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WO2025087935A1 - Récepteur antigénique chimérique spécifique du récepteur 1 du folate - Google Patents

Récepteur antigénique chimérique spécifique du récepteur 1 du folate Download PDF

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Publication number
WO2025087935A1
WO2025087935A1 PCT/EP2024/079876 EP2024079876W WO2025087935A1 WO 2025087935 A1 WO2025087935 A1 WO 2025087935A1 EP 2024079876 W EP2024079876 W EP 2024079876W WO 2025087935 A1 WO2025087935 A1 WO 2025087935A1
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seq
amino acid
acid sequence
car
cells
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Christoph Herbel
Maria BETHKE
Pierre ABRAMOWSKI
Miriam DROSTE
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Miltenyi Biotec GmbH
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Miltenyi Biotec GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector

Definitions

  • the invention relates to antigen binding proteins (such as antibodies or binding fragments) specific for Folate receptor alpha (FolRl).
  • the antigen binding proteins may be used for several applications, such as for the detection and/ or treatment of cancer.
  • FolRl is a membrane protein, which binds folic acid with high affinity and mediates the cellular uptake of this vitamin via receptor-mediated endocytosis 2 .
  • Folate is a basic component of cell metabolism, DNA synthesis and repair. Rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis.
  • FolRl levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression. It has been suggested that FolRl might confer a growth advantage to the tumor by modulating folate uptake from serum or by generating regulatory signals 3 .
  • 0varian cancer is defined as a group of tumors that originate in the ovaries. Ovarian cancer is the fourth most common cancer among women and is the leading cause of gynecological cancer-related death in women. Most clinical cases are epithelial ovarian cancers, which represent approximately 90% of diagnosed patients. Epithelial ovarian cancer has four main histologic subtypes: serous, endometrioid, mucinous, and clear cell. Approximately 70% of patients present with serous histology, and clinical trials have demonstrated that a patient’s subtype has prognostic importance. Other less common types of ovarian tumors include primary peritoneal, fallopian tube, and malignant germ cell tumors 1 .
  • [0005]0varian cancer patients have an approximate overall five-year survival rate of 49.1%. As with most cancers, the five-year survival rate can differ depending on the stage at which the cancer is diagnosed. If the cancer is caught early at a localized stage (Stage I— II), the five-year survival rate is 92.6%. Ovarian cancer that has spread to a different part of the body, also known as regional ovarian cancer (Stage III), has a five-year survival rate of 74.8%. The five-year survival rate decreases further to 30.3% in metastatic (Stage IV) ovarian cancer. Most patients present with advanced disease at diagnosis, and despite high responses to initial treatment, the majority will eventually relapse with incurable disease 1 .
  • chimeric antigen receptor provides a promising approach for adoptive cell immunotherapy for cancer.
  • a protein comprising the antigen binding domain of the protein that is used for immunotherapy should be used.
  • proteins comprising the antigen binding domain that is used for immunotherapy show poor binding properties for other applications such as imaging or flow cytometry. In most cases such a protein does not bind to patients tissue (that has been treated with fixatives) at all. So it cannot be used for the detection and prognosis of the outcome of such an immunotherapy.
  • the cell surface antigen FolRl is expressed on cancer cells such as triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer and can be used for targeted immunotherapy.
  • cancer cells such as triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer and can be used for targeted immunotherapy.
  • a protein comprising a specific antigen binding domain specific for FolRl can be used for detecting FolRl positive cells (especially cancer cells; figure 6) and simultaneously for immunotherapy such as CART cell therapy (Examples 4-5).
  • the first aspect of the invention is therefore a protein comprising an antigen binding domain comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of SEQ ID No. 20(GYSFTSYW), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No.
  • HCDR1 comprising an amino acid sequence of SEQ ID No. 20(GYSFTSYW)
  • HCDR2 comprising the amino acid sequence IYPGDSDT
  • HCDR3 comprising the amino acid sequence ARRKGPHYGSGAIFDY
  • said light chain variable region of an antibody comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).
