) from the transmembrane region of CD8, optionally wherein the transmembrane domain exhibits at least 90% sequence identity with SEQ ID No: 14, or (i) from the transmembrane region of CD28, optionally wherein the transmembrane domain exhibits at least 90% sequence identity with SEQ ID No: 15.

[0166] In embodiments, the transmembrane domain is from the transmembrane region of CD8. In embodiments, the transmembrane domain of CD8 exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 14. In embodiments, the transmembrane domain has the sequence as set forth in SEQ ID No: 14.

[0167] In other embodiments, the transmembrane domain is from the transmembrane region of CD28. In embodiments, the transmembrane domain of CD28 exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 15. In embodiments, the transmembrane domain has the sequence as set forth in SEQ ID No: 15. The use of a CD28 transmembrane domain is particularly preferred.

[0168] The CD4-specific CARs provided herein comprise a cytoplasmic domain comprising at least one signalling domain.

[0169] A “cytoplasmic domain” or “intracellular domain” is understood as domain which is located intracellularly, in the cytoplasm, upon expression of the CAR in an immune cell.

[0170] A cytoplasmic domain comprises at least one signalling domain. A signalling domain is understood as domain which is capable of mediating intracellular signalling when present in naturally occurring immune cell costimulatory or stimulatory molecules. A signalling domain encompasses a co-stimulatory and a stimulatory domain. In embodiments, the CD4-specific CAR comprises at least one stimulatory signalling domain. For example, the CD4-specific CAR comprises 1 , 2, 3, 4 or 5 stimulatory signalling domains. In embodiments, the CD4-specific CAR comprises at least one co-stimulatory signalling domain. For example, the CD4-specific CAR comprises 1 , 2, 3, 4 or 5 co-stimulatory domains.

[0171] Suitable examples of stimulatory or co-stimulatory domains include CD3 , 4-1 BB (CD137), DNAM1 and 2B4, ICOS (CD278), 0X40 (CD134), CD27, CD28, CD40, CD40L, DAP10, DAP12, Toll-like receptors (TLRs), IL-15Ra, IL-2Rp, STAT3 stimulatory domains or co-stimulatory domains and the like.

[0172] In embodiments, the CD4-specific CAR comprises a cytoplasmic domain comprising at least one co-stimulatory signalling domain and at least one stimulatory signalling domain, optionally wherein the at least one stimulatory signalling domain is selected from a CD3 , DAP10 and DAP12 stimulatory signalling domain, and / or wherein the at least one co-stimulatory signalling domain is selected from a 4-1 BB, a CD28, DNAM1 and 2B4 co-stimulatory signalling domain.

[0173] In embodiments, the CD4-specific CAR comprises a cytoplasmic domain comprising 1 or 2 co-stimulatory signalling domain(s) and 1 or 2 stimulatory signalling domain(s). In other embodiments, the CD4-specific CAR comprises a cytoplasmic domain comprising 2 co-stimulatory signalling domain(s) and 1 stimulatory signalling domain(s). In other embodiments, the CD4-specific CAR comprises cytoplasmic domain comprising 1 co-stimulatory signalling domain(s) and 2 stimulatory signalling domain(s).

[0174] In case of more than one co-stimulatory signalling domain being present, the co- stimulatory signalling domains may be identical or different from each other. In embodiments, the two or more co-stimulatory signalling domains are different from each other. In case of more than one stimulatory signalling domain being present, the stimulatory signalling domains may be identical or different from each other. In embodiments, the two or more stimulatory signalling domains are different from each other.

[0175] For example, the cytoplasmic domain may comprise DNAM1 , 2B4 and CD3 signalling domains, CD28, 2B4 and CD3 signalling domains, DNAM1 , CD28 and CD3 signalling domains, DNAM1 , 2B4 and DAP12 signalling domains, CD28, 2B4 and DAP12 signalling domains, DNAM1 , CD28 and DAP12 signalling domains, DNAM1 , 2B4, DAP12 and CD3 signalling domains, CD28, 2B4, DAP12 and CD3 signalling domains, 2B4, DAP12 and CD3 signalling domains, DNAM1 , DAP12 and CD3 signalling domains, CD28, DAP12 and CD3 signalling domains, DNAM1 , 2B4 and DAP10 signalling domains, CD28, 2B4 and DAP10 signalling domains, DNAM1 , CD28 and DAP10 signalling domains, DNAM1 , 2B4, DAP10 and CD3 signalling domains, CD28, 2B4, DAP10 and CD3 signalling domains, 2B4, DAP10 and CD3 signalling domains, DNAM1 , DAP10 and CD3 signalling domains, CD28, DAP10 and CD3 signalling domains, DAP10 and DAP12 signalling domains, 2B4, DAP10 and DAP12 signalling domains, DNAM1 , DAP10 and DAP12 signalling domains, or CD28, DAP10 and DAP12 signalling domains.

[0176] In embodiments, the CD4-specific CAR comprises a cytoplasmic domain comprising one co-stimulatory signalling domain and one stimulatory signalling domain.

[0177] In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 4-1 BB co-stimulatory domain and a CD3 signalling domain. In embodiments, the CD4- specific CAR comprises, from N- to C-terminus, a CD28 co-stimulatory domain and a CD3 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a DNAM1 co-stimulatory domain and a CD3 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 2B4 co-stimulatory domain and a CD3 signalling domain.

[0178] In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 4-1 BB co-stimulatory domain and a DAP10 or DAP12 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a CD28 co-stimulatory domain and a DAP10 or DAP12 signalling domain. In embodiments, the CD4- specific CAR comprises, from N- to C-terminus, a DNAM1 co-stimulatory domain and a DAP10 or DAP12 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 2B4 co-stimulatory domain and a DAP10 or DAP12 signalling domain.

[0179] In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 4-1 BB co-stimulatory domain and a DAP10 signalling domain. In embodiments, the CD4- specific CAR comprises, from N- to C-terminus, a CD28 co-stimulatory domain and a DAP10 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a DNAM1 co-stimulatory domain and a DAP10 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 2B4 co-stimulatory domain and a DAP10 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 4-1 BB co-stimulatory domain and a DAP12 signalling domain. In embodiments, the CD4- specific CAR comprises, from N- to C-terminus, a CD28 co-stimulatory domain and a DAP12 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a DNAM1 co-stimulatory domain and a DAP12 signalling domain. In embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a 2B4 co-stimulatory domain and a DAP12 signalling domain.

[0180] In embodiments, the sequence of the CD3 stimulatory domain is as set forth in SEQ ID No: 16. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No:

[0181] 16 may be used.

[0182] In embodiments, the sequence of the DAP12 stimulatory domain is as set forth in SEQ ID No: 23. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No:

[0183] 23 may be used.

[0184] In embodiments, the sequence of the DAP10 stimulatory domain is as set forth in SEQ ID No: 28. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 28 may be used.

[0185] The DAP10 stimulatory domain sequence is for example disclosed as topological domain in UNIPROT entry Q9UBK5 (HCST_HUMAN).

[0186] In embodiments, the sequence of the CD28 co-stimulatory domain is as set forth in SEQ ID No: 17. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No:

[0187] 17 may be used.

[0188] In embodiments, the sequence of the 2B4 co-stimulatory domain is as set forth in SEQ ID No: 24. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No:

[0189] 24 may be used.

[0190] In embodiments, the sequence of the 4-1 BB co-stimulatory domain is as set forth in SEQ ID No: 25. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 25 may be used.

[0191] In embodiments, the sequence of the DNAM1 co-stimulatory domain is as set forth in SEQ ID No: 26. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 26 may be used.

[0192] In particular embodiments, the CD4-specific CAR comprises, from N- to C-terminus, a CD28 co-stimulatory domain and a CD3 signalling domain.

[0193] Such CD4-specific CARs are particularly useful for expression and function in all three types of immune cells NK cells, macrophages and T cells.

[0194] In embodiments, the CD4-specific CAR has the following structure, from N- to C- terminus:

[0195] Anti-CD4 binding scFv - mutated lgG1 hinge - CD8 TM - CD28 Co-stimulatory domain - CD3 stimulatory domain.

[0196] A CD4-specific CAR having this structure was found to be particular advantageous for efficient transduction of primary NK cells, macrophages and T cells as well as for efficient recognition and killing of CD4-expressing cells.

[0197] The sequence of such CD4-specific CAR is as set forth in SEQ ID NO: 19.

[0198] In embodiments, the sequence of the CD4-specific CAR is as set forth in SEQ ID No: 19. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 19 may be used.

[0199] The sequence of such CD4-specific CAR including the sequence of the CD8 leader peptide (or leader sequence) is as set forth in SEQ ID NO: 20.

[0200] In embodiments, the sequence of the CD4-specific CAR including CD8 leader peptide (or leader sequence) is as set forth in SEQ ID No: 20. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 20 may be used. The leader peptide (also designated leader sequence or leader peptide) is typically removed by posttranslational processing upon expression and transport to the cell membrane of immune cells.

[0201] Table 1 : Sequences

[0202] The embodiments disclosed for the aspect of a CD4-specific chimeric antigen receptor (CAR) are understood to apply also for the other aspects of the invention herein. In particular, the embodiments disclosed for the aspect of a CD4-specific chimeric antigen receptor (CAR) are understood to apply also for the polynucleotides, vectors, expression vectors, modified cells, modified immune cells, pharmaceutical compositions, kit-of-parts, methods and uses provided herein.

[0203] In a further aspect, the invention relates to a polynucleotide encoding a CD4-specific CAR provided herein, optionally wherein the polynucleotide further encodes a leader sequence at the N-terminus of the CAR.

[0204] In a further aspect, the invention relates to a polynucleotide encoding a CD4-specific CAR provided herein, optionally wherein the polynucleotide further encodes a CD8 leader sequence at the N-terminus of the CAR.

[0205] As used herein, the terms “polynucleotide” or “nucleic acid” include messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single and double stranded polynucleotides. Polynucleotides include expression vectors, viral vectors, and transfer plasmids.

[0206] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of well-established techniques known and available in the art. In order to express a desired CAR polypeptide, a polynucleotide encoding the polypeptide, can be inserted into appropriate vector or expression cassette.

[0207] In embodiments, the polynucleotide of the invention encodes a CD4-specific CAR provided herein and further encodes a leader sequence at the N-terminus of the CAR. Suitable leader sequences or signal sequences are well-known in the art and direct the expression of proteins to the cell membrane. For example, a CD28 or CD8 leader sequence may be used.

[0208] In embodiments, the polynucleotide of the invention encodes a CD4-specific CAR provided herein and further encodes a CD8 leader sequence at the N-terminus of the CAR.

[0209] CAR constructs were generated herein with a CD8 leader sequence or a CD28 leader sequence.

[0210] The use of a CD8 leader sequence has been found advantageous for efficient expression of the CD4-specific CARs herein, in particular in primary NK cells.

[0211] In embodiments, the polynucleotide of the invention encodes a CD4-specific CAR that has the following structure, from N- to C- terminus:

[0212] Anti-CD4 binding scFv - mutated lgG1 hinge - CD8 TM - CD28 Co-stimulatory domain - CD3 stimulatory domain.

[0213] A CD4-specific CAR having this structure was found to be particular advantageous for efficient transduction of primary NK cells, macrophages and T cells as well as for efficient killing of CD4-expressing cells.

[0214] The sequence of a polynucleotide encoding such CD4-specific CAR is as set forth in SEQ ID NO: 21.

[0215] In embodiments, the sequence of a polynucleotide encoding such CAR is as set forth in SEQ ID No: 21 . For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 21 may be used.

[0216] The sequence of a polynucleotide encoding such CD4-specific CAR including the sequence encoding the CD8 leader sequence is as set forth in SEQ ID NO: 22.

[0217] In embodiments, the sequence of a polynucleotide encoding such CD4-specific CAR including CD8 leader sequence is as set forth in SEQ ID No: 22. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 22 may be used.

[0218] In embodiments, the CD8 leader sequence is as set forth in SEQ ID NO: 30 or is encoded by the sequence as set forth in SEQ ID NO: 31 . For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 30 may be used. For example, a sequence which exhibits at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No: 31 may be used.

[0219] In a further aspect, the invention relates to a vector comprising the polynucleotide provided herein.

[0220] In a further aspect, the invention relates to an expression vector comprising the polynucleotide provided herein, optionally wherein the expression vector:

[0221] (i) is a viral vector, optionally wherein the viral vector is selected from an SV40 vector, adenovirus vector, adeno-associated virus (AAV), lentiviral vector, and retroviral vector; or

[0222] (ii) the expression vector is a non-viral vector, optionally wherein the non-viral vector is selected from a transposon-based expression vector, or an expression vector for providing mRNA encoding the CD4-specific CAR.

