Antigen-binding domain binding to psma

Abstract

To provide: antigen-binding domains which bind to the antigen prostate-specific membrane antigen (PSMA); and chimeric antigen receptors (CARs) which comprise such antigen binding domains.SOLUTION: The present inventors have identified and characterized new PSMA binding domains with improved properties. The antigen-binding domain may comprise e.g., either: (a) a heavy chain variable region (VH) and a light chain variable region (VL) that are selected from among (i) VH-SEQ ID No. 7, VL-SEQ ID No. 8, (ii) VH-SEQ ID No. 15, VL-SEQ ID No. 16, (iii) VH-SEQ ID No. 23, VL-SEQ ID No. 24, and (iv) VH-SEQ ID No. 31, VL-SEQ ID No. 32; or (b) a domain antibody variable region selected from among (i) SEQ ID No. 36, (ii) SEQ ID No. 40, (iii) SEQ ID No. 44, (iv) SEQ ID No. 48, and (v) SEQ ID No. 52.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an antigen-binding domain that binds to the antigen prostate-specific membrane antigen (PSMA).The present invention also relates to a chimeric antigen receptor (CAR) that comprises such an antigen-binding domain.Cells that express CAR that binds to PSMA are useful for treating cancerous diseases such as solid tumors, including prostate cancer, kidney cancer, breast cancer and colon cancer. [Background technology]

[0002] 2. Background of the Invention Prostate cancer Prostate cancer is the most frequently diagnosed type of cancer in men and is estimated to be the second leading cause of cancer-related death. Radical prostatectomy remains one of the most common treatment options and may be combined with radiation therapy or chemotherapy.

[0003] Chimeric antigen receptors (CARs) Chimeric antigen receptors are proteins that transfer the specificity of antibodies to the effector functions of T cells. Their usual form is that of a type I transmembrane domain protein with an amino-terminal antigen-recognizing, spacer, and transmembrane domains, all connected to a compound endodomain that transmits T cell survival and activation signals (see Figure 1a).

[0004] The most common form of these molecules is a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody that recognizes a target antigen, fused to a signaling endodomain via a spacer and a transmembrane domain. Such molecules result in the activation of T cells in response to the recognition of their target by the scFv. When a T cell expresses such a CAR, the T cell recognizes and kills a target cell that expresses the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trials for the treatment of various cancers.

[0005] Prostate-specific membrane antigen (PSMA) One target of immunotherapeutic approaches to treat prostate cancer is prostate-specific membrane antigen (PSMA), also known as glutamic acid carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamic acid peptidase I (NAALADase I) or NAAG peptidase. PSMA is an 84 kDa class II transmembrane zinc metalloenzyme that catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to glutamate and N-acetylaspartate. The protein is primarily extracellular, with only a small intracellular region.

[0006] PSMA is highly expressed in the prostate and is overexpressed in more than 90% of all primary prostate cancers. PSMA expression in healthy tissue is minimal. Therefore, PSMA shows utility as a target for the diagnosis and treatment of prostate cancer. In addition, PSMA has potential utility as a target in kidney cancer, breast cancer and colon cancer.

[0007] An anti-PSMA CAR containing an antigen-binding domain based on the J591 monoclonal antibody has been previously described (Kloss CC. et al., Mol Ther. 2018 July 5;26(7):1855-1866.). The inventors sought to generate alternative PSMA-targeted CARs with improved properties. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Kloss CC. et al., Mol Ther. 2018 July 5;26(7):1855-1866 [Brief description of the drawings]

[0009] [Figure 1]Figure 1. Chimeric antigen receptor (CAR) design. (a) Generalized structure of CAR: the binding domain recognizes the antigen, the spacer elevates the binding domain from the cell surface, the transmembrane domain anchors the protein to the membrane, and the endodomain transmits the signal. (b)-(d): Different generations and permutations of CAR endodomains: (b) initial designs transmitted only ITAM signals via FcεR1-γ or CD3ζ endodomains, subsequent designs transmitted an additional (c) one or (d) two costimulatory signals in cis. [Diagram 2] Figure 2 shows the structure of prostate specific membrane antigen. Prostate specific membrane antigen is a class II membrane glycoprotein of 750 amino acids. The cytoplasmic (cytosolic) domain is residues 1-19. The signal anchor of the type II membrane protein is residues 20-43. The extracellular domain is residues 44-750. The extracellular domain is composed of the protease, apical and C-terminal domains. [Diagram 3] Figure 3. Different binding domain formats of chimeric antigen receptors: (a) Fab CAR format, (b) dAb CAR format, (c) scFv CAR format. [Figure 4] Figure 4: ELISA assay of cloned phage and scFv cultures. ELISA assay of cloned phage and scFv cultures in replicates after initial screening (n=3) with recombinant protein ELISA assay. [Diagram 5] Figure 5. Titration and enrichment of specific phage populations. (a) Titration and enrichment of specific phage populations after panning rounds 1 and 2 from recombinant human PSMA protein (Acro Biosystems) panning only, (b) Titration and enrichment of phage after one round of recombinant protein panning and one round of cell panning against SupT1 cells expressing PSMA. [Figure 6]Figure 6: ELISA screening of monoclonal C Myc tagged dAbs. ELISA screening of monoclonal C Myc tagged dAbs expressed by TG1 colonies from titer plates after cell panning. Expression was by IPTG induction and screening was performed against recombinant human PSMA. Detection was performed using anti-C Myc tag HRP conjugate. [Figure 7] Figure 7. ELISA screening of monoclonal C Myc tagged dAbs. Flow cytometry analysis of anti-PSMA dAbs binding to untransduced or transduced SupT1 cells with high (clone 30) or low (clone 83) PSMA expression. dAb clones B8 and G7 show high levels of specific binding. [Figure 8] FIG. 8. Flow cytometric analysis of the Fc-tagged dAbs depicted in FIG. [Figure 9] Flow cytometry analysis of NGS identified dAb sequences that bind to PSMA-expressing cell lines. dAbs 2 and 25 showed high binding to the appropriate cell lines. [Figure 10-1] Figure 10. IHC staining of PSMA expressing cell lines using cell panning and NGS derived dAbs. Data suggests that NGS derived clones 2 and 25 show comparable staining to the positive controls (J591 and 7A12 antibodies). [Figure 10-2] Figure 10. IHC staining of PSMA expressing cell lines using cell panning and NGS derived dAbs. Data suggests that NGS derived clones 2 and 25 show comparable staining to the positive controls (J591 and 7A12 antibodies). [Figure 10-3] Figure 10. IHC staining of PSMA expressing cell lines using cell panning and NGS derived dAbs. Data suggests that NGS derived clones 2 and 25 show comparable staining to the positive controls (J591 and 7A12 antibodies). [Figure 11-1]Figure 11. FACS-based killing assay. SupT1 cells engineered to express human PSMA antigen (SupT1-PSMA) were used as target cells. Non-engineered SupT1 cells (SupT1-NT) were used as a negative control. T cells were co-cultured with target cells at effector-to-target ratios of 1:1, 1:4 and 1:8. FBK was assayed after 24 hours of incubation and analyzed by cytofluorometric analysis. [Figure 11-2] Figure 11. FACS-based killing assay. SupT1 cells engineered to express human PSMA antigen (SupT1-PSMA) were used as target cells. Non-engineered SupT1 cells (SupT1-NT) were used as a negative control. T cells were co-cultured with target cells at effector-to-target ratios of 1:1, 1:4 and 1:8. FBK was assayed after 24 hours of incubation and analyzed by cytofluorometric analysis. [Figure 12-1] Figure 12: Cytokine release assay. Secretion of IL-2 (top) and IFNγ (bottom) by CAR T cells was measured by collecting supernatants at 24 hours from co-cultures of CAR-T cells and SupT1 cells engineered to express human PSMA antigen (SupT1-PSMA) with non-engineered SupT1 cells (SupT1-NT) as a negative control. T cells were co-cultured with target cells at effector-to-target ratios of 1:1, 1:4 and 1:8. Production of IL-2 and IFNγ was detected by ELISA. [Figure 12-2] Figure 12: Cytokine release assay. Secretion of IL-2 (top) and IFNγ (bottom) by CAR T cells was measured by collecting supernatants at 24 hours from co-cultures of CAR-T cells and SupT1 cells engineered to express human PSMA antigen (SupT1-PSMA) with non-engineered SupT1 cells (SupT1-NT) as a negative control. T cells were co-cultured with target cells at effector-to-target ratios of 1:1, 1:4 and 1:8. Production of IL-2 and IFNγ was detected by ELISA. [Figure 12-3]Figure 12: Cytokine release assay. Secretion of IL-2 (top) and IFNγ (bottom) by CAR T cells was measured by collecting supernatants at 24 hours from co-cultures of CAR-T cells and SupT1 cells engineered to express human PSMA antigen (SupT1-PSMA) with non-engineered SupT1 cells (SupT1-NT) as a negative control. T cells were co-cultured with target cells at effector-to-target ratios of 1:1, 1:4 and 1:8. Production of IL-2 and IFNγ was detected by ELISA. Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention The present inventors have identified and characterized a new PSMA-binding domain with improved properties.