  • LCDR1 comprising an amino acid sequence NIGSKS (SEQ ID No. 36)
  • LCDR2 comprising the amino acid sequence DDS
  • LCDR3 having the amino acid sequence QVWDSSSDPNYV
  • a protein comprising the antigen binding domain as disclosed herein can be used for immunotherapy such as CAR T cell therapy and protein (such as antibody) drug conjugates. They showed effective killing of cancer cells by said CAR in vivo and in vitro (figure 4 and 5). Therefore one aspect of the invention is a protein, which is a chimeric antigen receptor, comprising the antigen binding domain as defined by the first aspect of the invention, a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • the protein may be a protein drug conjugate (such as an antibody drug conjugate), wherein said protein comprises an antigen binding domain as described in the first aspect of the invention and a cytotoxic payload moiety.
  • a protein drug conjugate such as an antibody drug conjugate
  • said protein comprises an antigen binding domain as described in the first aspect of the invention and a cytotoxic payload moiety.
  • a construct may be used for immunotherapeutic applications.
  • the protein may comprise the antigen binding domain as defined in the first aspect and a detection moiety.
  • a protein may be used for detection of FolRl positive cells (especially cancer cells) in a sample (preferentially a patient sample). It may be used for flow cytometry and/or imaging applications. Such a method may be used for disease staging. Brief description of the drawings
  • Figure 1 CAR T cell binding domain candidates.
  • 189 human/mouse cross-reactive anti-FOLRl candidate binders were identified via phage display from a human antibody library. 19 candidates were further selected via flow cytometric screening of FOLR-variant expressing cells to select for FOLR1 -specific candidates. A CAR T cell screening approach was then used to assess functionality, specificity and cell expansion. 4 candidates were selected for further in vitro analysis, identifying one lead anti-FOLRl CAR candidate.
  • Figure 2 Unique binder sequences with cross-reactivity between hFOLRl and mFOLRl were identified via phage display.
  • Jurkat cells expressing the respective human or mouse FOLR variant were used to test for specificity of the candidates for FOLR1.
  • 1x105 FOLR-expressing Jurkat cells were plated and stained with 1 pg scFv-Fc for 30 minutes at 4°C.
  • An anti-IgG(Fc)-PE secondary antibody staining was performed for 10 minutes at 4°C.
  • Flow cytometric measurements were performed in triplicates and are displayed as % of stained cells.
  • Candidates staining for FOLR2 or FOLR4 and with low staining for FOLR1 were excluded. Selected candidates are marked in black, non-selected candidates in grey.
  • F), G Flow cytometric results of candidates in scFv-Fc format binding to mFOLRl overexpressing Jurkat cells.
  • H I) Flow cytometric results of candidates in scFv-Fc format binding to hFOLR2 overexpressing Jurkat cells.
  • J K) Flow cytometric results of candidates in scFv-Fc format binding to mF0LR2 overexpressing Jurkat cells.
  • L M) Flow cytometric results of candidates in scFv-Fc format binding to hFOLR4 overexpressing Jurkat cells.
  • N O) Flow cytometric results of candidates in scFv-Fc format binding to mF0LR4 overexpressing Jurkat cells.
  • Figure 4 CAR T cell screening reveals functional, specific anti-FOLRl CAR T cell candidates.
  • 2x104 GFP- and FOLR1 -expressing target cells (OV-90) were seeded and cocultured with 5x104 anti-FOLRl CAR T cells to measure antigen-dependent lysis of ovarian cancer cells.
  • 2x104 ovarian cancer cells (OV-90) were added in addition, followed by 5x104 ovarian cancer cells (OV-90) after 164h.
  • co-culture was performed with ovarian cancer cells deficient for FOLR1 (OV-90 FOLR1 KO).
  • Target cell lysis by the CAR T cell candidates was analyzed by decrease in GFP signal over time by measuring the Green Calibrated Units per pm2/image for 11 days.
  • target cells OV-90
  • Two positive control CAR T cell constructs known to successfully induce FOLR1 specific lysis of target cells were included.
  • IFN-y secretion was measured after 24h of each addition of ovarian cancer cells, respectively.
  • CAR T cell expansion was assessed by flow cytometric quantification after 92h, 164h as well as 260h of co-culture, i.e. before each addition of target cells and at end-point, respectively.
  • Figure 5 Advanced in vitro CAR T cell assays identify lead anti-FOLRl CAR T cell candidate.