[0223] The term “vector” is used herein to refer to a polynucleotide or nucleic acid molecule capable transferring or transporting another polynucleotide molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector polynucleotide molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposon-based vector, cosmids, bacterial artificial chromosomes and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses. Examples of useful plasmids include nano-S / MAR DNA vectors. These vectors are non-integrating, minimally sized vectors which can be used to genetically modify dividing cells in place of integrating vectors (e.g. Bozza M. et al. (Novel Non-integrating DNA Nano-S / MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models. Mol Ther Methods Clin Dev. 2020 Apr 25; 17:957-968). In particular embodiments, the vectors are expression vectors. Expression vectors may be used to produce CD4-specific CARs provided herein.

[0224] In addition, expression vectors include additional components which allow, in the case of viral vectors, for the production of the viral vectors, which in turn comprise a polynucleotide provided herein. Examples of expression vectors include, but are not limited to, plasmids, autonomously replicating sequences and transposable elements, including transposon-based expression vectors. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, transposons, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage, and animal viruses.

[0225] Examples of expression vectors are pCIneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DESTTM, pLenti6A / 5-DESTTM and pLenti6.2 / V5- GW / lacZ (Invitrogen)) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequences of the CARs provide herein can be ligated into such expression vectors for the expression of the CARs in mammalian cells.

[0226] In certain embodiments, the expression vectors provided herein are BACs which comprise a polynucleotide as provided herein. In particular embodiments, the BACs additionally comprise one or more polynucleotides encoding for proteins necessary to allow the production of a viral vector when expressed in a producer or packaging cell line. By way of example, PCT applications WO2017 / 089307 and WO201 7 / 089308 describe expression vectors used to produce retroviral vectors, in particular lentiviral vectors.

[0227] The “control elements” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence), introns, a polyadenylation sequence, 5' and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used. Transposon-based expression vectors are also known in the art and include for example the Sleeping Beauty (SB) transposon / transposase system and the piggyBAC transposon / transposase system. The SB transposon system as well as further transposon-based expression vectors are for example described in Magnani C.F. et al. (Cells 2020, 9, 1337). A transposon-based vector for expression is an example of a non-viral vector.

[0228] Both the SB and piggyBac transposon systems consist of two components: The engineered transposon, which carries the gene of interest to be inserted into the genome flanked by inverted terminal repeats (ITRs), and the transposase, which catalyzes the process of “cut-and-paste” transposition. Due to a “cut-and-paste” mechanism, mediated by the transposase recognition of the ITR elements, the transposon is mobilized from the plasmid DNA to an acceptor site within the genome. SB transposase inserts transposons into highly abundant TA sequences in the genome. PiggyBac transposase inserts transposons in TTAA sequences and was shown to have a higher activity for transposon mobilization than SB in mammalian cells. Single expression unit cassette, as well a multicistronic cassettes including multiple features (i.e. , genes and control regions) can be designed.

[0229] Transposon and transposase can be provided in the same molecule (cis configuration) or in two different molecules (trans configuration). In embodiments, the cis configuration is used. In this embodiment, a single plasmid is delivered into the cells. In embodiments, the trans configuration is used. In this embodiment, transposase plasmids can be independently controlled in order to enhance the transposition efficiency. The physical separation of the transposon from the transposase also provides the possibility of supplying the transposase in forms other than DNA, such as an mRNA molecule.

[0230] In embodiments, the expression vector is in particular used for delivery into the immune cells intended for administration to a subject, i.e., for delivery.

[0231] Examples of such vectors include, but are not limited to, plasmids, autonomously replicating sequences, transposable elements, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and viral vectors as well as non-viral vectors, as transposon-based vector, such as the Sleeping Beauty (SB) transposon vector. Examples of categories of animal viruses useful as viral vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus (AAV), herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).

[0232] Retroviruses are a common tool for gene delivery. In particular embodiments, a retrovirus is used, as retroviral vector, to deliver a polynucleotide encoding a CAR as provided herein to an immune cell. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a provirus. The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.

[0233] Retroviruses suitable for use herein, include, but are not limited to: Moloney murine leukaemia virus (M-MuLV), Koala Retrovirus (KoRV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumour virus (MuMTV), gibbon ape leukaemia virus (GaLV), feline leukaemia virus (FLV), spumavirus, Friend murine leukaemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentiviruses.

[0234] As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 , and HIV type 2); visna-maedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones (i.e. , HIV cis-acting sequence elements) are preferred.

[0235] Retroviral vectors and more particularly lentiviral vectors may be used in some embodiments. Accordingly, the term "retrovirus" or "retroviral vector" as used herein is meant to include "lentivirus" and "lentiviral vectors" respectively. Viral particles will typically include various viral components and sometimes also host cell components in addition to polynucleotide(s). The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.

[0236] The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus. The term "hybrid vector" refers to a vector, LTR or other nucleic acid containing both retroviral, e.g., lentiviral, sequences and non- lentiviral viral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid comprising retroviral e.g., lentiviral, sequences for reverse transcription, replication, integration and / or packaging. In particular embodiments, the terms "lentiviral vector" and "lentiviral expression vector" may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. Where reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements are present in RNA form in the lentiviral particles and are present in DNA form in the DNA plasmids.

[0237] At each end of the provirus are structures called "long terminal repeats" or "LTRs". The term "long terminal repeat (LTR)" refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally provide functions fundamental to the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTR comprises U3, R, and U5 regions and appears at both the 5' and 3' ends of the viral genome. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site). As used herein, the term "packaging signal" or "packaging sequence" refers to sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle. Several retroviral vectors use the minimal packaging signal (also referred to as the psi [W] sequence) needed for encapsidation of the viral genome. Thus, as used herein, the terms "packaging sequence", "packaging signal", "psi" and the symbol "W1are used in reference to the non-coding sequence required for encapsidation of retroviral RNA strands during viral particle formation. In various embodiments, vectors comprise modified 5' LTR and / or 3' LTRs. Either or both of the LTRs may comprise one or more modifications including, but not limited to, one or more deletions, insertions or substitutions. Modifications of the 3' LTR are often made to improve the safety of lentiviral or retroviral systems by rendering viruses’ replication defective. As used herein, the term “replication-defective” refers to virus that is not capable of complete, effective replication such that infective virions are not produced (e.g., replication-defective lentiviral progeny). The term “replication-competent” refers to wildtype virus or mutant virus that is capable of replication, such that viral replication of the virus is capable of producing infective virions (e.g., replication- competent lentiviral progeny).

[0238] "Self-inactivating" (SIN) vectors refers to replication-defective vectors, e.g., retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication and, thus, the viral transcript cannot be made without the U3 enhancerpromoter. In a further embodiment, the 3' LTR is modified such that the U5 region is replaced, for example, with an ideal poly(A) sequence. It should be noted that modifications to the LTRs such as modifications to the 3' LTR, the 5' LTR, or both 3' and 5' LTRs, are also included.

[0239] An additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukaemia virus (MoMLV), Rous sarcoma virus (RSV) and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters are able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus because there is no complete U3 sequence in the virus production system. In certain embodiments, the heterologous promoter has additional advantages in controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter can be inducible, such that transcription of all or part of the viral genome will occur only when the induction factors are present. Induction factors include, but are not limited to, one or more chemical compounds or the physiological conditions such as temperature or pH, in which the host cells are cultured.

[0240] In particular embodiments, the vector is a non-integrating vector, including but not limited to, an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is able to replicate without integration into chromosomal DNA of a host and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.

[0241] Preferred viral vectors include an SV40 vector, adenovirus vector, adeno- associated virus (AAV), lentiviral vector, and a retrovirus (or retroviral) vector.

[0242] In another embodiment, the expression vector is a non-viral vector wherein the non- viral vector is a transposon-based expression vector. In embodiments, the transposon-based vector is an SB transposon-based expression vector or piggyBAC expression vector, as described above.

[0243] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer or promoter / enhancer that continually or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence may be a "ubiquitous" promoter, enhancer or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a "cell-specific", "cell type-specific", "cell lineage-specific" or "tissue-specific" promoter, enhancer or promoter / enhancer that allows expression in a restricted variety of cell and tissue types, respectively.

[0244] Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukaemia virus (MoN4LV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1 -alpha (EF1a) promoter, early growth response 1 (EGR1 ), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3- phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1 ), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1 ), heat shock protein 70kDa (HSP70), B-kinesin (WIN), the human ROSA locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer / chicken beta- actin (CAG) promoter, a beta-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer binding site substituted (MND) promoter.

[0245] In an embodiment, it may be desirable to express a polynucleotide comprising a CAR from a constitutive promoter. Such constitutive promoters were used in the examples.

[0246] In an embodiment, it may be desirable to express a polynucleotide comprising a CAR from a NK cell-specific promoter. Such T cell-specific promoters are known in the art and are for example disclosed in Colamartino A et al. (A New Specific Promoter Allow Hematopoietic Stem Cell Immunotherapy Approach Against Acute Lymphoblastic Leukemia Using Chimeric Antigen Receptor, Biology of Blood and Marrow Transplantation, 25(3), Supplement, 2019, page S164) and Goodson- Gregg FJ et al. (Tuning of NK-Specific HLA-C Expression by Alternative mRNA Splicing. Front Immunol. 2020 Jan 10; 10:3034).

[0247] In an embodiment, it may be desirable to express a polynucleotide comprising a CAR from a macrophage-specific promoter.

[0248] In an embodiment, it may be desirable to express a polynucleotide comprising a CAR from a T cell-specific promoter. Such T cell-specific promoters are known in the art and are for example disclosed in Colamartino A et al. (A New Specific Promoter Allow Hematopoietic Stem Cell Immunotherapy Approach Against Acute Lymphoblastic Leukemia Using Chimeric Antigen Receptor, Biology of Blood and Marrow Transplantation, 25(3), Supplement, 2019, page S164).

[0249] Examples of inducible promoters and expression systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-I promoter (inducible by interferon), the "GeneSwitch" mifepristone- regulatable system, the cumate inducible gene switch, tetracycline-dependent regulatory systems, etc. In some embodiments, a polynucleotide or cell comprising the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and / or uncontrolled proliferation. In specific embodiments, the suicide gene is not immunogenic to the host comprising the polynucleotide or cell. A certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0250] In certain embodiments, vectors comprise gene segments that cause the immune effector cells, e.g., NK cells, macrophages or T cells, to be susceptible to negative selection in vivo. By "negative selection" is meant that the infused cell can be eliminated as a result of a change in the in vivo condition of the individual. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes are known in the art, and include, inter alia the following: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene which confers ganciclovir sensitivity; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase.

[0251] In some embodiments, modified immune effector cells, such as NK cells, macrophages or T cells, comprise a polynucleotide further comprising a positive marker that enables the selection of cells of the negative selectable phenotype in vitro. The positive selectable marker may be a gene which, upon being introduced into the host cell expresses a dominant phenotype permitting positive selection of cells carrying the gene. Genes of this type are known in the art, and include, inter alia, hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.

[0252] Preferably, the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also is accompanied by loss of the positive selectable marker. Even more preferably, the positive and negative selectable markers are fused so that loss of one obligatorily leads to loss of the other. An example of a fused polynucleotide that yields as an expression product a polypeptide that confers both the desired positive and negative selection features described above is a hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo. In addition, in embodiments, the polynucleotides encoding the CARs are in retroviral vectors containing the fused gene, particularly those that confer hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo, for example the HyTK retroviral vector.

[0253] In another embodiment, the expression vector is a non-viral vector wherein the non- viral vector an expression vector for providing mRNA encoding the CD4-specific CAR.

[0254] In this alternative method for preparing modified immune cells, the CAR immune cells are modified based on mRNA encoding the CD4-specific CAR.

[0255] The downregulation of CD4-positive T cells by CD4-targeted CAR immune cells constitutively expressing the CD4-binding CAR may become serious, as it may lead, similar to an HIV infection, to impaired cellular immunity and opportunistic infections.

[0256] Accordingly, transient expression of the CD4-specific CARs provided herein in immune cells may be advantageous in certain embodiments.

[0257] For example, mRNA may be prepared by methods known in the art using expression vectors for generating mRNA. For example, constitutive promoters such as the T7 promoter may be used. For example, in vitro transcription (IVT) systems well-known in the art may be used for generating mRNA from expression cassettes or vectors. The promotor and any other control elements or regulatory sequences are operably linked to the coding sequence, to allow transcription.

[0258] The term “promoter” as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In particular embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide. The term "enhancer" refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.

[0259] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a polynucleotide expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide-of- interest, wherein the expression control sequence directs transcription of the polynucleotide corresponding to the second sequence.