[0011] Thus, in a first aspect, the present invention provides an antigen binding domain that binds to a prostate specific membrane antigen and comprises any of the following: (a) a heavy chain variable region (VH) having complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3 selected from: and a light chain variable region (VL) having CDRs: LCDR1, LCDR2 and LCDR3 selected from: (i) HCDR1-SEQ ID NO: 1, HCDR2-SEQ ID NO: 2, HCDR3-SEQ ID NO: 3, LCDR1-SEQ ID NO: 4, LCDR2-SEQ ID NO: 5, LCDR3-SEQ ID NO: 6, (ii) HCDR1-SEQ ID NO: 9, HCDR2-SEQ ID NO: 10, HCDR3-SEQ ID NO: 11, LCDR1-SEQ ID NO: 12, LCDR2-SEQ ID NO: 13, LCDR3-SEQ ID NO: 14, (iii) HCDR1-SEQ ID NO: 17, HCDR2-SEQ ID NO: 18, HCDR3-SEQ ID NO: 19, LCDR1-SEQ ID NO: 20, LCDR2-SEQ ID NO: 21, LCDR3-SEQ ID NO: 22, (iv) HCDR1-SEQ ID NO:25, HCDR2-SEQ ID NO:26, HCDR3-SEQ ID NO:27, LCDR1-SEQ ID NO:28, LCDR2-SEQ ID NO:29, LCDR3-SEQ ID NO:30, or (b) a domain antibody variable region having complementarity determining regions (CDRs): VHHCDR1, VHHCDR2 and VHHCDR3 selected from: (i) VHHCDR1-SEQ ID NO: 33, VHHCDR2-SEQ ID NO: 34, VHHCDR3-SEQ ID NO: 35, (ii) VHHCDR1-SEQ ID NO: 37, VHHCDR2-SEQ ID NO: 38, VHHCDR3-SEQ ID NO: 39, (iii) VHHCDR1-SEQ ID NO: 41, VHHCDR2-SEQ ID NO: 42, VHHCDR3-SEQ ID NO: 43, (iv) VHHCDR1-SEQ ID NO: 45, VHHCDR2-SEQ ID NO: 46, VHHCDR3-SEQ ID NO: 47, and (v) VHHCDR1-SEQ ID NO: 49, VHHCDR2-SEQ ID NO: 50, VHHCDR3-SEQ ID NO: 51.

[0012] The antigen-binding domain may, for example, comprise any of the following: (a) a heavy chain variable region (VH), and a light chain variable region (VL) selected from: (i) VH-SEQ ID NO: 7, VL-SEQ ID NO: 8, (ii) VH-SEQ ID NO: 15, VL-SEQ ID NO: 16, (iii) VH-SEQ ID NO:23, VL-SEQ ID NO:24, and (iv) VH-SEQ ID NO:31, VL-SEQ ID NO:32, or (b) a domain antibody variable region selected from: (i) SEQ ID NO: 36, (ii) SEQ ID NO: 40, (iii) SEQ ID NO: 44, (iv) SEQ ID NO: 48, and (v) SEQ ID NO:52.

[0013] In a second aspect, the present invention provides a chimeric antigen receptor (CAR) comprising the antigen binding domain of the first aspect of the invention.

[0014] In a third aspect, the present invention provides a nucleic acid sequence encoding an antigen-binding domain according to the first aspect of the invention or a CAR according to the second aspect of the invention.

[0015] In a fourth aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence according to the third aspect of the invention and a nucleic acid sequence encoding a suicide gene.

[0016] In a fifth aspect, the present invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention or a nucleic acid construct according to the fourth aspect of the invention.

[0017] In a sixth aspect, the present invention provides a cell expressing a CAR according to the second aspect of the invention.

[0018] In a seventh aspect, the present invention provides a method of producing a cell according to the sixth aspect of the invention, the method comprising introducing a nucleic acid sequence encoding a CAR according to the second aspect of the invention into a cell ex vivo.

[0019] In an eighth aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells according to the sixth aspect of the invention, together with a pharma- ceutically acceptable carrier, diluent or excipient.

[0020] In a ninth aspect, the present invention provides a method of treating cancer comprising administering to a subject a cell according to the sixth aspect of the invention.

[0021] In a tenth aspect, the present invention provides a cell according to the sixth aspect of the invention for use in the treatment of cancer.

[0022] In an eleventh aspect, the present invention provides the use of a cell according to the sixth aspect of the invention in the manufacture of a medicament for treating cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description Antigen-binding domain The antigen binding domain of the present invention is an antibody-based binding domain that specifically binds to the target antigen PSMA.

[0024] The antigen-binding domain of an antibody, such as an IgG molecule, is composed of two variable domains, one derived from the heavy chain (VH) and one derived from the light chain (VL). Some of the antigen-binding domains of the invention contain both a VH domain and a VL domain.

[0025] The antigen-binding domain of the present invention, including the VH domain and the VL domain, can be, for example, a single chain variable fragment (scFv) or Fab format. The CAR of the present invention can include, for example, an scFv or Fab-based antigen-binding domain, as illustrated in Figure 3a (Fab) and Figure 3c (scFv).

[0026] An antigen binding domain may comprise a heavy chain variable region (VH) having complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3 selected from the following, and a light chain variable region (VL) having CDRs: LCDR1, LCDR2 and LCDR3 selected from the following: (i) HCDR1-SEQ ID NO: 1, HCDR2-SEQ ID NO: 2, HCDR3-SEQ ID NO: 3, LCDR1-SEQ ID NO: 4, LCDR2-SEQ ID NO: 5, LCDR3-SEQ ID NO: 6, (ii) HCDR1-SEQ ID NO: 9, HCDR2-SEQ ID NO: 10, HCDR3-SEQ ID NO: 11, LCDR1-SEQ ID NO: 12, LCDR2-SEQ ID NO: 13, LCDR3-SEQ ID NO: 14, (iii) HCDR1-SEQ ID NO: 17, HCDR2-SEQ ID NO: 18, HCDR3-SEQ ID NO: 19, LCDR1-SEQ ID NO: 20, LCDR2-SEQ ID NO: 21, LCDR3-SEQ ID NO: 22, and (iv) HCDR1-SEQ ID NO:25, HCDR2-SEQ ID NO:26, HCDR3-SEQ ID NO:27, LCDR1-SEQ ID NO:28, LCDR2-SEQ ID NO:29, LCDR3-SEQ ID NO:30, or

[0027] The position of the CDRs in a sequence can be determined by different numbering schemes, such as Kabat and IMGT. In this application, the Kabat numbering system is used, unless otherwise stated.