  • 2x104 GFP expressing cells were seeded and co-cultured with 1x104 anti- FOLRl CAR T cells to measure antigen-dependent lysis of ovarian cancer cells for five days. After 48h of co-culture, 2x104 ovarian cancer cells were added to the co-culture. Cell lysis by the CAR T cell candidates was tracked by measurement of the green area confluency. As negative controls, ovarian cancer cells were cultured without addition of CAR T cells or with untransduced T cells. A positive control CAR T cell construct known to successfully induce FOLR1 specific lysis of target cells was included.
  • the top four candidates identified by the CAR T cell screening were included in this assay, comprising three donors each. After 48h and 120h, flow cytometric measurement of activation marker expression CD 137 and CD69 was performed. In addition, activation markers on a fourth donor were measured upon co-culture with FOLRllow (OVCAR-3) expressing cells.
  • A) CAR architecture of the four anti-FOLRl directed CAR T cell candidates consisting of a FOLR1 directed scFv, a CD8 hinge and transmembrane region, a 4-1BB co-stimulatory region, and a CD3( ⁇ domain.
  • Figure 6 Immunofluorescence analysis of human cell lines and tissue revealed potential CAR T cell candidates in scFv-Fc format.
  • Cells were seeded at a concentration of 1E6 (Jurkat) or 2E5 (OV90) cells per 24-well and fixed with 4% PFA before pre-staining with DAPI. Staining of each conjugate was automatically performed, the signal was erased and the sample was washed. A monoclonal antibody directed to the target of interest was used as a positive reference. Cyclic imaging of scFv-Fc-PE conjugates on the cells was performed in a concentration of 5 pg/mL. The scale bar indicates 200 pm.
  • the invention provides a protein having an antigen binding domain specific for FolRl .
  • Specific binding of the target was shown in several different applications.
  • the protein of the present invention may comprise additional structural features and/or domains.
  • the protein having an antigen binding domain specific for FolRl may be e.g. a chimeric antigen receptor, protein drug conjugate (such as antibody drug conjugate), Bispecific T cell engager (BITE) or a detection reagent.
  • BITE Bispecific T cell engager
  • the present invention provides a protein comprising an antigen binding domain (specific for FolRl) comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20) , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No.
  • said light chain variable region of an antibody comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS
  • RECTIFIED SHEET (RULE 91) ISA/EP (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).
  • said protein comprising said antigen binding domain may comprise a heavy chain variable region of an antibody (VH) comprising the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No:35.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • said protein comprising said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80
  • the protein comprises an antigen binding domain.
  • Any molecule that binds specifically to a target antigen may be used as an antigen binding domain.
  • the antigen binding domain may be or may comprise, for example an antibody or antigen binding fragment thereof (e.g. Fab fragments or single chain Fv (scFv) fragments), VHH fragments, divalent single chain antibodies or diabodies.
  • the antigen binding domain may be an antibody or antigen binding fragment thereof.
  • the antigen binding domain may be a scfv.
  • the protein comprising said antigen binding domain may comprise at least one additional domain which may be selected from the group of: cytotoxic moiety, detection moiety and Fc terminus.
  • the protein comprising said antigen binding domain may comprise fragment crystallizable region of an antibody (Fc domain).
  • the protein comprising said antigen binding domain may be a chimeric antigen receptor (CAR) comprising said antigen binding domain specific for FoLRl, a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the invention provides a protein, which is a chimeric antigen receptor (CAR) comprising said antigen binding domain specific for FoLRl, a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • antigen binding domains may be or may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies.
  • the antigen binding domain is a scFv.
  • a linker may be for example the “(G4/S)3-linker” or a “whitlow linker, preferentially a “(G4/S)3-linker”.
  • Said CAR comprises the antigen binding domain (e.g. scfv) as defined by the first aspect of the invention.
  • the protein, of this aspect of the invention is a chimeric antigen receptor (CAR) comprising an antigen binding domain (e.g. scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No.
  • a second complementarity determining region comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).
  • LCDR1 comprising an amino acid sequence NIGSKS
  • LCDR2 comprising the amino acid sequence DDS
  • LCDR3 having the amino acid sequence QVWDSSSDPNYV
  • the protein( which is a CAR) comprises said antigen binding domain (e.g. scfv), wherein said antigen binding domain may comprise a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No.
  • VH heavy chain variable region of an antibody
  • LFR1 first framework region comprising an amino acid sequence of SEQ ID No.