[0260] In embodiments, immune cells are transfected with mRNA encoding the CD4- specific CAR provided herein (optionally wherein the mRNA further encodes a leader sequence at the N-terminus of the CAR). Suitable transfection methods are known in the art. For example, naked mRNA may be used for transfection, e.g., via electroporation. In embodiments, as shown in the examples, lipid nanoparticles were successfully used for efficient transient transfection via electroporation.

[0261] For example, lipid nanoparticles may be prepared wherein the lipid nanoparticles comprise the mRNA encoding the CD4-specific CAR provided herein (optionally wherein the mRNA further encodes a CD8 leader sequence at the N-terminus of the CAR). In embodiments, the mRNA is encapsulated in the nanoparticles. The lipid nanoparticles may be used for transfection of immune cells. As can be seen in the Examples, efficient transient transfection of immune cells was achieved.

[0262] Suitable lipid nanoparticles and methods for preparing mRNA-loaded lipid nanoparticles as well as methods for transfection are disclosed in the present examples as well as, e.g., in Sun D, Lu ZR (Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery. Pharm Res. 2023 Jan;40(1 ):27-46).

[0263] In a further aspect, the invention relates to a method for preparing the modified immune cell of the invention, comprising the steps of:

[0264] (i) providing mRNA encoding the CD4-specific CAR provided herein, optionally wherein the mRNA further encodes a leader sequence at the N-terminus of the CAR, (ii) transfecting immune cells with nanoparticles comprising the mRNA provided in (i), thereby obtaining the modified immune cell.

[0265] In embodiments, the nanoparticles are lipid nanoparticles.

[0266] In certain embodiments, the mRNA encoding the CD4-specific CAR provided herein (wherein the mRNA optionally further encodes a leader sequence at the N-terminus of the CAR) may be transfected into immune cells using transfection methods known in the art. For example, electroporation methods may be used. In other embodiments, lipid nanoparticles may be used for transfection. Suitable lipid nanoparticles (LNP) and methods for their preparation and methods for transfection are known in the art, and are described above.

[0267] For example, Billingsley MM et al. (Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering. Nano Lett. 2020 Mar 11 ;20(3):1578- 1589) discloses ionizable lipid nanoparticle including advantageous lipid nanoparticle formulations comprising ionizable lipids combined with set ratios of cholesterol, phospholipid, and lipid-anchored PEG, including a formulation designated C14-4. Further, nanoparticles, including lipid nanoparticles, which can be used are, for example, disclosed in Mukalel AJ et al. (Nanoparticles for nucleic acid delivery: Applications in cancer immunotherapy. Cancer Lett. 2019 Aug 28;458:102-112). For example, the lipid nanoparticle may comprise a liposome, an ionizable lipid, or may be a polymer-lipid nanoparticle. Examples of further ionizable lipids include DLinDMA, Lin-2-DMA, DLin-K-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, YSK13 and YSK15, ALC-0315 ((4- hydroxybutyl)azanediyl)bis(hexane-6, 1 -diyl)bis(2-hexyldecanoate) and SM-102 (heptadecan-9-yl 8-(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino octanoate). In other embodiments, multifunctional pH-sensitive amino lipids may be used.

[0268] Liposomes are typically composed of materials with polar head groups and nonpolar tails. They may spontaneously self-assemble into vesicles at low concentrations. Suitable materials for such cationic liposomes include, e.g., cationic lipids, such as DOTMA, DOTAP and zwitterionic DOPE. Lipids used for liposomal formulations include, e.g., DOTMA, DOSPA, DOTAP, DMRIE and DC-cholesterol. Ionizable LNP formulations are typically comprised of four components: ionizable lipids, such as C12- 200 lipids, phospholipids, such as DOPE and DSPC, cholesterol, and lipid-anchored PEG. Cationic polymers and biopolymers are preferably used as vectors for polynucleotide delivery. Further, chitosan-based nanoparticles, poly(beta-amino esters) or polyplexes may be used. In addition, polyethylenimine (PEI) or PLL (poly-L-lysine) are known as suitable nanoparticles.

[0269] A nanoparticle is understood as spherical or approximately spherical particle of a diameter of about 1 nm to about 1 pm, such as of about 10 nm to about 1 pm, of about 10 nm to about 100 nm, of about 50 nm to about 500 nm or any subrange thereof.

[0270] A lipid nanoparticle is understood as nanoparticle comprising at least one lipid.

[0271] The lipid nanoparticles herein are suitable for encapsulating a polynucleotide encoding the CD4-specific CAR provided herein (optionally wherein the mRNA further encodes a leader sequence at the N-terminus of the CAR). In preferred embodiments, the polynucleotide is RNA, in particular mRNA. In other embodiments, the polynucleotide is DNA, such as an expression cassette or vector.

[0272] In a further aspect, the invention relates to a method for preparing the modified immune cell of the invention, comprising the steps of:

[0273] (i) providing an expression vector comprising a polynucleotide encoding the CD4- specific CAR provided herein, optionally wherein the polynucleotide further encodes a leader sequence at the N-terminus of the CAR encoding the CD4- specific CAR,

[0274] (ii) introducing the expression vector of (i) into an immune cell, thereby obtaining the modified immune cell.

[0275] Preferably, the leader sequence is a CD8 leader sequence. In embodiments, the CD8 leader sequence is as set forth in SEQ ID NO: 30 or is encoded by the sequence as set forth in SEQ ID NO: 31 .

[0276] The method comprises in step providing an expression vector provided herewith and introducing the expression vector in an immune cell.

[0277] Methods of introducing an expression vector in an immune cell are known in the art.

[0278] In some embodiments, an immune cell, as exemplary and preferred host cell, is transduced with a retroviral vector, e.g., a lentiviral vector, encoding a CD4-specific CAR. Such expression vectors are described above. For example, an immune cell is transduced with a vector encoding a CD4-specific CAR as described herein. These transduced cells can elicit a CAR-mediated cytotoxic response.

[0279] A “host cell” includes cells electroporated, transfected, infected or transduced in vivo, ex vivo or in vitro with a vector or a polynucleotide. Host cells may include packaging cells, producer cells and cells transduced with viral vectors.

[0280] In particular embodiments, host cells transduced with viral or non-viral vectors are administered to a subject in need of therapy. In preferred embodiments, the host cell is a NK cell, macrophage or T cell.

[0281] Large scale viral vector production is often necessary to achieve a suitable viral titre. Viral particles may be produced by transfecting a transfer vector into a packaging cell line that comprises viral structural and / or accessory genes.

[0282] As used herein, the term “packaging vector" refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four or more viral structural and / or accessory genes. Typically, the packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known by those of skill in the art. In particular embodiments, a retroviral / lentiviral transfer vector is introduced into a packaging cell line, via transfection, transduction or infection, to generate a producer cell or cell line. In particular embodiments, packaging vectors are introduced into human cells or cell lines by standard methods including, e.g., calcium phosphate transfection, lipofection or electroporation.

[0283] In some embodiments, the packaging vectors are introduced into the cells together with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be linked physically to genes encoding by the packaging vector, e.g., by IRES or self-cleaving viral peptides.

[0284] As used herein, the term "packaging cell lines" is used in reference to cell lines that do not contain a packaging signal but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which are necessary for the correct packaging of viral particles. Any suitable cell line can be employed to prepare packaging cells. Generally, the cells are mammalian cells. In a particular embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines which can be used include, for example, CHO cells, BHK cells, NOCK cells, C3H IOT1 / 2 cells, FLY cells, Psi2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W 138 cells, MRCS cells, A549 cells, HT1080 cells, HEK293 cells, HEK293T cells, B- 50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W 163 cells, 211 cells and 21 IA cells.

[0285] As used herein, the term "producer cell line" refers to a cell line which is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal. The production of infectious viral particles and viral stock solutions may be carried out using conventional techniques.

[0286] Infectious virus particles may be collected from the packaging cells using conventional techniques. For example, the infectious particles can be collected by cell lysis, or collection of the supernatant of the cell culture, as is known in the art. Optionally, the collected virus particles may be purified if desired. Suitable purification techniques are well known to those skilled in the art. Viral envelope proteins (env) determine the range of host cells which can ultimately be infected and transformed by recombinant retroviruses generated from the cell lines. In the case of lentiviruses, such as HIV-1 , HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gp120.

[0287] The terms "pseudotype" or "pseudotyping" as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4 presenting cells.

[0288] In one preferred embodiment, lentiviral envelope proteins are pseudotyped with VSV-G, BaEV or KoRV. In one embodiment, packaging cells produce a recombinant retrovirus, e.g., lentivirus, pseudotyped with the VSV-G, BaEV or KoRV envelope glycoprotein. For example, lentiviral envelope proteins pseudotyped with a KoRV envelope protein are disclosed in W02023 / 083760.

[0289] For example, lentiviral or retroviral envelope proteins pseudotyped with a BaEV envelope protein are disclosed in WO2013 / 045639 A1 and WO2019 / 121945 A1 .

[0290] For the data in NK cells in the Examples, gammaretroviral envelope proteins pseudotyped with a BaEV were used.

[0291] In other embodiments, viral vectors may be pseudotyped with an envelope protein from either another retrovirus or an unrelated virus. The skilled person will appreciate that the viral vectors described herein may be pseudotyped with any suitable envelope protein. The delivery of a gene(s) or other polynucleotide sequence using a retroviral or lentiviral vector by means of viral infection rather than by transfection is referred to as "transduction".

[0292] In one embodiment, retroviral vectors are transduced into a cell through infection and provirus integration. In certain embodiments, an immune cell, is "transduced" if it comprises a gene or other polynucleotide sequence delivered to the cell by infection using a viral or retroviral vector. In particular embodiments, a transduced cell comprises one or more genes or other polynucleotide sequences delivered by a retroviral or lentiviral vector in its cellular genome.

[0293] In a further aspect, the invention relates to a modified immune cell

[0294] (i) comprising a polynucleotide provided herein, or an expression vector provided herein, and / or

[0295] (ii) expressing a CD4-specific CAR provided herein.

[0296] The embodiments disclosed for the aspects of CD4-specific CAR, polynucleotides, vectors, expression vectors, are understood to apply also for the modified immune cells and modified cells provided herein.

[0297] As used herein, the terms “modified cell”, “genetically engineered cell” or “genetically modified cell” refers to a cell wherein extra genetic material was added in the form of DNA or RNA into the total genetic material of the originator cell. The “immune cell”, “effector immune cell”, or “immune effector cell” is any cell of the immune system that has one or more effector functions, (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC).

[0298] In embodiments, the immune cell herein is selected from a T cell, a Natural Killer (NK) cell or a macrophage.

[0299] In embodiments, modified immune cells or modified immune effector cells are genetically modified to express a CAR provided herein, for use in the treatment of a CD4-mediated and / or CD4-associated diseases.

[0300] The immune cell may be selected from the group consisting of a T cell, a natural killer T lymphocyte (NKT) cell, a neutrophil, a macrophage, and a natural killer (NK) cell. Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into an immune effector cell in vivo or in vitro. An immune cell includes progenitors of immune effectors cells such as hematopoietic stem cells (HSCs) contained within the CD34 population of cells derived from cord blood, bone marrow or mobilized peripheral blood which upon administration in a subject differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells.

[0301] In preferred embodiments, immune cells which can be used with the CD4-specific CARs provided herein are selected from NK cells, macrophages and T cells.

[0302] In a more preferred embodiment, immune cells which can be used with the CD4- specific CARs provided herein are selected from NK cells and macrophages.

[0303] In one preferred embodiment, immune cells which can be used with the CD4- specific CARs provided herein are NK cells. In a further preferred embodiment, immune cells which can be used with the CD4-specific CARs provided herein are primary NK cells. In one preferred embodiment, immune cells which can be used with the CD4-specific CARs provided herein are primary autologous NK cells.

[0304] In another preferred embodiment, immune cells which can be used with the CD4- specific CARs provided herein are macrophages. In a further preferred embodiment, immune cells which can be used with the CD4-specific CARs provided herein are primary macrophages. In one preferred embodiment, immune cells which can be used with the CD4-specific CARs provided herein are primary autologous macrophages.

[0305] NK cells and macrophages are particularly advantageous for subjects which are immunocompromised, such as subjects infected with HIV, HTLV-1 or HLTV-2 infection, subjects which are heavily pre-treated with chemotherapy regimens and subjects suffering from T cell malignancies, and which therefore have a significantly reduced pool of suitable T cells.

[0306] Thereby, sufficient amounts of viable starting immune cell cells can be obtained from such subjects for autologous CAR immune cell therapy.

[0307] In addition, T cells, especially in the context of an anti-CD4 CAR product, are committing "fratricide" by killing normal and therapeutic, CAR-carrying CD4 positive T cells. Such "fratricide" is also avoided when using NK cells and / or macrophages.

[0308] In another embodiment, immune cells which can be used with the CARs provided herein are T cells. In a further preferred embodiment, immune cells which can be used with the CARs provided herein are primary T cells. In one preferred embodiment, immune cells which can be used with the CARs provided herein are primary autologous T cells.