[0028] The antigen-binding domain is (i) VH-SEQ ID NO: 7, VL-SEQ ID NO: 8, (ii) VH-SEQ ID NO: 15, VL-SEQ ID NO: 16, (iii) VH-SEQ ID NO:23, VL-SEQ ID NO:24, and (iv) It may comprise one of the VH / VL domain pairs: VH-SEQ ID NO:31, VL-SEQ ID NO:32. [ka] [ka] [ka] [ka]

[0029] The following alternative humanized light chains for 7A12 are also provided (with the CDRs determined according to the Kabat numbering system): [ka] [ka] [ka] [ka]

[0030] Additionally, a humanized heavy chain of 7A12 is provided (with the CDRs determined according to the Kabat numbering system). [ka] [ka]

[0031] From these sequences, the following consensus sequences for the CDRs can be determined, where "X" can be any naturally occurring amino acid: [Table A-1] [Table A-2]

[0032] Alternatively, the antigen-binding domain of the invention may comprise a single variable domain.

[0033] Single domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain.

[0034] The first single domain antibodies were engineered from heavy chain antibodies found in camelids. These are called VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR, "immunoglobulin novel antigen receptor") from which single domain antibodies called VNAR fragments can be derived. Most research on single domain antibodies is currently based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind target epitopes.

[0035] An antigen binding domain of the invention may comprise a domain antibody variable region having complementarity determining regions (CDRs): VHHCDR1, VHHCDR2 and VHHCDR3 selected from the following: VHHCDR1 - SEQ ID NO: 33, VHHCDR2 - SEQ ID NO: 34, VHHCDR3 - SEQ ID NO: 35, (ii) VHHCDR1-SEQ ID NO: 37, VHHCDR2-SEQ ID NO: 38, VHHCDR3-SEQ ID NO: 39, (iii) VHHCDR1-SEQ ID NO: 41, VHHCDR2-SEQ ID NO: 42, VHHCDR3-SEQ ID NO: 43, (iv) VHHCDR1-SEQ ID NO: 45, VHHCDR2-SEQ ID NO: 46, VHHCDR3-SEQ ID NO: 47, and (v) VHHCDR1-SEQ ID NO: 49, VHHCDR2-SEQ ID NO: 50, VHHCDR3-SEQ ID NO: 51.

[0036] The antigen binding domain may, for example, comprise an antibody variable region domain selected from: (i) SEQ ID NO: 36, (ii) SEQ ID NO: 40, (iii) SEQ ID NO: 44, (iv) SEQ ID NO: 48, and (v) SEQ ID NO:52. [ka] [ka]

[0037] The inventors have found that the antigen-binding domains of the invention have improved stability at acidic pH (see Example 7). Because the tumor microenvironment is acidic, these antigen-binding domains are proposed to have beneficial properties in the treatment of solid tumors.

[0038] Chimeric Antigen Receptor (CARS) Classical chimeric antigen receptors (CARs) are chimeric type I transmembrane proteins that connect an extracellular antigen recognition domain (binder) to an intracellular signaling domain (endodomain). Binders are typically single-chain variable fragments (scFv) derived from monoclonal antibodies (mAbs), but can be based on other formats that contain antibody-like antigen-binding sites. A spacer domain is usually required to isolate the binder from the membrane and allow for proper orientation. A common spacer domain used is the Fc of IgG1. A more compact spacer, for example a stalk from CD8α or even just an IgG1 hinge, may be sufficient depending on the antigen. The transmembrane domain anchors the protein to the cell membrane and connects the spacer to the endodomain.

[0039] Early CAR designs had endodomains derived from the intracellular portion of either FcεR1 or the γ-chain of CD3ζ. As a result, these first-generation receptors delivered immunological signal 1, which was sufficient to trigger T cell killing of cognate target cells, but failed to fully activate T cells to proliferate and survive. To overcome this limitation, compound endodomains have been constructed. Fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ results in second-generation receptors that can simultaneously deliver activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28. It provides the most potent costimulatory signal, i.e., immunological signal 2, and induces T cell proliferation. Several receptors have also been described that contain endodomains from the TNF receptor family, such as the closely related OX40 and 41BB, which transmit survival signals. An even more potent third generation CAR is now described, with endodomains that can transmit activation, proliferation and survival signals (Figure 1).

[0040] When the CAR binds to a target antigen, this results in the transmission of an activation signal to the T cell in which the CAR is expressed. Thus, the CAR directs the specificity and cytotoxicity of the T cell to tumor cells expressing the target antigen.

[0041] Thus, a CAR typically comprises: (i) an antigen-binding domain, (ii) a spacer, (iii) a transmembrane domain, and (iii) an intracellular domain that comprises or associates with a signaling domain.

[0042] A CAR may have the following general structure:

[0043] Antigen-binding domain-spacer domain-transmembrane domain-intracellular signaling domain (endodomain).

[0044] CAR antigen-binding domain The antigen-binding domain is the part of the chimeric receptor that recognizes the antigen. In classical CARs, the antigen-binding domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody (see FIG. 3c). CARs have also been produced using domain antibodies (dAbs) or VHH antigen-binding domains (see FIG. 3b).

[0045] CAR may comprise an antibody Fab fragment (see FIG. 3a). FabCAR comprises two chains with an antibody-like light chain variable region (VL) and constant region (CL). One chain comprises a heavy chain variable region (VH) and constant region (CH). One chain also comprises a transmembrane domain and an intracellular signaling domain. The association between CL and CH causes the receptor to assemble.

[0046] The two chains of a Fab CAR have the general structure: VH-CH-spacer-transmembrane domain-intracellular signaling domain, and VL-CL or VL-CL-spacer-transmembrane domain-intracellular signaling domain, and VH-CH may have:

[0047] In the case of a Fab-type chimeric receptor described herein, the antigen-binding domain is composed of a VH from one polypeptide chain and a VL from another polypeptide chain.

[0048] The polypeptide chain may include a linker between the VH / VL domain and the CH / CL domain. The linker may be flexible and may serve to spatially separate the VH / VL domain from the CH / CL domain.

[0049] Alternatively, the CAR may comprise a single chain variable fragment (scFv) fragment of an antibody (see FIG. 3c). In these "classical" CARs, the VH and VL domains can be in either orientation in the molecule and have the general structure: VH-VL-spacer-transmembrane domain-intracellular signaling domain, or VL-VH-spacer-transmembrane domain-intracellular signaling domain Gives.

[0050] The polypeptide chain may include a linker between the VH and VL domains to provide sufficient flexibility to allow VH / VL pairing and formation of an antigen-binding site.

[0051] Alternatively, the CAR may comprise a domain antibody (dAb) type antigen-binding domain (Figure 3b). Such a CAR has the general structure: dAb-spacer-transmembrane domain-intracellular signaling domain may have:

[0052] Spacer CARs generally contain a spacer sequence or hinge region that connects the antigen-binding domain with the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. The flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

[0053] The spacer may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular domain of CD8, CD4 or CD28, or all or part of an antibody constant domain. In particular, the spacer may be derived from the human CD8 alpha chain, the FcγRIIIα receptor or IgG1. The spacer may be a synthetic sequence corresponding to a naturally occurring hinge sequence or may be a completely synthetic sequence. The spacer may contain up to 300 amino acids, for example 10-100 amino acids and / or 25-50 amino acids.

[0054] Transmembrane domain The transmembrane domain is the part of the chimeric receptor that spans the membrane. The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. It is typically an alpha helix that is composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the chimeric receptor. The presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Alternatively, an artificially designed TM domain can be used.

[0055] End Domain The endodomain is the signaling part of the chimeric receptor. It can be part of or associated with the intracellular domain of the chimeric receptor. After antigen recognition, the receptor cluster, native CD45 and CD148 are excluded from the synapse and the signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta, which contains three ITAMs. It transmits the activation signal to the T cell after antigen binding. CD3-zeta may not provide a fully competent activation signal and additional costimulatory signaling may be required. Costimulatory signals promote the proliferation and survival of T cells. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and those belonging to the TNF family (OX40, 41BB, CD27, GITR, etc.). For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0056] The endodomain is (i) an ITAM-containing endodomain, such as an endodomain derived from CD3ζ; and / or (ii) a costimulatory domain, such as an endodomain from CD28 or ICOS, and / or (iii) domains that transmit survival signals, e.g., TNF receptor family endodomains such as OX-40, 4-1BB, CD27, or GITR. may include.