  • LFR2 comprising an amino acid sequence of SEQ ID No. 40
  • LFR3 comprising an amino acid sequence of SEQ ID No. 41
  • LFR4 comprising an amino acid sequence of SEQ ID No. 42.
  • Said protein (which is a CAR) comprises said antigen binding domain (e.g. scfv), wherein said antigen binding domain may comprise a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 35.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • said protein which is a CAR comprises said antigen binding domain (e.g. scfv), wherein said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80.
  • said protein (which is a CAR) may comprise a hinge domain as spacer, wherein the hinge domain may comprise e.g. a sequence of the hinge of CD8alpha (SEQ ID No:2) or IGG4 (SEQ ID No:72).
  • the CAR may comprise the hinge domain of CD8alpha
  • the hinge domain of CD8alpha may comprise SEQ ID NO:2.
  • the protein described in this aspect of the invention, is a chimeric antigen receptor (CAR) comprising an antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No.
  • CAR chimeric antigen receptor
  • HCDR3 comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38) and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.
  • Said protein (which is a CAR) may comprise said antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No.
  • VH heavy chain variable region of an antibody
  • said light chain variable region of an antibody may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42 and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.
  • Said protein (which is a CAR) may comprise said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No:35 and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • CD8alpha preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.
  • said protein which is a CAR and comprises said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80 and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.
  • said protein which is a CAR may comprise a transmembrane domain comprising a sequence of the transmembrane domains from CD8alpha (SEQ ID No:4) or CD28 (SEQ ID No:76), preferentially CD8alpha (SEQ ID No:4).
  • the protein, of this aspect of the invention may comprise an antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No.
  • HCDR1 comprising an amino acid sequence GYSFTSYW
  • HCDR2 second complementarity determining region
  • HCDR3 comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence of SEQ ID No. 37 (DDS) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No.
  • the hinge domain of CD8alpha preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2 and the transmembrane domain of CD8alpha, preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NO:4.
  • Said protein (which is a CAR) may comprise said antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No.
  • VH heavy chain variable region of an antibody
  • said light chain variable region of an antibody may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42
  • LFR1 first framework region
  • LFR2 comprising an amino acid sequence of SEQ ID No. 39
  • LFR2 comprising an amino acid sequence of SEQ ID No. 40
  • LFR3 comprising an amino acid sequence of SEQ ID No. 41
  • LFR4 fourth framework region
  • Said protein (which is a CAR) may comprise said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 35, the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2 and the transmembrane domain of CD8alpha, preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • said protein (which is a CAR) comprises intracellular signaling domains e.g. a sequence of the intracellular signaling domains of CD3zeta (SEQ ID No:8) and/or one or more of CD28 (SEQ ID NO:78), CD137 (41BB; SEQ ID N0:6) and 0X40 (SEQ ID NO:74).
  • the CAR may comprise or consist of a transmembrane domain of CD8alpha, an intracellular primary (stimulatory) signaling domain of CD3 zeta and an intracellular costimulatory signaling domain of CD137 (41BB).
  • the protein, of this aspect of the invention may comprise or consist of an antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No.
  • HCDR3 comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No.
  • the hinge domain of CD8alpha preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2
  • the transmembrane domain of CD8alpha preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON
  • an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.
  • Said protein (which is a CAR) may comprise or consist of said antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No.
  • VH heavy chain variable region of an antibody
  • said light chain variable region of an antibody may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No.
  • the hinge domain of CD8alpha (preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2)
  • the transmembrane domain of CD8alpha preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON
  • an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.
  • Said protein (which is a CAR) may comprise or consist of said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 35 and the hinge domain of CD8alpha (preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2), the transmembrane domain of CD8alpha (preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON) and an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • CD8alpha preferentially the hinge domain of CD8alpha
  • said protein (which is a CAR) may comprise or consist of said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80; the hinge domain of CD8alpha (preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2), the transmembrane domain of CD8alpha (preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON) and an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.
  • said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80
  • the hinge domain of CD8alpha preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2
  • the transmembrane domain of CD8alpha preferentially the transmembrane domain of CD8alpha may comprise SEQ
  • said protein (which is a CAR) may comprise or consist of the amino acid sequence SEQ ID No:44.
  • This aspect of the invention further provides isolated nucleic acid sequences which encode the FolRl-CARs as disclosed herein (especially for this aspect of the invention).