[0309] In one embodiment, the immune cell is a cytotoxic T lymphocyte (CD8 positive).

[0310] For CD8 positive T cells as CAR immune cells, no “fratricide” is expected.

[0311] The term “T cell” is known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper I (Th1 ) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 positive T cell), a cytotoxic T cell (CTL; CD8-positive T cell), CD4 positive CD8 positive T cell, CD4 negative CD8 negative T cell or any other subset of T cells. Other exemplary populations of T cells suitable for use herein include naive T cells and memory T cells.

[0312] Methods for making or generating and preparing the modified immune cells which express the CD4-specific CARs are provided herein. In various embodiments, such methods comprise introducing into an immune cell a polynucleotide and / or expression vector as described herein.

[0313] In one embodiment, the method comprises transfecting or transducing immune cells isolated from an individual such that the immune cells express a CD4-spcific CAR provided herein. In certain embodiments, the immune effector cells are isolated from an individual and are genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In further embodiments, the immune cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a CAR. In this regard, the immune effector cells may be cultured before and / or after being genetically modified (i.e., transduced or transfected to express a CAR provided herein). Thus, in certain embodiments, the immune cells may be stimulated and induced to proliferate by contacting the cell with antibodies or antigen-binding fragments that bind CD3 and / or antibodies or antigen binding fragments that bind to CD28; thereby generating a population of immune effector cells. In further embodiments, the method of generating immune effector cells contemplated herein comprises stimulating the immune effector cell and inducing the cell to proliferate by contacting the cell with antibodies or antigen binding fragments that bind CD3 and antibodies or antigen binding fragments that bind to CD28; thereby generating a population of immune effector cells. Such stimulation is in particular suitable for T cells as immune effector cells. Suitable protocols for stimulating NK cells are known in the art and comprise stimulation with IL-2 and / or IL-15. Suitable protocols for macrophages are known in the art and comprise differentiation with GM-CSF.

[0314] In particular embodiments, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the immune cells are obtained from a subject. In particular embodiments, the modified cells comprise NK cells. In other particular embodiments, the modified cells comprise macrophages. In other particular embodiments, the modified immune cells comprise T cells.

[0315] In particular embodiments, peripheral blood mononuclear cells (PBMCs) may be directly genetically modified to express CD4-specific CARs using methods contemplated herein. In certain embodiments, after isolation of PBMCs, NK cells are further isolated. In certain embodiments, after isolation of PBMCs, macrophages are further isolated. In certain embodiments, after isolation of PBMCs, T cells are further isolated. The immune cells, in particular NK cells, macrophages and / or T cells, may be obtained by leukapheresis from a subject. Thereby, primary immune cells, in particular primary NK cells, primary macrophages and / or primary T cells are obtained.

[0316] The immune cells, such as NK cells or macrophages, or T cells, can be genetically modified following isolation using known methods described herein, or the immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In a particular embodiment, the immune effector cells, such as NK cells or macrophages, or T cells, are genetically modified with the CARs provided herein (e.g., transduced with a viral or non-viral vector, or mRNA comprising a polynucleotide encoding a CAR) and then are activated and expanded in vitro. Alternatively, the cells can be activated and expanded before or after genetic modification to express a CAR.

[0317] In particular embodiments, successfully transduced NK cells, macrophages or T cells that comprise the vector or mRNA can be sorted using flow cytometry to isolate immune cells, e.g., NK cells, macrophages or T cells, using an appropriate cell surface expression marker (e.g. CD3 for T cells) and can then be further propagated to increase the number of these CAR protein expressing NK cells, macrophages or T cells in addition to cell activation (e.g. using anti-CD3 antibodies and or anti-CD28 antibodies and IL-2 in the case of T cells; combinations of IL-2, IL-12, IL-15, IL-18, IL-21 and / or irradiated feeder cells based on K562 or primary immune cells for NK cells; or GM-CSF or M-CSF for macrophages).

[0318] For example, CAR protein expression can be determined by antibody staining in flow cytometry, as in the Examples.

[0319] Further, functionality of CAR immune cells can be determined by methods known in the art. For example, cytotoxicity assays may be performed to determine functionality. In the examples, a Calcein-based cytotoxicity assay was used to determine cytotoxicity of CAR immune cells towards target cells. Further, in the examples, an assay for determining cytotoxicity of CAR immune cells towards primary PBMC cells was used. After incubation, cells are stained with antibody panels.

[0320] Further, alternatively, a flow cytometry-based cytotoxicity assay may be used. Such assay was used successfully for CAR T cells in the examples. For determining functionality of CAR macrophages, a macrophage phagocytosis assay may be determined.

[0321] For example, it is possible to determine a threshold value of functionality of a candidate CAR immune cell, as determined by the value of cytotoxicity, or phagocytosis. For example, a value or threshold value may be determined as compared to a control value. Such control value may for example be the value of a CAR immune cell or other cytotoxic agent of known cytotoxicity.

[0322] Standard procedures are used for cryopreservation of immune cells, e.g., NK cells, macrophages or T cells expressing the CAR protein, for storage and / or preparation for use in a human subject.

[0323] In embodiments, the immune cell is selected from a T cell, NK cell and macrophage, optionally wherein the immune cell is a primary cell and / or autologous cell or an allogeneic cell.

[0324] “Autologous” as used herein, refers to cells from the same subject. “Allogeneic” as used herein, refers to cells of the same species that differ genetically to the cell in comparison.

[0325] In embodiments, the immune cell is a primary cell obtained from a patient. Such primary cell is obtained from a donor subject, which may be the same subject as the subject to be treated. In such embodiment, the primary cell is an autologous primary cell. The use of primary cells is preferred according to the invention. The present CD4-specific CARs surprisingly exhibit both efficient expression and functionality in primary NK cells, macrophages and T cells.

[0326] In other embodiments, such primary cell is obtained from a donor subject, which is a subject different from the subject to be treated. In such embodiment, the primary cell is an allogeneic primary cell. In an embodiment, the allogeneic primary cell is H LA-matched.

[0327] In embodiments, basal or tonic signalling is determined prior to administering the CAR immune cell to a patient. Thereby, antigen-independent signalling is determined. The CAR’s propensity to antigen-independent (basal) signalling might indicate a self-aggregation leading to antigen-independent CAR activation that in turn could cause early CAR exhaustion, resulting in loss of therapeutic potency. Basal activation of CAR-immune cells may be determined through the levels of the activation marker CD69, the exhaustion markers PD1 and TIM3, the phosphorylation of intracellular signalling domain (CD3 , DAP10 or DAP12), and the ability of CAR-immune cells to secret IFN-y, IL-6 or other pro-inflammatory cytokines in the absence of antigen.

[0328] In a yet further aspect, the invention relates to a modified cell

[0329] (i) comprising a polynucleotide provided herein, or an expression vector provided herein, and / or

[0330] (ii) expressing a CD4-specific CAR provided herein.

[0331] In embodiment, the modified cell is an immune cell. In embodiments, the modified cell is a hematopoietic stem cell. In other embodiments, the modified cell is an induced pluripotent stem cell (iPSC).

[0332] In a further aspect, the invention relates to a pharmaceutical composition comprising a modified immune cell and at least one pharmaceutically acceptable carrier and / or excipient, wherein the modified immune cell is

[0333] (i) comprising a polynucleotide provided herein, or an expression vector provided herein, and / or

[0334] (ii) expressing a CD4-specific CAR provided herein.

[0335] In a further aspect, the invention relates to a modified immune cell or pharmaceutical composition provided herein, for use in the treatment of a CD4-mediated and / or CD4-associated disease in a subject.

[0336] Such pharmaceutical compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice, see e.g., Remington's Pharmaceutical Sciences, 16thedition (1980) Mack Publishing Co.

[0337] Pharmaceutical compositions may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular and intraportal). In one embodiment, the composition is suitable for intravenous administration.

[0338] A “therapeutically effective amount” of a modified immune cell may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, tumour size, extent of infection or metastasis and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune cells, e.g., NK cells, macrophages or T cells, described herein, may be administered at a dosage of 102to 1010cells / kg body weight, preferably 105to 106cells / kg body weight, including all integer values within those ranges. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein. For uses provided herein, the cells are generally in a volume of a litre or less, can be 500 mL or less, even 250 mL or 100 mL or less. Hence the density of the desired cells is typically greater than 106cells / ml, e.g., greater than 106, 107, 108or 109cells / ml. The clinically relevant number of immune cells, e.g., NK cells, macrophages or T cells, can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108109, 1010, 1011or 1012cells. In some embodiments, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of 106 / kilogram (106to 1011per patient) may be administered. In a particular embodiment, between 1 x 107and 9 x 107CAR-immune cells may be administered. Compositions comprising modified immune cells, e.g., NK cells, macrophages or T cells, may be administered multiple times at dosages within these ranges. For example, modified immune cells, e.g., NK cells, macrophages or T cells may be administered every 7 days. Alternatively, compositions comprising modified immune cells, e.g., NK cells, macrophages or T cells may be administered as a single dose. The cells may be allogeneic, syngeneic, xenogeneic or autologous to the patient undergoing therapy. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-y, IL-2, IL-12, IL-15, TNFa, IL-18, and TNFI3, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1 a, etc.), to enhance induction of the immune response. Generally, compositions comprising the cells activated and expanded as described herein may be utilised in the treatment of diseases that arise in individuals who are immunocompromised. In particular embodiments, modified immune cells, e.g., NK cells, macrophages or T cells may be administered either alone, or as a pharmaceutical composition in combination with carriers, diluents, excipients and / or with other components such as IL-2 or other cytokines or cell populations. In particular embodiments, pharmaceutical compositions comprise an amount of modified immune cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0339] Pharmaceutical compositions comprising modified immune cells, e.g., NK cells, macrophages or T cells, may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminium hydroxide); and preservatives. In particular embodiments, compositions are preferably formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerine, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0340] In one embodiment, the modified immune cells, e.g., NK cells, macrophages or T cells, are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In particular embodiments, the pharmaceutically acceptable cell culture medium is a serum free medium. In another preferred embodiment, modified immune cells, e.g., NK cells, macrophages or T cells, are formulated in a solution comprising a cryopreservation medium. For example, cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw. Examples of cryopreservation media which may be used include CRYOSTOR CS10, CRYOSTOR CS5, and CRYOSTOR CS2.

[0341] Modified immune cells, e.g., NK cells, macrophages or T cells, may be formulated for administration alone, or in combination with one or more therapeutic agents. Thus, the modified immune cells, e.g., NK cells, macrophages or T cells, may be administered alone or in combination with other known treatments for the respective disease, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, anti-retroviral therapy etc., depending on the disease treated. The compositions may also be administered in combination with antibiotics.

[0342] Further provided is a kit of parts comprising the pharmaceutical composition comprising the modified immune cells, e.g., NK cells, macrophages or T cells, and other medicaments, optionally and / or with instructions for use. For convenience, the kit of parts may comprise the reagents in predetermined amounts with instructions for use. The kit may also include devices used for administration of the pharmaceutical composition.

[0343] The terms "individual", "subject" and "patient" are used herein interchangeably and refer to any animal that exhibits a symptom of a disease, disorder or condition that can be treated with the CAR immune cells and methods and uses provided herein. In preferred embodiments, a subject includes any animal that exhibits symptoms of any CD4-mediated and / or CD4-associated disease. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0344] In preferred embodiments, the subject is a human.

[0345] Subjects include human patients that have been diagnosed with, or are suspected to have a CD4-mediated and / or CD4-associated disease.

[0346] As used herein "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction of the disease or condition, or the delaying of the progression of the disease or condition, e.g., delaying tumour outgrowth, slowing down cancer progression, or slowing down progression of an autoimmune disease or slowing down reoccurrence of detectable virus titres in case of an HIV, HTLV-1 or HLTV-2 infection. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. “Treating” also includes ameliorating or mitigating one or more disease symptoms, such as ameliorating one or more autoimmune disease symptoms, one or more symptoms of an HIV, HTLV- 1 or HLTV-2 infection or one or more symptoms of a CD4 positive cancer.

[0347] As used herein, "prevent" and similar words such as "prevented", "preventing" etc., indicate an approach for preventing, inhibiting or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition.

[0348] In a further aspect, the invention relates to a modified immune cell provided herein, for use in the treatment of a CD4-mediated and / or CD4-associated disease in a subject.

[0349] A “CD4-mediated and / or CD4-associated disease” is understood as a disease which can be treated or ameliorated by killing, reducing the number and / or inactivating CD4-expressing cells, and / or a disease wherein CD4-expressing cells are involved in mediating, the onset of, the progression of, the recurrence of, or the therapyresistance of a disease.