[0057] Several systems have been described in which the antigen recognition moiety is on a separate molecule from the signalling moiety, such as those described in WO 015 / 150771, WO 2016 / 124930 and WO 2016 / 030691. Thus, the chimeric receptor of the present invention may comprise an antigen-binding component comprising an antigen-binding domain and a transmembrane domain, which may interact with a separate intracellular signaling component comprising a signaling domain. The vector of the present invention may express such a chimeric receptor signaling system comprising an antigen-binding component and an intracellular signaling component.

[0058] The chimeric receptor may contain a signal peptide so that, when expressed intracellularly, the nascent protein is targeted to the endoplasmic reticulum and subsequently expressed at the cell surface. The signal peptide may be at the amino terminus of the molecule.

[0059] Prostate-specific membrane antigen (PSMA) Prostate-specific membrane antigen is a class II membrane glycoprotein that catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to glutamate and N-acetylaspartate (NAA). It is a zinc metalloenzyme that in humans is encoded by the FOLH1 gene.

[0060] The amino acid sequence of human PSMA is available from Uniprot under accession number Q04609 (https: / / www.uniprot.org / uniprot / Q04609). In this 750 amino acid sequence, the cytoplasmic domain is residues 1-19. The type II membrane protein signal anchor is residues 20-43. The extracellular domain is residues 44-750. The extracellular domain consists of the protease, apical and C-terminal domains (see FIG. 2).

[0061] Nucleic acid sequence The present invention also provides a nucleic acid sequence encoding the antigen binding domain or CAR of the present invention.

[0062] As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0063] It will be understood by those skilled in the art that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art will understand that, using routine techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein can be made to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0064] Nucleic acids according to the present invention may include DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that contain synthetic or modified nucleotides therein. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It is understood that for the purposes of the uses described herein, polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or life span of the polynucleotide of interest.

[0065] The terms "variant," "homolog" or "derivative" with respect to a nucleotide sequence include any substitution, mutation, modification, replacement, deletion or addition of one (or more) nucleic acid from or to the sequence.

[0066] The nucleic acid sequence may be codon optimized for expression in a particular host cell.

[0067] nucleic acid construct The present invention also provides a nucleic acid construct encoding a Fab-type CAR of the present invention.

[0068] A nucleic acid construct encoding a FabCAR (Figure 3A) may have the following structure: VH-CH-spacerTM-endo-coexpr-VL-CL or VL-CL-spacerTM-endo-coexpr-VH-CH (In the formula, VH is a nucleic acid sequence encoding the heavy chain variable region; CH is a nucleic acid sequence encoding the heavy chain constant region, The spacer is a nucleic acid encoding the spacer, TM is a nucleic acid sequence encoding a transmembrane domain, endo is a nucleic acid sequence encoding an endodomain, coexpr is a nucleic acid sequence enabling simultaneous expression of the first and second polypeptides, VL is a nucleic acid sequence encoding a light chain variable region, and CL is a nucleic acid sequence encoding the light chain constant region.

[0069] For both of the above constructions, the nucleic acid sequences encoding the two polypeptides may be in any order in the construct.

[0070] In the above structure, "coexpr" is a nucleic acid sequence that allows for the simultaneous expression of two polypeptides as separate entities. It may be a sequence that encodes a cleavage site, so that the nucleic acid construct produces both polypeptides linked by the cleavage site. The cleavage site may be self-cleaving, and when the polypeptides are produced, they are immediately cleaved into individual peptides without the need for any external cleavage activity.

[0071] The cleavage site may be any sequence that allows the two polypeptides to be separated.

[0072] The term "cleavage" is used herein for convenience, however, the cleavage site may separate the peptide into individual entities by mechanisms other than classical cleavage. For example, for the foot and mouth disease virus (FMDV) 2A autocleaving peptide (see below), various models have been proposed to explain the "cleavage" activity: proteolysis by host cell proteinases, autoproteolysis or translational effects (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such "cleavage" is not important for the purposes of the present invention, as long as the cleavage site, when located between nucleic acid sequences encoding proteins, allows the proteins to be expressed as separate entities.

[0073] The cleavage site may be, for example, a furin cleavage site, a tobacco etch virus (TEV) cleavage site, or may encode a self-cleaving peptide.

[0074] "Autocleaving peptide" refers to a peptide that functions such that once a protein and polypeptide comprising the autocleaving peptide is produced, it is immediately "cleaved" or separated into separate and independent first and second polypeptides without the need for any external cleavage activity.

[0075] The self-cleaving peptide may be a 2A self-cleaving peptide from an aphthovirus or a cardiovirus. Primary 2A / 2B cleavage in aphthoviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In apotoviruses such as foot and mouth disease virus (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), represents an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al. (2001) supra).

[0076] "2A-like" sequences have been found in picornaviruses other than apto- or cardioviruses, in "picornavirus-like" insect viruses, type C rotaviruses, and in repeat sequences within trypanosome species and bacterial sequences (Donnelly et al., 2001, supra).

[0077] The cleavage site may include a 2A-like sequence shown as SEQ ID NO: 161 (RAEGRGSLLTCGDVEENPGP).

[0078] The present invention also provides nucleic acid constructs comprising one or more nucleic acid sequences encoding a CAR of the present invention together with a nucleic acid sequence encoding another molecule, such as a suicide gene.

[0079] Suicide genes are genetically encoded mechanisms that allow for the selective destruction of adoptively transferred cells, such as T cells, in the face of unacceptable toxicity.

[0080] The suicide gene may be "RQR8", described in WO 2013 / 153391, having the sequence set forth as SEQ ID NO:4 therein, or "Rapcasp9", described in WO 2016 / 135470.

[0081] vector The present invention also provides a vector or kit of vectors comprising one or more nucleic acid sequences encoding an antigen binding domain or chimeric antigen receptor according to the invention. Such vectors can be used to introduce the nucleic acid sequences into a host cell so as to express the antigen binding domain or chimeric antigen receptor.

[0082] The vector can be, for example, a plasmid or a viral vector, such as a retroviral or lentiviral vector, or a transposon-based vector or a synthetic mRNA.

[0083] The vector may be capable of transfecting or transducing a cell, such as a T cell or a NK cell.

[0084] cell The present invention provides a cell comprising the chimeric antigen receptor of the present invention.

[0085] The cell may comprise a nucleic acid sequence, a nucleic acid construct or a vector of the invention.

[0086] The cell can be a cytolytic immune cell, such as a T cell or a NK cell.

[0087] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. There are various types of T cells, as summarized below.

[0088] T helper cells (TH cells) assist other white blood cells in immunological processes including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes including TH1, TH2, TH3, TH17, Th9 or TFH, which secrete different cytokines to promote different kinds of immune responses.

[0089] Cytolytic T cells (TC cells or CTLs) destroy virus-infected and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0090] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has resolved. They expand into large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with a "memory" against past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0091] Regulatory T cells (Treg cells), previously known as suppressor T cells, are crucial for the maintenance of immune tolerance. Their main role is to shut off T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus.

[0092] Two major classes of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells.

[0093] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+Treg cells) arise in the thymus and are associated with interactions between developing T cells and both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can disrupt the development of regulatory T cells and lead to the fatal autoimmune disease IPEX.

[0094] Adaptive Treg cells (also known as Tr1 or Th3 cells) can arise during a normal immune response.

[0095] The cell may be a natural killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0096] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute the third type of cell differentiated from a common lymphoid precursor that gives rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus, and then enter the circulation.

[0097] The cells of the invention can be any of the cell types described above.