  • the nucleic acid sequence encoding the CAR as disclosed herein may be contained in a vector, such as a viral vector.
  • the vector may be a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, adenoviral vector, or a retrovirus vector, or a combination thereof.
  • said nucleic acid sequence encoding the CAR as disclosed herein may be contained in a lentiviral vector.
  • the vector further comprises a promoter wherein the promoter is an inducible promoter, a tissue specific promoter, a constitutive promoter, a suicide promoter or any combination thereof.
  • the vector encoding the CAR can be further modified to include one or more operative elements to control the expression of CAR T cells, or to eliminate CAR-T cells by virtue of a suicide switch.
  • the suicide switch can include, for example, an apoptosis inducing signaling cascade or a drug that induces cell death.
  • the vector expressing the CAR can be further modified to express an enzyme such thymidine kinase (TK) or cytosine deaminase (CD).
  • This aspect of the invention further provides a composition comprising a lentiviral vector comprising a nucleic acid sequence coding for the FolRl-CAR as disclosed herein (especially for this aspect of the invention).
  • Said composition may be used in immunotherapy.
  • Said composition may be used for treatment of a disease such as cancer, autoimmune disease or infections disease.
  • This aspect of the invention further provides a composition for use in a method of treating a subject in need thereof comprising administering a lentiviral vector comprising a nucleic acid sequence coding for the FolRl-CARs as disclosed herein (especially for this aspect of the invention).
  • This aspect of the invention further provides an engineered cell expressing said protein which is CAR specific for FolRl as disclosed herein (especially in this aspect of the invention).
  • Said cell may be an immune cell.
  • said cell may be a T cell, tumor infiltrating lymphocytes (TILs) or NK cell.
  • TILs tumor infiltrating lymphocytes
  • NK cell In a more preferred embodiment of the invention said cell is a T cell.
  • This aspect of the invention further provides an engineered cell expressing said protein which is a FolRl CAR as disclosed herein (especially in this aspect of the invention) for use in immunotherapy.
  • the immunotherapy may be for treatment of cancer in a subject suffering from cancer, wherein the cancerous cells of said cancer express FolRl.
  • the immunotherapy may be for treatment of cancer in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.
  • Said subpopulation of cancerous cells expressing FolRl may comprise at least one cell which expresses FolRl out of all cancerous cells in the subject suffering from said cancer.
  • Preferentially said subpopulation of cancerous cells expressing FolRl may comprise at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% of all cancerous cells of a subject suffering from said cancer.
  • This aspect of the invention further provides a population of cells comprising cells expressing said protein which is a FolRl -CAR as disclosed herein (especially in this aspect of the invention).
  • Said population or isolated population of engineered cells may be expanded to therapeutically effective amount of cells before use in said immunotherapy.
  • Said cancer may be selected from the group consisting of triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer.
  • Said cancer may be ovarian cancer.
  • Said cells may be immune cells or immune cell subsets, preferentially T cell, tumor infiltrating lymphocytes (TILs) or NK cell, more preferentially T-cells.
  • This aspect of the invention further provides a method for treating cancer comprising administering to a subject in need thereof engineered cells expressing a protein which is a FolRl -CAR as disclosed herein (especially in this aspect of the invention).
  • the treatment of cancer may be in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.
  • This aspect of the invention further provides a composition comprising genetically modified cells expressing a CAR specific for the antigen FoLRl as disclosed herein (especially in this aspect of the invention) for use in treatment of disease.
  • a disease may be selected from the group of cancer, autoimmune disease and infectious diseases.
  • said composition may be used for the treatment of cancer (FolRl expressing cancer).
  • said composition may be used for the treatment of cancer in a subject in need thereof.
  • This aspect of the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising genetically modified cells expressing a CAR specific for the antigen FoLRl as disclosed herein (especially in this aspect of the invention) and optional a pharmaceutically acceptable carrier.
  • Pharmaceutical acceptable carriers, diluents or excipients 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., aluminum hydroxide); and preservatives.
  • This aspect of the invention further provides a composition for use in a method of treating a subject in need thereof comprising administering genetically modified cells expressing a CAR specific for the antigen FoLRl as disclosed herein (especially in this aspect of the invention).
  • the third aspect of the invention provides a composition comprising the protein as disclosed in the first aspect of the invention, wherein said composition comprises said protein which is a tagged protein, and an anti-Tag CAR comprising an antigen binding domain specific for said tag of said tagged protein, a transmembrane domain and an intracellular signaling domain.