[0350] In embodiments, a modified CAR immune cell is used which is selected from an NK cell, macrophage and a T cell.

[0351] In embodiments, two or more different modified CAR immune cells are used, preferably selected from NK cells, macrophages and T cells.

[0352] For example, a modified CAR NK cell and a modified CAR macrophage of the present invention may be used. For example, a modified CAR NK cell and a modified CAR T cell of the present invention may be used. For example, a modified CAR macrophage and a modified CAR T cell of the present invention may be used.

[0353] In embodiments, the CD4-mediated and / or CD4-associated disease is selected from:

[0354] (i) a CD4 positive cancer, optionally wherein the CD4 positive cancer is a CD4 positive T cell lymphoma or CD4 positive T cell leukemia or a CD4 positive malignancy, or wherein the CD4 positive cancer is selected from anaplastic large cell lymphoma, peripheral T cell lymphoma (PTL), or acute myeloid leukemia (AML), cutaneous T cell lymphoma, and Sezary Syndrome; optionally wherein the subject treated exhibits a blast crisis, a minimal residual disease, has undergone lymphodepletion or depletion of CD4 positive cells and / or has undergone stem cell transplantation and / or will undergo stem cell transplantation;

[0355] (ii) a CD4 negative cancer, in which non-malignant CD4 positive T cells represent a population within or surrounding the tumour tissue;

[0356] (iii) a T cell-mediated autoimmune disease, optionally wherein the T cell-mediated autoimmune disease is rheumatoid arthritis or ulcerative colitis; and

[0357] (iv) an HIV, HTLV-1 or HLTV-2 infection, optionally wherein the subject has undergone lymphodepletion or depletion of CD4 positive cells.

[0358] In one embodiment, the CD4-mediated and / or CD4-associated disease is a CD4 positive cancer.

[0359] A CD4 positive cancer is understood as a cancer wherein at least a proportion of the cancer cells expresses CD4 on the cell surface.

[0360] In embodiments, at least 20%, 25%, 30%, 35%, 40%, 45, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, such as 100%, of the cancer cells in a sample expresses CD4 on the cell surface.

[0361] CD4 cell surface expression can be determined, e.g., by IHC or flow cytometry using a labelled anti-CD4 antibody. For example, an anti-CD4 antibody which is labelled with a fluorescent dye may be used, in particular for FACS analysis methods. Alternatively, an indirect label may be used which is detectable by a secondary antibody, e.g., for use in immunoassays such as ELISA or IHC methods.

[0362] For example, staining with dye-conjugated antibodies may be performed, as in the Examples herein.

[0363] A “T cell lymphoma” is a cancerous lymphoma affecting T cells. Lymphoma arises mainly from the uncontrolled proliferation of T cells and can become cancerous. T cell lymphoma is a type of Non-Hodgkin lymphoma (NHL) and represents less than 15% of all Non-Hodgkin's diseases in the category. T cell lymphomas are typically further categorised based on their growth patterns as either aggressive (i.e., fastgrowing) or indolent (i.e., slow-growing).

[0364] Known common T cell lymphoma subtypes include Peripheral T cell lymphoma (PTCL), Angioimmunoblastic T cell lymphoma (AITL), Anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic large cell lymphoma, ALK-negative anaplastic large cell lymphoma, Primary cutaneous anaplastic large cell lymphoma, Breast cancer-associated anaplastic large cell lymphoma, Adult T cell leukemia / lymphoma (ATL), Extranodal NK / T cell lymphoma, nasal type (ENKTL), and Cutaneous T cell lymphoma (CTCL).

[0365] CD4 positive T cell lymphoma include Peripheral T cell lymphoma (PTCL), Angioimmunoblastic T cell lymphoma (AITL), Anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic large cell lymphoma, ALK-negative anaplastic large cell lymphoma, Primary cutaneous anaplastic large cell lymphoma, Breast cancer- associated anaplastic large cell lymphoma, Adult T cell leukemia / lymphoma (ATL), Extranodal NK / T cell lymphoma, nasal type (ENKTL), and Cutaneous T cell lymphoma (CTCL).

[0366] Cutaneous T cell lymphoma (CTCL) includes subtypes Mycosis fungoides and Sezary syndrome.

[0367] A “T cell leukemia” or “TCL” is understood as a lymphoid leukemia which affects T cells. A “CD4 positive T cell leukemia” is understood as T cell leukemia wherein at least a proportion of the leukemia cells expresses CD4 on the cell surface.

[0368] For example, at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40% or 50% of the leukemia cells in a sample, such as a blood sample, expresses CD4 on the cell surface.

[0369] T cell leukemia include large granular lymphocytic leukemia, Adult T cell leukemia / lymphoma and T cell prolymphocytic leukemia.

[0370] CD4 positive T cell leukemia include large granular lymphocytic leukemia, Adult T cell leukemia / lymphoma and T cell prolymphocytic leukemia.

[0371] A “CD4 positive malignancy” is understood as malignant disorder wherein the malignant cells include CD4 positive cells. Examples of CD4 positive malignancies are CD4 positive T cell leukemia and CD4 positive T cell lymphoma described above.

[0372] The present modified CAR immune cells are particularly advantageous for treating a CD4 positive cancer, optionally wherein the CD4 positive cancer is a CD4 positive T cell lymphoma or CD4 positive T cell leukemia or a CD4 positive malignancy in a subject. In particular, modified CAR NK cells or CAR macrophages, or CAR CD8 positive T cells may be used.

[0373] This applies in particular for a subject which exhibits a blast crisis, a minimal residual disease, has undergone lymphodepletion or depletion of CD4 positive cells and / or has undergone stem cell transplantation and / or will undergo stem cell transplantation.

[0374] Blast crisis is characterized by an increase in blast cells in the marrow, which in turn results in blood hyperviscosity and relative reduction of other cell lines. Blast crises is a life-threatening hematologic emergency.

[0375] Also, for treating minimal residual disease, the present modified CAR immune cells may be used advantageously, to eliminate any remaining CD4 positive malignant cell.

[0376] For example, such patients may undergo hematopoietic stem cell transplantation (HSCT) after CAR immune cell therapy, to restore a healthy immune system.

[0377] Patients which have undergone or will undergo stem cell transplantation typically have to undergo prior lymphodepletion (typically applying standard lymphodepleting protocols, such as chemotherapy regimen with busulfan and fludarabine). Depletion of CD4 positive cells may be achieved by administering a depleting anti-CD4 antibody to a patient. For such subjects, there is typically not enough starting CD4 positive T cell material available. Using primary, autologous NK cells and / or macrophages may be advantageous.

[0378] In one embodiment, the CD4-mediated and / or CD4-associated disease is a CD4 negative cancer, in which non-malignant CD4 positive T cells represent a population within or surrounding the tumour tissue. A “CD4 negative cancer” is understood is understood as a cancer wherein only a minor proportion of the cancer cells expresses CD4 on the cell surface or wherein the cancer cells do not express CD4 on the cell surface.

[0379] In embodiments, less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 7%, 6%, 5%, 4%, 3%, 2% or 1 %, such as 0%, of the cancer cells in a sample expresses CD4 on the cell surface.

[0380] In some embodiments, the CD4 negative cancer is a solid malignancy, such as colorectal cancer, breast cancer, ovarian cancer, lung cancer, melanoma, hepatocellular carcinoma, glioblastoma or pancreatic cancer.

[0381] In the CD4 negative cancer herein, non-malignant CD4 positive T cells represent a population within or surrounding the tumour tissue.

[0382] In embodiments, non-malignant CD4 positive T cells represent a significant population within or surrounding the tumour tissue. For example, the number of tumor-resident CD4-positive cells can be assessed by histology of tumor biopsies or by flow cytometry of singularized tissue samples using CD4-specific staining reagents and may represent more than 0%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of total cells.

[0383] In preferred embodiments, the non-malignant CD4 positive T cells within or surrounding the tumour tissue are regulatory T cells (Tregs). The presence of non- malignant CD4 positive T cells within or surrounding the tumour tissue can be determined by methods known in the art, e.g., by using immunological and immunohistological methods using antibodies binding to cell surface markers for the cells. In particular, cell surface markers for Tregs are well-known in the art.

[0384] As shown in the examples, the CD4-specific CAR immune cells of the invention are capable of killing Treg cells.

[0385] In such embodiments, CD4 negative cancer may be treated which contain Tregs within and / or surrounding the tumour tissue.

[0386] In embodiments, the CD4-specific CAR immune cell herein may be used for treating the CD4 negative cancer in combination with at least one additional therapeutic agent. The at least one additional therapeutic agent may be for example a chemotherapeutic or immunotherapeutic agent. For example, an immunotherapeutic agent includes a tumour vaccine, a further CAR immune cell comprising a cancer antigen-binding domain, TILs or an immune checkpoint inhibitor, or any combination thereof.

[0387] Suitable immune checkpoint inhibitors are known in the art. In embodiments, the immune checkpoint inhibitor is selected from an antagonist anti-PD-1 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, an antagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-VISTA antibody, an antagonist anti-TIGIT antibody, an antagonist anti-CEACAM1 antibody, an antagonist anti-CD96 antibody, an agonist anti-GITR antibody, an agonist anti-OX40 antibody, or a combination thereof. In another embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody, optionally wherein the anti-PD-1 antibody is pembrolizumab or nivolumab. In certain embodiments, the immunotherapeutic agent is an inhibitor of indoleamine-2,3- dioxygenase (IDO). In another embodiment, the inhibitor is selected from the group consisting of epacadostat, F001287, indoximod, and NLG919.

[0388] In certain embodiments, the additional therapeutic agent is a tumour vaccine. In embodiment, the tumour vaccine comprises a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. In another embodiment, the heat shock protein is hsc70 and is complexed with a tumor-associated antigenic peptide. In another embodiment, the heat shock protein is gp96 protein and is complexed with a tumour-associated antigenic peptide, optionally wherein the HSPPC is derived from a tumour obtained from a subject.

[0389] The antigen of the further CAR immune cell comprising a cancer antigen-binding domain binds to an antigen different from CD4. In embodiments, the antigen is expressed on the surface of the cancer cells of the subject to be treated. Exemplary antigens include, for example, CD19 and BCMA.

[0390] In other embodiments, tumour-infiltrating lymphocytes (TILs) may be used. For example, autologous TILs may be used. TILs are used in adoptive lymphocyte transfer therapy. Methods for providing and adoptively transferring TILs are known in the art. For example, TILs are for example expanded ex vivo from surgically resected tumours that have been cut into small fragments or from single cell suspensions isolated from the tumour fragments. Multiple individual cultures may be established, grown separately and assayed for specific tumour recognition. TILs may be expanded over the course of a few weeks with a high dose of IL-2 in 24-well plates. Selected TIL lines with appropriate tumour recognition may then be further expanded, e.g., in a “rapid expansion protocol” (REP), which uses anti-CD3 activation for a typical period of two weeks. The therapeutic TIL population is infused back into the patient. Adoptive transfer of the TILs may include prior chemotherapy for lymphodepletion, e.g., with a combination of fludarabine and cyclophosphamide.

[0391] In a further aspect, the present invention relates to (a) a CD4-specific CAR immune cell of the present invention, and (b) an additional therapeutic agent for, use in a method for the treatment of CD4-mediated and / or CD4-associated disease. In a further aspect, the present invention relates to a pharmaceutical composition, kit or kit-of-parts comprising (a) a CD4-specific CAR immune cell of the present invention, and (b) an additional therapeutic agent herein. In certain embodiments, the additional therapeutic agent is a chemotherapeutic or immunotherapeutic agent. In certain embodiments, the immunotherapeutic agent includes a tumour vaccine, a further CAR immune cell comprising a cancer antigen-binding domain, TILs or an immune checkpoint inhibitor, or any combination thereof.

[0392] The CD4-specific CAR immune cell and the additional therapeutic agent may be provided spatially separate, e.g. in separate containers, or as a single composition.

[0393] The CD4-specific CAR immune cell and the additional therapeutic agent may be administered at the same time point(s) or at different time point(s).

[0394] Dosages and dosage regimens for the additional therapeutic agent depend on the additional therapeutic agent and can be determined by the person skilled in the art. For example, in the case of approved therapeutic agents, dosages and dosage regimens corresponding to or similar to the approved dosage and dosage regimen may be used.

[0395] In another embodiment, the CD4-mediated and / or CD4-associated disease is a T cell-mediated autoimmune disease, optionally wherein the T cell-mediated autoimmune disease is rheumatoid arthritis or ulcerative colitis. An “autoimmune disease” is a condition that results from an anomalous response of the adaptive immune system, wherein it mistakenly targets and attacks healthy, functioning parts of the body. A “T cell-mediated autoimmune disease” is understood as autoimmune disease wherein the disease is at least partially mediated by T cells.