[0098] Cells according to the first aspect of the invention may be generated ex vivo either from the patient's own peripheral blood (first party), or in the context of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0099] Alternatively, cells according to the first aspect of the invention may be derived from ex vivo differentiation of inducible or embryonic precursor cells into T cells or NK cells. Alternatively, immortalized T cell lines may be used which retain their lytic function and can act as therapeutic agents.

[0100] In all these embodiments, the chimeric polypeptide-expressing cells are generated by introducing DNA or RNA encoding the chimeric polypeptide by one of a number of means, including transduction with a viral vector, transfection with DNA or RNA.

[0101] The cells of the present invention may be ex vivo cells from a subject. The cells may be from a peripheral blood mononuclear cell (PBMC) sample. The cells may be activated and / or expanded, for example by treatment with an anti-CD3 monoclonal antibody, before being transduced with a nucleic acid encoding a molecule that provides a chimeric polypeptide of the first aspect of the present invention.

[0102] The cell of the present invention is (i) isolation of a cell-containing sample from a subject or other source as listed above, and (ii) transducing or transfecting a cell with one or more nucleic acid sequences encoding the chimeric polypeptide. It can be produced by

[0103] The cells can then be obtained, for example by purification, selected based on expression of the antigen-binding domain of the antigen-binding polypeptide.

[0104] Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition comprising a plurality of cells according to the present invention.

[0105] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient.The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds.Such preparations may be, for example, in a form suitable for intravenous injection.

[0106] Treatment The present invention provides a method for treating and / or preventing a disease, the method comprising the step of administering a cell of the present invention (eg, in a pharmaceutical composition described above) to a subject.

[0107] The method for treating disease relates to the therapeutic use of the cells of the present invention. Herein, the cells can be administered to a subject with an existing disease or condition to relieve, reduce or ameliorate at least one symptom associated with the disease, and / or to slow down, reduce or block the progression of the disease.

[0108] The method of preventing disease relates to the prophylactic use of the cells of the present invention.Herein, such cells can be administered to subjects who have not yet suffered from disease and / or have not shown any symptoms of disease, to prevent or impair the cause of disease, or to reduce or prevent the occurrence of at least one symptom associated with disease.Subjects can be considered to have a predisposition to disease or to be at risk of developing disease.

[0109] The method comprises: (i) isolating a cell-containing sample; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the invention; (iii) administering the cells from (ii) to a subject; may be involved.

[0110] The cell-containing sample may be isolated from a subject or other source, for example, as described above. Cells may be isolated from the subject's own peripheral blood (first party), or in the context of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0111] The present invention provides a CAR-expressing cell of the present invention for use in the treatment and / or prevention of disease.

[0112] The present invention also relates to the use of the CAR-expressing cells of the present invention in the manufacture of a medicament for treating and / or preventing a disease.

[0113] The disease treated and / or prevented by the methods of the present invention may be a cancerous disease such as prostate cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, and thyroid cancer.

[0114] In particular, the disease may be prostate cancer.

[0115] The cells of the present invention may be capable of killing target cells, such as cancer cells. Target cells may be characterized by the presence of tumor-secreted or chemokine ligands in the vicinity of the target cells. Target cells may be characterized by the presence of soluble ligands along with the expression of tumor-associated antigens (TAA) on the target cell surface.

[0116] The cells and pharmaceutical compositions of the present invention may be for use in the treatment and / or prevention of the above diseases.

[0117] The present invention will now be further described by examples, which are intended to serve to aid those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way. EXAMPLES

[0118] Example 1 - Preparation of PSMA binders Following immunization, a rat phage display library was generated and panned against in-house recombinant human PSMA immobilized on beads. After demonstrating enrichment of specific phages, the library was screened using a phage ELISA assay and isolated clones were sequenced. Binding of clones was re-evaluated to confirm positive selection before cloning into a mouse IgG2A Fc vector for further analysis.

[0119] material and method Generation of Strep2-tagged PSMA StrepII-tagged PSMA (MP27034) was produced in-house and transfected into 30 mL of HEK293F cells according to standard protocols. After 4 days, culture supernatants were harvested, sterilized through a 0.22 μm filter, aliquoted, and stored at 4°C.

[0120] Bead-based capture of recombinant PSMA and FACS analysis protocol (QC) 25 μl of Magstrep beads (IBA) were removed from a storage vial. The beads were washed with 1 mL PBS.T (0.05% Tween®) x 3, with magnetic separation between washes. 1.5 mL of MP27034 supernatant (PSMA was not quantified) was incubated with the beads for 30 min at room temperature with agitation. The beads were washed with 1 mL (x3) of PBS.T with separation via magnet after each wash. The beads were blocked in 1 mL of 1% BSA PBS for 30 min at room temperature with 25 μl of unconjugated beads for negative selection / control in QC experiments. The beads are washed with 1 mL (x3) of PBS.T with separation via magnet after each wash. Stain with 1 / 1000 dilution of anti-PSMA Alexaflour 488 (Abcam ab187570) for 10 min at room temperature with agitation. Flow confirmation of binding was performed using conjugated beads on a Milteni MACSquant.

[0121] Phage panning PSMA St2 (27034) in the supernatant from transiently transfected HEK293T cells (RJ) was conjugated to beads as described above (without staining). Both IBA type 2 and type 3 strep-tactin beads were used for panning in a KingFisher Flex. A 100-fold representation of the phage library (1 × 10) was added to the beads in order to remove non-specifically bound phages for 30 min at room temperature with agitation. 10 phages / ml) were incubated with BSA-conjugated beads. The beads were magnetically separated and the phage-containing supernatant was transferred directly to the PSMA-conjugated beads.

[0122] Panning and washing steps were performed on a Kingfisher Flex using "Phage Display Protocol 2" with 5 cycles of 3 min washes in PBS.T 0.05%. Elution from PSMA St2 conjugated beads was performed with 500 μl 2× Biotin solution in PBS for 20 min.

[0123] Phages were then titrated and amplified according to standard protocols. Briefly, eluted phages were re-infected into log phase TG1 cells (5 ml) and amplified by plating on Amp / Gluc agar plates. Titrations were performed to establish phage numbers and enrichment. Binders were generated using several rounds of phage panning and amplification.

[0124] Monoclonal phage expression ELISA A standard phage ELISA protocol was used. Briefly, individual bacterial colonies were picked and cultured at 37°C to an OD of 0.5 in 2TY Gluc / Amp medium. 600nm After incubation for 3 h, phage expression was induced by addition of helper phage (M13KO7) for 40 min of superinfection, resuspended in 2TY supplemented with ampicillin and kanamycin (100 μg / ml and 70 μg / ml, respectively), and incubated overnight at 30° C. with agitation. Cells were pelleted by centrifugation, and the supernatant was used directly for screening ELISA and flow cytometry.

[0125] ELISA All incubations were performed at room temperature for 1 h with agitation and washed three times with 0.05% PBS.tween. PSMA was seeded at 1 μg / ml (50 μl) into nunc 96-well plates, incubated for 1 h at room temperature, plates washed, blocked with 2% milk for 1 h, and washed again before adding supernatants. The detection antibody was an anti-M13-HRP conjugate and plates were read at 450 nm after developing with TMB substrate.

[0126] Deviations from this ELISA protocol include scFv detection using anti-c-Myc HRP or anti-Flag-HRP as appropriate.

[0127] result PSMA batch QC before panning begins Before starting the panning, a batch of PSMA was generated from a transient 293 transfection. This QC showed that PSMA ST2 could be successfully bound to and eluted from type 3 beads using biotin. Therefore, type 3 beads were used for all of the following bead-based panning strategies. [Table 1-1] [Table 1-2]

[0128] Panning enrichment showed positive results for phage titers after two pans, so single colonies were selected for screening for specific antibodies.