[0396] T cell-mediated autoimmune diseases include rheumatoid arthritis and ulcerative colitis.

[0397] Rheumatoid arthritis (RA) is a chronic inflammatory disease of the joints that results in disability and premature mortality. In RA patients, an abnormal T cell development and a negative role of CD4 positive T cells was observed.

[0398] Currently available treatment options include NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide and Tumor Necrosis Factor (TNF) Inhibitors, including etanercept and infliximab.

[0399] Many patients affected by ulcerative colitis need immunosuppressant therapies, such as with corticosteroids, which may be associated with a higher risk of contracting opportunistic infectious diseases. Also, the treatment with therapeutic antibodies such as ustekinumab, is associated with considerable side effects. Also, there is currently no complete cure possible.

[0400] Ulcerative colitis is an inflammatory bowel disease (IBD) that causes inflammation and ulcers in the tract. Also in ulcerative colitis subjects, abnormal changes in CD4 positive T cell subsets for observed.

[0401] Many patients affected by ulcerative colitis need immunosuppressant therapies, such as with corticosteroids, which may be associated with a higher risk of contracting opportunistic infectious diseases. Also, the treatment with therapeutic antibodies such as ustekinumab, is associated with considerable side effects. Also, there is currently no complete cure possible.

[0402] Treatment with modified CAR immune cells is useful for reducing the number and / or killing autoreactive CD4 positive cells. In one embodiment, the CD4-mediated and / or CD4-associated disease is an HIV, HTLV-1 or HLTV-2 infection, optionally wherein the subject has undergone lymphodepletion or depletion of CD4 positive cells.

[0403] HTLV-1 and HLTV-2 are retroviruses of the human T-lymphotropic virus (HTLV) family. A subgroup of HTLV-1 infected subjects suffers from adult T cell lymphoma (ATL) or HTLV-l-associated myelopathy / Tropical spastic paraparesis (HAM / TSP). Both diseases are only diagnosed in individuals testing positive to HTLV-1 infection. HTLV-2 may be linked to Cutaneous T cell lymphoma (CTCL).

[0404] The human immunodeficiency virus (HIV) is a lentivirus. Two types of HIV have been characterized, HIV-1 and HIV-2. HIV can infect a variety of immune cells including as CD4 positive T cells. HIV infection leads to low levels of CD4 positive T cells through a number of mechanisms, including pyroptosis of abortively infected T cells, apoptosis of uninfected bystander cells, direct viral killing of infected cells, and killing of infected CD4 positive T cells by CD8 positive cytotoxic lymphocytes that recognize infected cells. When CD4 positive T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections, leading to the development of AIDS. In particular, over time, an HIV infection causes acquired immunodeficiency syndrome (AIDS) in infected subjects, a condition in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. Without treatment, the average survival time after infection with HIV is estimated to be 9 to 11 years, depending on the HIV subtype. The present treatment option of HIV / AIDS normally includes the use of multiple antiretroviral drugs. A complete cure is, however, not possible by multiple antiretroviral drugs, as residual HIV-infected CD4 positive T cells remain in infected subjects and HIV particles can be reproduced by the infected CD4 positive T cells.

[0405] Therefore, eliminating and / or killing CD4 positive cells by administering modified CAR immune cells provided herein may be useful for treating subject infected with HIV, HTLV-1 or HLTV-2.

[0406] For example, such patients may undergo hematopoietic stem cell transplantation (HSCT) after CAR immune cell therapy, to restore a healthy immune system.

[0407] In a yet further aspect, the invention relates to method of treating a CD4-mediated and / or CD4-associated disease in a subject in need thereof, the method comprising administering to said patient a therapeutically effective amount of a modified immune cell provided herein.

[0408] In embodiments thereof, the CD4-mediated and / or CD4-associated disease is selected from:

[0409] (i) a CD4 positive cancer, optionally wherein the CD4 positive cancer is a CD4 positive T cell lymphoma or CD4 positive T cell leukemia or a CD4 positive malignancy, or wherein the CD4 positive cancer is selected from anaplastic large cell lymphoma, peripheral T cell lymphoma (PTL), or acute myeloid leukemia (AML), cutaneous T cell lymphoma, and Sezary Syndrome; optionally wherein the subject treated exhibits a blast crisis, a minimal residual disease, has undergone lymphodepletion or depletion of CD4 positive cells and / or has undergone stem cell transplantation and / or will undergo stem cell transplantation;

[0410] (ii) a CD4 negative cancer, in which non-malignant CD4 positive T cells represent a population within or surrounding the tumour tissue;

[0411] (iii) a T cell-mediated autoimmune disease, optionally wherein the T cell-mediated autoimmune disease is rheumatoid arthritis or ulcerative colitis; and

[0412] (iv) an HIV, HTLV-1 or HLTV-2 infection, optionally wherein the subject has undergone lymphodepletion or depletion of CD4 positive cells.

[0413] In a yet further aspect, the invention relates to method of treating a CD4 positive cancer in a subject in need thereof, the method comprising administering to said patient a therapeutically effective amount of a modified immune cell provided herein.

[0414] In embodiments, the CD4 positive cancer is a CD4 positive T cell lymphoma or CD4 positive T cell leukemia or a CD4 positive malignancy, or the CD4 positive cancer is selected from anaplastic large cell lymphoma, peripheral T cell lymphoma (PTL), or acute myeloid leukemia (AML), cutaneous T cell lymphoma, and Sezary Syndrome.

[0415] Figure 6(B) demonstrates CAR-mediated cytotoxicity of CD4-CAR-NK cells towards diverse AML and TCL cell lines assessed by a Calcein-based cytotoxicity assay. Figure 7 demonstrates cytotoxicity of CD4-specific CAR NK cells towards patient- derived, primary samples of (A) T cell malignancies and (B) acute myeloid leukemia, assessed in a flow cytometry-based cytotoxicity assay. The data demonstrate that CD4-specific CAR NK cells provided herein can be used for treating a CD4 positive cancer such as a CD4 positive T cell lymphoma or CD4 positive T cell leukemia or a CD4 positive malignancy, including AML or TCL.

[0416] In embodiments, the subject treated exhibits a blast crisis, a minimal residual disease, has undergone lymphodepletion or depletion of CD4 positive cells and / or has undergone stem cell transplantation and / or will undergo stem cell transplantation.

[0417] In a yet further aspect, the invention relates to a method of treating a CD4 negative cancer, in which non-malignant CD4 positive T cells represent a population within or surrounding the tumour tissue, in a subject in need thereof, the method comprising administering to said patient a therapeutically effective amount of a modified immune cell provided herein.

[0418] In embodiments, the non-malignant CD4 positive T cells are Treg cells. In embodiments, the Treg cells represent a significant population within or surrounding the tumour tissue.

[0419] In a yet further aspect, the invention relates to method of treating a T cell-mediated autoimmune disease in a subject in need thereof, the method comprising administering to said patient a therapeutically effective amount of a modified immune cell provided herein.

[0420] In embodiments, the T cell-mediated autoimmune disease is rheumatoid arthritis or ulcerative colitis.

[0421] In a yet further aspect, the invention relates to method of treating an HIV, HTLV-1 or HLTV-2 infection in a subject in need thereof, the method comprising administering to said patient a therapeutically effective amount of a modified immune cell provided herein.

[0422] In embodiments, the subject has undergone lymphodepletion or depletion of CD4 positive cells.

[0423] The embodiments disclosed for any aspect provided herein are understood to apply also for the other aspects of the invention herein. “Percent identity” between a query nucleic acid sequence and a subject nucleic acid sequence is understood as the “Identities" value, expressed as a percentage, that is calculated using a suitable algorithm or software, such as BLASTN, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle or EMBOSS infoalign, over the entire length of the query sequence after a pairwise global sequence alignment has been performed using a suitable algorithm or software, such as BLASTN, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene. A query nucleic acid sequence may be described by a nucleic acid sequence provided herein.

[0424] “Percent identity” between a query amino acid sequence and a subject amino acid sequence is understood as the “Identities” value, expressed as a percentage, that is calculated using a suitable algorithm or software, such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle or EMBOSS infoalign, over the entire length of the query sequence after a pairwise global sequence alignment has been performed using a suitable algorithm / software such as BLASTP, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene. A query amino acid sequence may be described by an amino acid sequence provided herein.

[0425] The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid or nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the subject sequence. Such alterations include at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acids or nucleotides in the query sequence or in one or more contiguous groups within the query sequence.

[0426] The term “about” is understood as the respective value ± 10% or as the respective value ± 5 %.

[0427] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings contemplated herein that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognise a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

[0428] Examples

[0429] Example 1 : Cells and cultivation

[0430] Isolation and cultivation of NK cells

[0431] Human NK cells were isolated from healthy donor blood samples from the Institute for Transfusion Medicine of the University Clinic in Leipzig, Germany (ethics vote number 327 / 22-ek), using the NK cell isolation RosetteSep™ Human NK Cell Enrichment Cocktail (Stemcell Technologies, Canada). After isolation, NK cells were activated and expanded by culturing in NK MACS® basal medium (Miltenyi Biotec, Germany), 5% human serum (Sigma Aldrich, USA), 140 U / mL IL-15 and 500 U / mL IL-2 (PeproTech, USA). NK cells were cultured in 24 well suspension plates (Greiner Bio-One GmbH, Germany) or 24 well G-Rex plates (Wilson Wolf, USA).

[0432] Isolation and cultivation of CD8+ T cells

[0433] Primary human CD8+T cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained by density gradient centrifugation of blood from healthy donors (purchased from the Institute for Transfusion Medicine of the University Clinic of Leipzig, Germany, ethics vote number: 272-12-13082012) using Ficoll-Paque PLUS (Th. Geyer GmbH & Co. KG, Germany) followed by magnetic-activated cell sorting using the CD8+T cell isolation kit in combination with magnetic LS columns (Miltenyi Biotec) according to manufacturer's protocol. After isolation, cell concentration was adjusted to 1x106cells / mL in lmmunoCultTM-XF T Cell Expansion Medium (Stemcell Technologies) supplemented with 100 ng / mL IL-2 (PeproTech). T cells were stimulated by the addition of 25 pL / mL ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (Stemcell Technologies). For the duration of culture, cell concentration was adjusted to 1 -3x105cells / mL by addition of fresh medium with IL-2 on days 3, 6, 8, and 10.

[0434] Isolation and cultivation of macrophages

[0435] Primary human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) obtained by density gradient centrifugation of blood from healthy donors (purchased from the Institute for Transfusion Medicine of the University Clinic of Leipzig, Germany, ethics vote number: 272-12-13082012) using RosetteSep™ Human Monocyte Enrichment Cocktail (StemCell Technologies), SepMateTM-50 (IVD) Tubes (StemCell Technologies) and Ficoll-Paque PLUS (Th. Geyer GmbH & Co. KG, Germany) according to manufacturer's protocol. After isolation, cell concentration was adjusted to 1x106cells / mL in RPMI + 5% FBS supplemented with 10 ng / pL GMCSF (Bio-techne) and seed at 0.5x1061 Well in a 48 Well adherent cell plate. Medium was changed at day 5 and as necessary. Cell culture of target cells

[0436] All cell lines used to test the functionality of the CD4 CAR, characteristics, and culture conditions are listed in Table . Cell lines were stained for CD4 expression with CD4 Antibody, anti-human, REAfinity™ (Miltenyi, Germany). Table 2: Cell lines used in functionality testing of the CD4 CAR

[0437] Example 2: Generation of CAR-modified immune cells

[0438] CAR T cells

[0439] CAR T cells were generated either by lentiviral transduction or via transfection of T cells with CAR-encoding mRNA.

[0440] For the generation of stable CAR T cells by lentiviral transduction, first, the lentiviral vector was prepared by means of HEK-293T cells as production cell line. Therefore, HEK-293T cells were cotransfected with the respective transfer plasmid encoding the CD4 CAR construct as well as with pMD2.G (VSV-G envelope), pRSV-Rev, and pMDLg / pRRE (HIV-1 gag / pol) packaging plasmids (Addgene, UK) using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. The supernatant containing viral particles was collected up to 72 h later and concentrated by centrifugation using Amicon® Ultra- 15 Centrifugal Filter Units (Merck KGaA, Germany). The viral titer was determined by addition of different volumes of the concentrated supernatant to 1x105HEK-293T cells plated in 24 well plates and flow cytometric analysis of the cells expressing the CAR three days later. For transduction of T cells, 24 h after T cell isolation and activation, the lentiviral vector was added to the cells at a ratio of one viral particle per T cell (multiplicity of infection = 1 ) followed by centrifugation at 1800 rpm for 90 min at 32°C and over-night incubation at 37°C and 5% CO2. This step was repeated next day. Transduced T cells were expanded for five additional days in T cell expansion medium with IL-2 until usage. For the generation of transient CAR T cells by mRNA-transfection, CAR-encoding mRNA was produced in a three-stage process starting with polymerase chain reaction (PCR) for the generation of the DNA template with the T7 promotor region using Q5 High-Fidelity DNA Polymerase kit (New England Biolabs, USA), followed by in vitro transcription (IVT) using HighYield T7 RNA Synthesis Kit (Jena Bioscience, Germany) and CleanCap Reagent AG (3' OMe) (TriLink BioTechnologies, USA), and subsequent polyadenylation using E. coli Poly(A) Polymerase kit (New England Biolabs). For the delivery of generated CAR-mRNA into T cells, isolated and expanded CD8+T cells were coincubated with mRNA- loaded lipid nanoparticles (mRNA-LNPs), which were formulated using the GenVoy- ILM T Cell Kit for mRNA by means of the NanoAssemblr® Spark device (Precision NanoSystems (now part of Cytiva), Canada) according to manufacturer's instructions.

[0441] CAR NK cells

[0442] CAR NKs were generated via gammaretroviral transduction. For the generation of viral particles, HEK293T cells were cotransfected with the respective CD4 CAR construct, the gag-pol plasmid pHIT60 (Soneoka et al. 1995), and the envelope derived from the baboon endogenous virus, short BaEV (Girard-Gagnepain et al. 2014). Transfection was done using TransIT-VirusGEN Transfection Reagent (Mirus Bio, USA). Viral supernatants were harvested up to 72 h post-transfection and stored at -80 °C until transduction. Activated NK cells were transduced at day three up to day seven past isolation as described by others (Kim et al. 2023), using 10 pg / mL Vectofusin®-1 (Miltenyi Biotec) as transduction enhancer. If not indicated otherwise, CAR expression was analyzed on day three post-transduction.

[0443] CAR Macrophages

[0444] For the generation of transient CAR macrophages by mRNA-transfection, CAR- encoding mRNA was produced in a three-stage process starting with polymerase chain reaction (PCR) for the generation of the DNA template with the T7 promotor region using Q5 High-Fidelity DNA Polymerase kit (New England Biolabs, USA), followed by in vitro transcription (IVT) using HighYield T7 RNA Synthesis Kit (Jena Bioscience, Germany) and CleanCap Reagent AG (3' OMe) (TriLink BioTechnologies, USA), and subsequent polyadenylation using E. coli Poly(A) Polymerase kit (New England Biolabs). Purified mRNA was then frozen in aliquots at -80°C. To transfect mRNA into macrophages, a LipoMM:Opti-MEM mastermix was first prepared. Lipofectamine MessengerMAX reagent was diluted in Opti-MEM medium (or MEMalpha as an alternative) at a 1 :50 volume ratio, with 2.5pl Lipofectamine and 122.5pl Opti-MEM used per well. This mixture was incubated for at least 10 minutes at room temperature. mRNA was thawed from -80°C on ice and diluted in Opti-MEM medium to a concentration of 4-8 ng of mRNA per pL of Opti- MEM. For each mRNA, the diluted mRNA solution was immediately mixed with the Lipofectamine solution at a 1 :1 ratio and incubated for 5 minutes at room temperature for encapsulation. The encapsulated mRNA mixtures was then added drop-wise to each well containing macrophages, delivering a fixed amount of mRNA per well. Finally, the transfected cells are to be incubated at 37°C and 5% CO2.

[0445] Example 3: Functional testing of CAR-immune cells

[0446] A. Analysis of CAR expression

[0447] CD4-CAR expression on T cells, NK cells and macrophages was analyzed by F(ab')2(Jackson ImmunoResearch Labs, UK) or FLAG tag (BioLegend, USA) antibody staining in flow cytometry. Specificity was confirmed by staining with recombinant biotinylated human CD4 protein (ACRO Biosystems) which showed virtually identical results.

[0448] B. Phenotyping of CAR immune cells

[0449] CAR expressing immune cells were analyzed for purity as well as for the expression of activation markers, typical immune cell receptors or exhaustion markers by flow cytometry. Directly conjugated antibodies (see Table 3) were added for 30 minutes on ice in a dilution of 1 :200 in PBS buffer with 2% fetal bovine serum and 1 mM EDTA, and washed prior to analysis.

[0450] Table 3: Antibody panels for characterization of CAR immune cells compared to unmodified cells

[0451]

[0452] C. Cytotoxicity assays

[0453] Calcein cytotoxicity assay

[0454] Cell Lines of interest were stained with Calcein-AM (Life Technologies, Germany). Labeled target cells and effector cells were cocultured in RPMI 1640, Gibco™ (Life Technologies, Germany) with 10 % FCS (BioSell, Germany) at indicated E:T ratios in 96 well format, whereby the target cell count was set to 10.000 cells per well and the effector cell count was adapted respectively. Spontaneous release was obtained by measuring supernatants without effector cells, maximum release was obtained with 1 % Triton-X-100 (VWR International GmbH, Germany) in the medium. After 2,5 h of coculture, the fluorescence (F) of supernatants was measured at excitation 485 nm and emission 535 nm in a Tecan infinite M200 microplate reader (Tecan, Switzerland).

[0455] Specific lysis was calculated by: [F(sample)-F(spontaneous)] / [F(maximum)-F(spontaneous)] x100%.

[0456] Cytotoxicity against primary PBMC samples

[0457] PBMCs were isolated either from healthy, AML or TCL Patient blood samples. All PBMCs were frozen and thawed upon respective assays. AML or TCL PBMC samples were thawed one day prior to the assay and cultured at densities of 1 -2 mio cells / mL. Healthy and TCL patient-derived samples and assays were performed in RPMI with 10 % FCS. AML samples and assays were cultured and performed in RPMI with 5 % Human serum (Sigma Aldrich). TCL samples and assays were cultured and performed in RPMI with 10 % FCS. If not indicated otherwise Effector to target ratios were set 1 :1 . CAR effector cells (identified by GFP expression) were cocultured with TCL patient derived PBMCs for 4h, with AML derived PBMCs for 12h and with healthy PBMCs for 24 h. After incubation, cells were stained with antibody panels in Table 4 (TCL) or 5 (AML).

[0458] Table 4: Antibody panels for PBMC and TCL coculture assays

[0459] Table 5: Antibody panels for AML coculture assays

[0460] Flow cytometry-based cytotoxicity assay

[0461] To analyze CAR T cell functionality, a coculture of CAR T cells and target cells was prepared. Therefore, target cells (cell lines or primary CD3+T cells) were labeled using CellTrace Violet Cell Proliferation Kit (Thermo Fisher Scientific) allowing discrimination of effector and target cells in subsequent analysis. Subsequently, 25,000 of the labeled target cells were plated in a 96 Il-bottom well plate together with different numbers of CAR T effector cells to achieve various effector-to-target (E:T) ratios. Irrespective of the proportion of CAR+cells upon T cell modification, the whole T cell population was considered as effector cells, resulting in identical total cell numbers for every co-culture of the same E:T ratio. Target cells incubated in RPMI + 10% FBS without effector cells were used as reference sample, and target cells treated with 0.1 % Tween 20 were used as positive control that indicates 100% cytotoxicity. Upon incubation for 24 h, the cells were harvested, incubated with 7- AAD (BD, Germany) to stain dead cells, and analyzed on BD FACSCanto™ II. Cytotoxicity was calculated with the following formula: cytotoxicity [%] = 100% - (CellTrace Violet+7-AAD~ cells in co-culture sample [%] I CellTrace Violet+7-AAD~ cells in reference sample [%]) * 100%.

[0462] Macrophage Phagocytosis Assay

[0463] To analyze phagocytosis of target cells by CAR-macrophages, the former are labeled with CellTrace Violet. SUP-M2 cells are counted, and the volume needed for 1 million cells of each is calculated. CellTrace DMSO stock solution (5mM) is diluted to 5pM in pre-warmed PBS. Cells are pelleted by centrifugation, and the supernatant is removed. The cell pellet is gently resuspended in the PBS dye solution and incubated for 20 minutes at room temperature or 37°C, protected from light. Culture medium containing at least 1 % protein is added at five times the original staining volume, and cells are incubated for 5 minutes. Cells are then pelleted again and resuspended in fresh, pre-warmed RPMI + 10% FCS.

[0464] For the phagocytosis assay, supernatant from macrophage wells is aspirated and discarded. Target cell suspension (500pl) or RPMI+10%FCS for controls is added to each (CAR-) macrophage well, maintaining an effectortarget ratio of 1 :1. Cells are incubated for 4 hours at 37°C.

[0465] After incubation, cells are prepared for FACS staining. Supernatant is aspirated and collected, followed by a PBS wash. Macrophages are detached by incubation in Accutase for two 10-m inute periods. Cells are washed once in FACS buffer.

[0466] Antibody master mixes are prepared: for non-EGFP samples, 1200pl FACS buffer is combined with 6pl each of aFLAG-tag-APC REA, aCD14-PE-Vio770, and aCD4- PE. For EGFP samples, 600pl FACS buffer is mixed with 3pl CD14-APC. Cells are pelleted and resuspended in the respective antibody mastermix, then incubated for 30 minutes on ice in the dark.

[0467] After incubation, cells are washed twice in FACS buffer. Samples are analyzed by flow cytometry, with events double positive for CD14 and CellTrace Violet considered as macrophages that have phagocytosed target cells.

[0468] Example 3: Experimental Results CAR encoding constructs, including CAR-encoding mRNA and / or CAR encoding DNA constructs (including viral particles, plasmids etc) generated herein encode, from N-terminus to C-terminus, a leader sequence, an anti-CD4 binding domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain and at least one stimulatory domain. The use of a CD8 leader sequence has been found advantageous for efficient expression of the CD4-specific CARs herein in primary NK cells.

[0469] We generated CAR constructs herein, wherein the anti-CD4 binding domain is an anti-CD4 scFv wherein the sequence of the VH is as set forth in SEQ ID NO: 7, and the sequence of the VL is as set forth in SEQ ID NO: 8 and the VL and VH are linked by a peptide linker (Gly4Ser)3 or Whitlow linker. An scFv of structure VL- Whitlow linker - VH having a sequence as set forth in SEQ ID NO: 18 has been found suitable and advantageous for CAR immune cells herein.

[0470] We generated CAR constructs having a human lgG1 hinge region, mutated human lgG1 hinge region, human CD28 hinge region and short human lgG4 hinge region. The results are shown in Figure 3. In Figure 3A, a comparison of the impact of different hinge domains on the CD4-specific CAR expression in transduced NK cells is provided. The ratio of expression was determined by assessing % CAR positive cells in flow cytometry, relative to the unmutated lgG1 hinge. In Figure 3B, an assessment of functionality of CAR NK cells transduced with CD4-specific CARs carrying the different hinge regions and an unmutated hinge region from IgG 1 (“aCD4norm”) in a flow cytometry based PBMC cocultivation assay is provided. The relative decrease of CD4+ T cells are determined when cocultured with CAR NK cells of two independent healthy donors (black). The absolute CAR expression for these two donors is presented in grey in this Figure. It was found that expression is advantageous for a mutated human lgG1 hinge region. Also, functionality was found advantageous for mutated human lgG1 hinge region or native human lgG1 hinge region.

[0471] We generated CAR constructs with CD8 transmembrane domain or a CD28 transmembrane domain. Such transmembrane domains were successfully used in CD4-specific CARs provided in the Examples (data not shown for CD28 transmembrane domain).

[0472] We generated CAR constructs with different co-stimulatory domains and stimulatory domains. For example, CAR constructs were generated with a stimulatory domain from CD3 , DAP10 or DAP12, and a co-stimulatory signalling domain from 4-1 BB, CD28, DNAM1 or 2B4. Various combinations of a co-stimulatory and a stimulatory domain were generated and analyzed for expression and functionality (data not shown). It was found that constructs with a CD28 co-stimulatory domain and a CD3 stimulatory domain were advantageous in expression and functionality.

[0473] The results shown in Figures 4 to 10 were generated with CAR constructs encoding from N-terminus to C-terminus, a CD8 leader sequence, an anti-CD4 binding scFv, a IgG 1 hinge region, a CD8 transmembrane domain, a CD28 Co-stimulatory domain and a CD3 stimulatory domain. In these Examples, the CD8 leader sequence is as set forth in SEQ ID NO: 30 (or is encoded by the sequence as set forth in SEQ ID NO: 31 ). In these examples, the amino acid sequence of the anti-CD4 binding scFv is as set forth in SEQ ID NO: 18. The scFv is also designated as MAX.16H5 scFv herein. In these examples, the amino acid sequence of the CD8 transmembrane domain is as set forth in SEQ ID NO: 14. In these constructs, the CD28 co- stimulatory domain has the sequence as set forth in SEQ ID NO: 17, and the CD3 stimulatory domain has the sequence as set forth in SEQ ID NO: 16. The sequence of the complete encoded CAR is provided in SEQ ID NO: 19 (without leader sequence) and SEQ ID NO: 20 (with leader sequence). The nucleic acid sequence of the complete CAR construct is provided in SEQ ID NO: 21 (without leader sequence) and SEQ ID NO: 22 (with leader sequence).