[0129] Based on the selection criteria of PSMA binding over background, colonies were stored as glycerol stocks in 50% glycerol and cultured to allow for repeated screening. [Table 2]

[0130] Repeated ELISA results from M13 phage expression by monoclonal selection (following phage precipitation) showed that the signal was replicable and significant compared to binding to background proteins.

[0131] FIG. 4 shows ELISA assays of cloned phage and scFv cultures in replicates after initial screening (n=3) in recombinant protein ELISA assays.

[0132] overview A selection of binders was generated from this phage display campaign along with additional sequences using hybridoma generation from the same animals.

[0133] 7A12 Humanized In silico humanization and antibody modeling 7A12 humanization was performed by in silico modeling and CDR grafting using BioLuminate software (Schrodinger).

[0134] In silico immunogenicity analysis Objective: To characterize MHCI binding and MHCI immunogenic properties (Nielsen et al., 2003 Protein Sci. 12, 1007-1017; Bui et al., 2005 Immunogenetics 57, 304-314; Peters & Sette 2005 BMC Bioinformatics 6,132;Karosiene et al., 2012 Immunogenetics 64,177-186;Zhanget et al., 2009 Bioinformatics 25,1293-1299) to predict the T cell epitopes of the newly designed humanized constructs. Furthermore, the T20 score analyzer (https: / / dm.lakepharma.com / bioinformatics / ) is used to predict the humanness of the grafted frameworks (Gao et al., 2013, BMC Biotechnol. 13,55).

[0135] IEDB This tool allows the analysis of peptides derived from antibody sequences for binding affinity to MHCI. It analyzes the sequences of grafted antibody frameworks by a proteasome cleavage / TAP transport / MHC class I combined predictor (http: / / tools.iedb.org / processing / ) for the human HLA allele A*02:01 using all peptide lengths (8-14). A specific cutoff of 274 nM is applied to differentiate binding peptides (Paul, S. et al., 2013 J. Immunol. 191, 5831-5839).

[0136] IEDB MHC-I immunogenicity All peptides with an IC50 below 274 nm were then analyzed with the Class I Immunogenicity Tool (http: / / tools.iedb.org / immunogenicity / ) to determine an immunogenicity score (positive values ​​indicate immunogenicity) (Calis et al., 2013 PLoS Comput. Biol. 9, e1003266).

[0137] Since an affinity threshold of 50 nm is required for an efficient CTL response (Sette, A. et al., 1994 J. Immunol. Baltim. 153, 5586-5592), all peptides with an affinity of 50 nm or less for MHC-I are considered to be potentially immunogenic.

[0138] T20 Scores The T20 score analyzer is used to calculate the humanness score of monoclonal antibodies from antibody variable sequences (Gao et al., 2013, BMC Biotechnol. 13, 55). The sequences of the grafted antibody frameworks are analyzed by the T20 score analyzer (https: / / dm.lakepharma.com / bioinformatics / ). A score of 80 or above is associated with a human sequence.

[0139] Example 2 - Generation of anti-PSMA single domain antibodies Using a combination of immobilized antigen and cell-based phage display panning, single domain antibodies (dAbs) with specificity for PSMA were isolated from a phage display library of immunized llamas.

[0140] Screening assays were first performed on the recombinant proteins before confirmation of cellular binding to transduced endogenous PSMA-expressing cells was confirmed by flow cytometry.

[0141] SPR analysis (Biacore) showed that the antibodies bound with affinities in the nM range in IHC staining and that selected antibodies bound to PSMA (human and rhesus monkey).

[0142] material and method Immunization and library preparation Two llamas (9240, 9241) were genetically immunized with pCMV-PSMA via subcutaneous injection (days 0 and 21) to prime the immune response prior to protein immunization (days 42 and 63) with recombinant His-tagged PSMA. Peripheral blood lymphocytes (PBLs) were isolated from a 150 ml blood sample taken 70 days after primary immunization. RNA was isolated from PBLs using an RNeasy maxi kit (Qiagen). cDNA was synthesized from each llama using oligo-dT priming and Superscript III reverse transcriptase. dAb encoding cDNAs were amplified by PCR using specific primers, analyzed to ensure the presence of a 400 bp fragment, and purified (Zymoclean). The resulting clean PCR products were cut by restriction enzyme digestion (PST1 and NOT1) and ligated into the M13 phage vector viral coat protein (pRL144). TG1 E. coli were electroporated multiple times and after selection on ampicillin agar plates samples were pooled and frozen.

[0143] Phage carrying antibodies expressed as protein coats were amplified from TG1 E. coli. Briefly, TG1 cells were inoculated into 2TY medium supplemented with glucose (2%) / ampicillin (1 μg / ml) and grown to an OD of 0.5 before adding M13KO7 helper phage. 600nm The cells were grown at 37°C with agitation until 40 min. After 40 min, the cells were resuspended in 2YT medium supplemented with kanamycin and ampicillin (70 / 100 μg / ml) and incubated overnight at 30°C with agitation. Phages were purified using PEG-based precipitation and titrated on TG1 E. coli to determine PFU / ml.

[0144] Phage display panning Recombinant Proteins Recombinant PSMA was immobilized at 1 μg / ml on Nunc immunotubes overnight at 4° C., followed by blocking with 2% milk in PBS. Phages were blocked for 1 h in 2% milk in PBS (2 ml) before being added to the PSMA-coated immunotubes. After 1 h at room temperature, the tubes were washed 15 times with 3×1 min incubations using PBS 0.05% Tween20. Elution of specific phages was performed by adding rewarmed trypsin (2 ml) to the tube and incubating at 37°C for 10-15 min. Eluted phages were reinfected into log-phase TG1 cells (5 ml) and amplified by plating on Amp / Gluc agar plates. Titrations were performed to establish phage numbers and enrichment.

[0145] Cell-based panning Cell-based panning was used to enrich and isolate binders with specificity for PSMA as displayed on the cell surface. PSMA stably transduced and non-transduced SupT1 cells were cultured and a total of 5 × 10 per library were cultured. 6 Cells were harvested, resuspended in 4 ml ice-cold milk / PBS 2% and kept on ice. Phages (2 × 10 ) from pan1 on the recombinant protein were then transferred to the phage buffer prior to centrifugation (400 × g / 5 min / 4 °C) and resuspension of SupT1 NT cells in the phage solution. 11 PFU / ml) were blocked for 1 hour in 2 ml PBS / milk 2%. Phage and SupT1 NT cells were incubated at 4°C for 1 h with end-to-end rotation to remove non-specific binders, after which the process was repeated with SupT1-PSMA cells. Washing consisted of successive centrifugations and PBS washes prior to incubation with trypsin-EDTA at 37°C (10 min) and phage titration and amplification into E. coli TG1 cells as described above. This was performed by resuspension in 0.05% Tween 20 (adjusted to pH 5).

[0146] screening Individual bacterial colonies were picked and cultured at an OD of 0.5 in 2TY Gluc / Amp medium at 37°C before induction of dAb expression by addition of IPTG (1 mM) and overnight incubation at 30°C. 600nm The cells were pelleted by centrifugation and the supernatant was used directly for screening ELISA and flow cytometry.

[0147] ELISA All incubations were performed for 1 h at room temperature with agitation and washing was performed three times with PBS 0.05% Tween 20. PSMA was seeded at 1 μg / ml (50 μl) into Nunc 96-well plates and incubated for 1 h at room temperature, plates were washed and blocked with 2% milk for 1 h and washed again before adding supernatants. The detection antibody was an anti-C Myc-HRP conjugate and plates were read at 450 nm after developing with TMB substrate.

[0148] Flow cytometry Transduced or non-transduced SupT1 cells expressing high or low levels of human PSMA were stained in various flow cytometry experiments together with the tumor cell lines LnCAP, PC3 and 22RV1. Briefly, cells were incubated with screening antibodies tagged with either mouse Fc or C-Myc tags, followed by washing with PBS.T 0.05% and the appropriate secondary antibodies (anti-C Myc PE and anti-mouse Fc AF647). The flow cytometers used were Miltenyi MACSquant (Figure 5) and IntelliCyt (Figure 6).