[0474] As shown in Figure 4, advantageous expression data were found for a CAR construct of the invention in primary T cells, primary NK cells and primary macrophages. For primary T cells, this was found in Figure 4(A) both for either stably transduced cells (left) or transiently transfected cells with mRNA (right). For NK cells, this was found in primary NK cells in a G-rex cell culture system (Figure 4(B)). For macrophages, this was found in primary macrophages transiently modified with CD4-CAR mRNA 24 hours post infection (Figure 4(C)).

[0475] Figure 5 shows growth kinetics and phenotyping of CD4-specific CAR NK cells in a G-rex 24-well-plate, which can be scaled up to GMP-compliant bioreactors. As shown in Figure 5(B), the expression of NK cell markers on day 14 post-isolation on CAR- and unmodified NK cells show normal NK cell receptor expression and cellular activation. Figure 5(A) shows that CAR NK cells can be expanded, allowing upscaling. This demonstrates that CAR NK cells herein exhibit advantageous properties for therapeutic purposes. Figure 6 demonstrates that CD4-CAR-NK cells are highly functional and specific. Figure 6(A) shows expression of CD4 on diverse AML (left) and TCL (right) cell lines. Figure 6(B) demonstrates CAR-mediated cytotoxicity of CD4-CAR-NK cells towards diverse cell lines assessed by a Calcein-based cytotoxicity assay in an E:T ratio of 2.5:1. To assess CAR-mediated cytotoxicity, natural cytotoxicity (% killing executed by unmodified NK cells) was subtracted. Figure 6(C) shows cytotoxicity in E:T ratios 2.5:1 and 1 :1 towards selected cell lines comparing CD4-CARs and irrelevant CD19-CARS.

[0476] Figure 7 demonstrates cytotoxicity of CD4-specific CAR NK cells towards patient- derived, primary samples of (A) T cell malignancies and (B) acute myeloid leukemia, assessed in a flow cytometry-based cytotoxicity assay.

[0477] The data demonstrate that CD4-specific CAR NK cells provided herein can be used for treating a T cell malignancy and / or a CD4 positive malignancy, such as an acute myeloid leukemia.

[0478] Figure 8 shows the cytotoxicity of CD4-specific CAR NK cells towards activated, primary T cells from unmatched donors in a flow-cytometry based cytotoxicity assay.

[0479] Figure 9 shows the cytotoxicity of (A) stably transduced and mRNA-transfected CD4-specific CAR T cells towards primary activated T cells and (B) CD4-specific viral ly transduced CAR T cells towards AML and TCL cell lines.

[0480] Figure 10 shows phagocytosis of mRNA-transfected CD4-specific CAR macrophages towards the CD4-positive cell line Sup-M2 in an E:T ratio of 1 :1 for 4 hours.

[0481] References

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[0484] 3. W. Nawaz, B. Huang, S. Xu, Y. Li, L. Zhu, H. Yiqiao, Z. Wu, X. Wu, AAV- mediated in vivo CAR gene therapy for targeting human T-cell leukemia. Blood cancerjournal 11, 119 (2021 ).

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[0486] Ma, Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells. Leukemia 30, 701-707 (2016).

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[0488] 6. J. Feng, H. Xu, A. Cinquina, Z. Wu, W. Zhang, L. Sun, Q. Chen, L. Tian, Le Song, K. G. Pinz, M. Wada, X. Jiang, W. M. Hanes, Y. Ma, H. Zhang, Treatment of aggressive T-cell lymphoma / leukemia with anti-CD4 CART cells. Frontiers in Immunology 13, 997482 (2022).

[0489] 7. H. Zhang, J. Feng, W. Zhang, Q. Chen, Y. Cao, K. Pinz, F. Liu, Y. Ma, M. Wada, Y. Ma, First-in-Human CD4 CAR Clinical Trial on Peripheral T-Cell Lymphoma. Blood 134, 2881 (2019).

[0490] 8. Huda Salman, iCell Gene Therapeutics, CD4CAR for CD4+ Leukemia and Lymphoma.

[0491] 9. Legend Biotech USA Inc, Study of CD4-Targeted Chimeric Antigen Receptor T- Cells (CDA- CAR-T) in Subjects With Relapsed or Refractory T-Cell Lymphoma.

[0492] 10.R. K. Das, L. Vernau, S. A. Grupp, D. M. Barrett, Naive T-cell DeficitsatDiagnosisand after Chemotherapy Impair Cell Therapy Potential in Pediatric Cancers. Cancer discovery 9, 492-499 (2019).

[0493] 11.T. Tonn, D. Schwabe, H. G. Klingemann, S. Becker, R. Esser, U. Koehl, M. Suttorp, E. Seifried, O. G. Ottmann, G. Bug, Treatment of patients with advanced cancer with the natural killer cell line NK-92. Cytotherapy 15, 1563-1570 (2013).

[0494] 12.Y. Hu, Z.-G. Tian, C. Zhang, Chimeric antigen receptor (CAR)-transduced natural killer cells in tumor immunotherapy. Acta pharmacologica Sinica 39, 167- 176 (2018).

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[0496] Kang, Chung Hyo; Park, Chi Hoon (2023): Optimized conditions for gene transduction into primary immune cells using viral vectors. In: Scientific reports 13

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Claims

Fraunhofer-Gesellschaft zur Fbrderung August 5, 2025 der angewandten Forschung e.V. F74640PC PIN / FLZ / PINCLAIMS1 . A CD4-specific chimeric antigen receptor (CAR) comprising:(i) an extracellular CD4 binding-domain comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) CDRH1 , CDRH2 and CDRH3 and a light chain variable region (VL) comprising complementarity determining regions CDRL1 , CDRL2 and CDRL3, wherein:(a) CDRH1 comprises the amino acid sequence of NYWMH (SEQ ID NO: 1 );(b) CDRH2 comprises the amino acid sequence of ALYPGNVDTTYNQKFKD (SEQ ID NO: 2);(c) CDRH3 comprises the amino acid sequence of MGTTLEAPLDY (SEQ ID NO: 3);(d) CDRL1 comprises the amino acid sequence of SARSSVSYLY (SEQ ID NO: 4);(e) CDRL2 comprises the amino acid sequence of DTSNLAS (SEQ ID NO: 5); and / or(f) CDRL3 comprises the amino acid sequence of QQWSDYPLT (SEQ ID NO: 6);(ii) a hinge domain (hinge),(iii) a transmembrane domain (TM); and(iv) a cytoplasmic domain comprising at least one signalling domain.

2. The CD4-specific CAR of claim 1 , wherein the CD4 binding-domain: comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and / or comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

3. The CD4-specific CAR of claim 1 or 2, wherein the sequence of the VH is as set forth in SEQ ID NO: 7, and the sequence of the VL is as set forth in SEQ ID NO: 8.

4. The CD4-specific CAR of any one of claims 1 to 3, wherein the CD4 bindingdomain is a single-chain variable fragment (scFv), optionally wherein the amino acid sequence of the scFv is as set forth in SEQ ID NO: 18.

5. The CD4-specific CAR of any one of claims 1 to 4, wherein the VH and VL are joined by flexible peptide linker, optionally wherein the peptide linker is a glycine-linker (Gly)n, a glycine-serine linker (Glyi-sSen-sjn, such as (Gly4Ser)3 (SEQ ID NO: 9), a glycine-alanine linker, or a linker with the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10), wherein n is an integer of 1 , 2, 3, 4 or 5.

6. The CD4-specific CAR of any one of claims 1 to 5, wherein the hinge domain is selected from a lgG1 hinge domain, lgG4 hinge domain and a CD28 hinge domain, optionally wherein the hinge domain is a native human domain, codon-optimized domain or a domain comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations as compared to a native domain.

7. The CD4-specific CAR of any one of claims 1 to 6, wherein the hinge domain is a mutated lgG1 hinge domain, wherein(i) the mutated IgG 1 hinge domain is a human IgG 1 domain wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions are mutated in the CH2 region, and / or(ii) the mutated lgG1 hinge domain exhibits substantially abolished binding to the FcyRs and / or substantially abolished effector function, optionally wherein the sequence of the mutated lgG1 hinge domain is as set for the in SEQ ID NO: 13.

8. The CD4-specific CAR of any one of claims 1 to 7, wherein the transmembrane domain is (i) from the transmembrane region of CD8, optionally wherein the transmembrane domain exhibits at least 90% sequence identity with SEQ ID No: 14, or (i) from the transmembrane region of CD28, optionally wherein the transmembrane domain exhibits at least 90% sequence identity with SEQ ID No: 15.

9. The CD4-specific CAR of any one of claims 1 to 8, wherein the CAR comprises cytoplasmic domain comprising at least one co-stimulatory signalling domain and at least one stimulatory signalling domain, optionally wherein the at least one stimulatory signalling domain is selected from a CD3 , DAP10 and DAP12 stimulatory signalling domain, and / or wherein the at least one co-stimulatory signalling domain is selected from a 4- 1 BB, a CD28, DNAM1 and 2B4 co-stimulatory signalling domain.

10. The CD4-specific CAR of any one of claims 1 to 9, wherein the CD4-specific CAR has the following structure, from N- to C- terminus:Anti-CD4 binding scFv - mutated IgG 1 hinge - CD8 TM - CD28 Co-stimulatory domain - CD3 stimulatory domain.

11. A polynucleotide encoding a CD4-specific CAR according to any one of claims 1 to 10, optionally wherein the polynucleotide further encodes a CD8 leader sequence at the N-terminus of the CAR.

12. An expression vector comprising the polynucleotide of claim 11 , optionally wherein the expression vector:(i) is a viral vector, optionally wherein the viral vector is selected from an SV40 vector, adenovirus vector, adeno-associated virus (AAV), lentiviral vector, and retroviral vector; or(ii) the expression vector is a non-viral vector, optionally wherein the non- viral vector is selected from a transposon-based expression vector, or an expression vector for providing mRNA encoding the CD4-specific CAR.

13. A modified immune cell (i) comprising a polynucleotide according to claim 11 , or an expression vector according to claim 12, and / or (ii) expressing a CD4- specific CAR according to any one of claims 1 to 10.

14. The modified immune cell of claim 13, wherein the immune cell is selected from a T cell, NK cell and macrophage, optionally wherein the immune cell is a primary cell and / or autologous cell or an allogeneic cell.

15. A method for preparing the modified immune cell of claim 13 or 14, comprising the steps of (A) or (B):(A)(i) providing mRNA encoding the CD4-specific CAR according to any one of claims 1 to 10, optionally wherein the mRNA further encodes a CD8 leader sequence at the N-terminus of the CAR,(ii) preparing lipid nanoparticles comprising the mRNA of (ii), and(iii) transfecting immune cells with lipid nanoparticles comprising the mRNA obtained in (ii), thereby obtaining the modified immune cell;(B)(i) providing an expression vector comprising a polynucleotide encoding the CD4-specific CAR according to claim 13, optionally wherein the polynucleotide further encodes a CD8 leader sequence at the N-terminus of the CAR encoding the CD4-specific CAR,(ii) introducing the expression vector of (i) into an immune cell, thereby obtaining the modified immune cell.

16. A modified immune cell according to claim 13 or 14, for use in the treatment of a CD4-mediated and / or CD4-associated disease of a subject.

17. The modified immune cell for use according to claim 16, wherein the CD4- mediated and / or CD4-associated disease is selected from:(i) a CD4 positive cancer, optionally wherein the CD4 positive cancer is a CD4 positive T cell lymphoma or CD4 positive T cell leukemia or a CD4 positive malignancy, or wherein the CD4 positive cancer is selected from anaplastic large cell lymphoma, peripheral T cell lymphoma (PTL), or acute myeloid leukemia (AML), cutaneous T cell lymphoma, and Sezary Syndrome; optionally wherein the subject treated exhibits a blast crisis, a minimal residual disease, has undergone lymphodepletion or depletion of CD4 positive cells and / or has undergone stem cell transplantation and / or will undergo stem cell transplantation;(ii) a CD4 negative cancer, in which non-malignant CD4 positive T cells represent a population within or surrounding the tumour tissue;(iii) a T cell-mediated autoimmune disease, optionally wherein the T cell- mediated autoimmune disease is rheumatoid arthritis or ulcerative colitis; and(iv) an HIV, HTLV-1 or HLTV-2 infection, optionally wherein the subject has undergone lymphodepletion or depletion of CD4 positive cells.

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