[0149] result To allow for the investigation of the antibody repertoire, two phage display libraries were constructed from two DNA- and protein-immunized llamas. Panning was performed against recombinant human PSMA (Acro Biosystems) showing initial high enrichment of specific clones (Figure 5(A)). After screening, both of these libraries were panned against SupT1 cells expressing PSMA to generate cell membrane-bound PSMA specificities. The cell-panned libraries yielded similar enrichment titers as those from the recombinant protein panning (Figure 5(B)).

[0150] After evaluation of the initial panning enrichment, monoclonal phage expressing TG1 colonies were selected from the titration plates, cultured and dAb expression was induced by IPTG addition and glucose starvation. ELISA analysis showed a mixture of positive clones and background binders (Figure 6). Positive clones were selected, sequenced and further screened against recombinant PSMA in a titration assay (data not shown). Selected binders were taken forward to flow cytometry, showing that clones B8 and G7 from Llama 2 had the highest affinity and specificity for the high expressing PSMA clones (Figures 7 and 8).

[0151] The enriched repertoire of dAbs within the library of cell-panned phages shown by the initial screen was significant enough to warrant further screening of similar sequences by Next gen Sequence analysis (NGS). Both llamas 1 and 2 were analyzed by NGS (PacBio) and sequences were assessed for similarity to high affinity isolated sequences for confirmed cell binders. A set of 36 additional binders was generated by G Block gene synthesis (IDT technologies). These were cloned into a mouse IgG2A-Fc format and expressed from HEK293 cells and then screened against a variety of cell types including transduced and endogenous expression (Figure 9).

[0152] Two binders of particular interest were isolated from the NGS array (clones 2 and 25). Immunohistochemistry was performed on transduced cells or human tumor and rhesus monkey samples to establish the feasibility of in vivo model validation and IHC analysis of colocalization (Figure 10).

[0153] The results showed that the B8 and G7 clones (49110 and 49112) bound to PSMA-positive cell lines, but they either bound less well than the gold standard control or did not bind to PSMA-transduced rhesus monkey cell lines. Based on this data, the two candidates selected for further analysis were clones 2 and 25 from the NGS selection.

[0154] Example 3 - Biophysical characterization of anti-PSMA VHH antibodies method Anti-PSMA clones were expressed in expi-CHO cells by transient transfection. Supernatants from transfected CHO cells were purified using Protein A affinity chromatography. A HiTrap MabSelect SuRE 1 ml column was equilibrated with 5 column volumes of PBS pH 7.4. The supernatant was applied to the column at a flow rate of 1 mL / min using the Akta™ Pure system. After applying the supernatant, the column was washed with 20 column volumes of PBS. The sample was then eluted from the column at 1 mL / min using 3 ml of IgG elution buffer (Pierce-21004) and loaded directly onto two HiTrap 5 ml desalting columns, pre-equilibrated in PBS, and collected onto a 96-well plate using a fraction collector device.

[0155] Fractions containing protein were pooled and analyzed by SDS-PAGE. The material was divided into aliquots and stored at -80°C.

[0156] Size Exclusion Chromatography 20 μl of purified recombinant antibody was loaded onto a Superdex 200 increase 5 / 150 GL column on an Akta Pure system. Briefly, the column was equilibrated with 1.5 column volumes of PBS at 0.3 ml / min. Samples were loaded at 0.3 ml / min by direct loop injection and run in PBS over 1.5 column volumes. Eluted proteins were detected at OD280nm. VHH-Fc constructs were expected to elute at 2 ml.

[0157] thermal stability Samples were normalized to 1 mg / ml and loaded into standard glass capillary tubes (Nanotemper). Thermal stability was determined by linear temperature increments of 1 °C / min from 20 °C to 95 °C in a Nanotemper Prometheus NT.48 instrument. Data were analyzed as the first derivative of the 350 nm / 330 nm ratio.

[0158] Surface plasmon resonance Kinetic affinity for soluble PSMA was determined using a Biacore T200 instrument. Recombinant purified antibodies were captured on Protein A chip #7 for 60 seconds at 10 μl / min with a target of 100 RU. Recombinant PSMA-His (sino biologicals) dialyzed in HBS-P+ buffer was used as analyte in a 1:2 serial dilution of 10 points from 1 μM, run at 30 μl / min for 150 seconds, with a dissociation time of 300 seconds. Double reference subtraction was applied. Sensogram fitted with a 1:1 Langmuir model.

[0159] Anti-PSMA antibody J591 (UPenn) was used as a reference.

[0160] result The antibodies were transiently expressed in VHH-Fc format in ExpiCHO and purified by Protein A affinity chromatography. Protein purity was assessed by SDS-PAGE and molecular distribution was measured by size exclusion chromatography. The antibodies showed low aggregation tendency. Thermal stability was assessed by DSF, showing melting temperatures above 57° C. for both antibodies tested.

[0161] Affinities were determined by surface plasmon resonance on a Biacore T200 instrument against recombinant soluble PSMA. Affinities were measured in the nM range for both antibodies (Table 3). [Table 3]

[0162] Example 4 - Constructs of anti-PSMA CAR A panel of nucleic acid constructs encoding CARs is generated as follows. fmc63_BBz:SFGmR.RQR8-2A-FMC63-CD8STK-TyrpTM-41BBz mu7A12_BBz:SFGmR.RQR8-2A-aPSMA_mu7A12_CD8STK-TyrpTM-41BBz hu7A12_BBz:SFGmR.RQR8-2A-aPSMA_hu7A12_CD8STK-TyrpTM-41BBz hu7A12_28z:SFGmR.RQR8-2A-aPSMA_hu7A12_CD8STK-TyrpTM-41BBz J591_BBz:SFGmR.RQR8-2A-aPSMA_hu7A12_CD8STK-TyrpTM-41BBz SFGmR is the vector backbone, RQR8 is the suicide gene RQR8 described in WO 2013 / 153391; 2A is a member of the 2A self-cleaving peptide family that allows for the simultaneous expression of RQR8 and CAR polypeptides. CD8 STK is the CD8 stalk spacer; Tryp™ is a transmembrane domain derived from Tryp-1; 41BBz is a second generation endodomain that contains the endodomains from CD3 zeta and 4-1BB.

[0163] Example 5 - FAC-based killing assay (FBK) The ability of CAR-T cells to kill target cells was examined using a FACS-based killing assay. SupT1 cells engineered to express human PSMA antigen (SupT1-PSMA) were used as target cells. Non-engineered SupT1 cells (SupT1-NT) were used as a negative control. T cells were co-cultured with target cells at effector-to-target ratios of 1:1, 1:4 and 1:8. FBK was assayed after 24 hours of incubation and analyzed by cytofluorometric analysis.

[0164] Example 6 - Cytokine Release Secretion of IL-2 and IFNγ by CAR T cells was measured by harvesting supernatants at 24 hours from the co-cultures described in Example 5. IL-2 and IFNγ production was detected by ELISA.

[0165] methodology cell line SupT1 cell lines (NT and PSMA+) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMAX. T cells were isolated from peripheral blood mononuclear cells (PBMCs) and maintained in RPMI-1640 medium supplemented with 10% FBS, 1% GlutaMAX, and 100U / mL IL-2.

[0166] Transduction Retroviruses were generated by transiently transfecting HEK293T cells with RDF plasmid (RD114 envelope), gag / pol plasmid and CAR plasmid using GeneJuice. Retroviral viral supernatants were harvested at 48 and 72 hours. T cells were stimulated in T175 TC-treated flasks with 0.5 μg / mL anti-CD3 and anti-CD28 antibodies and maintained in 100 U / mL IL-2. Non-TC treated 6-well plates were coated with Retronectin and incubated at 4°C for 24 h before T cell transduction. 6 3 ml of viral supernatant was seeded before adding 1 ml of activated T cells at a concentration of cells / ml, followed by addition of 100 U / mL IL-2, centrifugation at 1000xg for 40 min at room temperature, and incubation at 37°C and 5% CO2 for 2-3 days.

[0167] Deficiency of NK cells and NKT cells CD56 depletion was performed using the EasySep™ human CD56 positive selection kit.

[0168] Cytotoxicity assay CAR T cells were co-cultured with SupT1-NT and SupT1-PSMA at effector-to-target ratios (E:T ratios) of 1:1, 1:4 or 1:8 in TC-treated 96-well plates (target cells at a constant concentration of 50,000 cells per condition). Readout was performed 24 h after co-culture by staining with anti-CD3-PeCy7 and Qben10-APC to differentiate effector T and target cells, and 7-AAD dead cell stain was used to exclude dead cells. Cytotoxicity readout was accessed by flow cytometry.

[0169] Cytokine ELISA Human IL-2 ELISA MAX™ Deluxe and Human IFN-γ ELISA MAX™ Deluxe kits were used to access the levels of cytokines secreted into co-culture supernatants harvested from the cytotoxicity assays.

[0170] Example 7 - pH Scout The stability and affinity kinetics of the binders at different levels of pH were investigated.

[0171] methodology Surface plasmon resonance Kinetic affinity for soluble PSMA at different pH was determined using a Biacore T200 instrument. Recombinant purified antibodies were captured on a Protein A chip #6 and pre-equilibrated with buffer HBS-P+ at the required pH (pH 7.4, pH 7, pH 6.5 and pH 6) at 10 μl / min for 60 s, targeting 100 RU. Recombinant PSMA-His (sino biologicals) dialyzed in HBS-P+ buffer and adjusted to the required pH was used as analyte in a 1:2 serial dilution of 10 points starting from 1 μM, run at 30 μl / min for 150 s, with a dissociation time of 300 s. Double reference subtraction was applied. Sensogram fitted with a 1:1 Langmuir model.

[0172] thermal stability Samples were normalized to 1 mg / ml in PBS buffered to the required pH (pH 7.4, pH 7, pH 6.5 and pH 6) and loaded into standard glass capillary tubes (Nanotemper). Thermal stability was determined by linear temperature increments of 1 °C / min from 20 °C to 95 °C in a Nanotemper Prometheus NT.48 instrument. Data were analyzed as the first derivative of the 350 nm / 330 nm ratio.

[0173] result All antibodies tested showed increased affinity for the target protein at lower pH. Humanized clones 7A12 and 7A12 showed higher thermal stability that was hardly affected by changes in pH conditions. These results support the conclusion of clone 7A12 being a more stable binder in physiological and tumor-specific pH microenvironments compared to J591 (Table 4). [Table 4-1] [Table 4-2]

[0174] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. The present invention provides, for example, the following items. (Item 1) An antigen-binding domain that binds to prostate-specific membrane antigen (PSMA), (a) a heavy chain variable region (VH) having complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3 selected from the following, and a light chain variable region (VL) having CDRs: LCDR1, LCDR2 and LCDR3 selected from the following: (i) HCDR1-SEQ ID NO: 1, HCDR2-SEQ ID NO: 2, HCDR3-SEQ ID NO: 3, LCDR1-SEQ ID NO: 4, LCDR2-SEQ ID NO: 5, LCDR3-SEQ ID NO: 6, (ii) HCDR1-SEQ ID NO: 9, HCDR2-SEQ ID NO: 10, HCDR3-SEQ ID NO: 11, LCDR1-SEQ ID NO: 12, LCDR2-SEQ ID NO: 13, LCDR3-SEQ ID NO: 14, (iii) HCDR1-SEQ ID NO: 17, HCDR2-SEQ ID NO: 18, HCDR3-SEQ ID NO: 19, LCDR1-SEQ ID NO: 20, LCDR2-SEQ ID NO: 21, LCDR3-SEQ ID NO: 22, (iv) HCDR1-SEQ ID NO: 25, HCDR2-SEQ ID NO: 26, HCDR3-SEQ ID NO: 27, LCDR1-SEQ ID NO: 28, LCDR2-SEQ ID NO: 29, LCDR3-SEQ ID NO: 30, (v) HCDR1-SEQ ID NO: 33, HCDR2-SEQ ID NO: 34, HCDR3-SEQ ID NO: 35, LCDR1-SEQ ID NO: 36, LCDR2-SEQ ID NO: 37, LCDR3-SEQ ID NO: 38, (vi) HCDR1-SEQ ID NO: 41, HCDR2-SEQ ID NO: 42, HCDR3-SEQ ID NO: 43, LCDR1-SEQ ID NO: 44, LCDR2-SEQ ID NO: 45, LCDR3-SEQ ID NO: 46, and (vii) HCDR1-SEQ ID NO: 49, HCDR2-SEQ ID NO: 50, HCDR3-SEQ ID NO: 51, LCDR1-SEQ ID NO: 52, LCDR2-SEQ ID NO: 53, LCDR3-SEQ ID NO: 54, or (b) a domain antibody variable region having complementarity determining regions (CDRs) selected from: VHHCDR1, VHHCDR2 and VHHCDR3; (i) VHHCDR1-SEQ ID NO: 57, VHHCDR2-SEQ ID NO: 58, VHHCDR3-SEQ ID NO: 59, (ii) VHHCDR1-SEQ ID NO: 61, VHHCDR2-SEQ ID NO: 62, VHHCDR3-SEQ ID NO: 63, (iii) VHHCDR1-SEQ ID NO: 65, VHHCDR2-SEQ ID NO: 66, VHHCDR3-SEQ ID NO: 67, (iv) VHHCDR1-SEQ ID NO: 69, VHHCDR2-SEQ ID NO: 70, VHHCDR3-SEQ ID NO: 71, and (v) an antigen-binding domain comprising any one of VHHCDR1-sequence number 73, VHHCDR2-sequence number 74, and VHHCDR3-sequence number 75. (Item 2) (a) a heavy chain variable region (VH), and a light chain variable region (VL) selected from: (i) VH-SEQ ID NO: 7, VL-SEQ ID NO: 8, (ii) VH-SEQ ID NO: 15, VL-SEQ ID NO: 16, (iii) VH-SEQ ID NO: 23, VL-SEQ ID NO: 24, (iv) VH-SEQ ID NO: 31, VL-SEQ ID NO: 32, (v) VH-SEQ ID NO: 39, VL-SEQ ID NO: 40, (vi) VH-SEQ ID NO: 47, VL-SEQ ID NO: 48, and (vii) VH-SEQ ID NO:55, VL-SEQ ID NO:56, or (b) a domain antibody variable region selected from: (i) SEQ ID NO: 60, (ii) SEQ ID NO: 64, (iii) SEQ ID NO: 68, (iv) SEQ ID NO: 72, and (v) SEQ ID NO: 76 2. The antigen-binding domain according to item 1, comprising any one of the following: (Item 3) A chimeric antigen receptor (CAR) comprising the antigen-binding domain of item 1 or 2. (Item 4) A nucleic acid sequence encoding the antigen-binding domain of item 1 or 2 or the CAR of item 3. (Item 5) A nucleic acid construct comprising the nucleic acid sequence according to item 4 and a nucleic acid sequence encoding a suicide gene. (Item 6) A vector comprising the nucleic acid sequence according to item 4 or the nucleic acid construct according to item 5. (Item 7) A cell expressing the CAR described in item 3. (Item 8) A method for producing the cell of item 7, comprising introducing a nucleic acid sequence encoding the CAR of item 3 into a cell ex vivo. (Item 9) 8. A pharmaceutical composition comprising a plurality of cells according to item 7 together with a pharma- ceutically acceptable carrier, diluent or excipient. (Item 10) A method for treating cancer, comprising administering the cell of item 7 to a subject. (Item 11) The cell according to item 7 for use in treating cancer. (Item 12) 8. Use of the cells according to item 7 in the manufacture of a medicament for treating cancer.

Claims

[Claim 1] The invention described in the specification.

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