  • this aspect of the invention provides a composition comprising the protein as disclosed in the first aspect of the invention, wherein said protein is a tagged protein, and an anti-Tag CAR comprising an antigen binding domain specific for said tag of said tagged protein, a transmembrane domain and an intracellular signaling domain.
  • Said tagged protein comprises an antigen binding domain (specific for FolRl) comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No.
  • a second complementarity determining region comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38)
  • the antigen binding domain of said tagged protein may comprise said heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42.
  • VH heavy chain variable region of an antibody
  • HFR1 comprising an amino acid sequence of SEQ ID No. 23
  • said tagged protein may comprise said antigen binding domain comprising a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • said tagged protein may comprise said antigen binding domain and the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80.
  • the tagged protein may be any protein or protein fragment that specifically binds to a given antigen.
  • Exemplary such proteins may be: an antibody, antigen binding fragment of an antibody, scfv, heterologous antibody (fusion scfv-IGG) VHH fragments, divalent single chain antibodies or diabodies or alike.
  • said tagged protein may be an antibody or an antigen binding fragment.
  • antigen binding domain is fused to an Fragment crystallizable region of an antibody (Fc domain)
  • the tagged protein may comprise a tag.
  • Said tag may be selected from a variety of known tags, proteins and molecules. However, it is essential that the tag can be bound/ recognized by the antigen binding domain of the anti Tag CAR.
  • Said tag may be or may comprise parts of a protein. It may be selected from standard tags commonly known in the art. Exemplary tags are e.g. c-Myc-tag, Strep-Tag II, Flag-Tag, Polyhistidine-tag, Avi-Tag, Calmodulin binding protein-Tag, Yol-tag (derived from alpha-tubulin), E-tag, HA-Tag, S-tag, SBP-tag or V5 tag.
  • other tags may be chosen such as biotin.
  • the composition of this aspect of the invention comprises two parts: the tagged protein and the anti-tag CAR.
  • Said anti-tag CAR comprises an antigen binding domain specific for said tag of said tagged protein, a transmembrane domain and an intracellular signaling domain.
  • the antigen binding domain needs to be specific for the tag of the tagged protein.
  • the antigen binding domain may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. Any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain. Often the antigen binding domain is a scFv.
  • variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv.
  • a linker may be for example the “(G4/S)3-linker” or a “whitlow linker.
  • the anti-tag CAR as disclosed herein may comprise a spacer domain (also known as hinge domain).
  • exemplary spacer domains may be selected from the group of CD8alpha and IGG4
  • the anti-tag CAR may comprise a transmembrane domain, which may be selected from the group of CD28 or CD8alpha.
  • the transmembrane domain may be CD8alpha.
  • the anti-Tag CAR may comprise an intracellular signaling domain e.g. a sequence of the intracellular signaling domains of CD3zeta (SEQ ID No: 8) and/or one or more of CD28 (SEQ ID NO:78), CD137 (41BB; SEQ ID NO:6) and 0X40 (SEQ ID NO:74).
  • the CAR may comprise or consist of a transmembrane domain of CD8alpha, an intracellular primary (stimulatory) signaling domain of CD3 zeta and an intracellular costimulatory signaling domain of CD137 (41BB) and CD28.
  • One aspect of the invention provides a protein, which is a protein drug conjugate (such as an antibody drug conjugate) comprising an antigen binding domain as described in the first aspect of the invention and a cytotoxic payload moiety.
  • the protein, which is a protein drug conjugate comprises an antigen binding domain (specific for FolRl) and a cytotoxic payload moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No.
  • a second complementarity determining region comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38)
  • Said protein (protein drug conjugate) which comprises an antigen binding domain and a cytotoxic payload moiety, wherein said antigen binding domain comprises wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No.
  • LFR2 comprising an amino acid sequence of SEQ ID No. 40
  • LFR3 comprising an amino acid sequence of SEQ ID No. 41
  • LFR4 comprising an amino acid sequence of SEQ ID No. 42.
  • said protein (which is a protein drug conjugate) may comprise said antigen binding domain and said cytotoxic payload moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35.
  • said protein (which is a protein drug conjugate) may comprise said antigen binding domain and said cytotoxic payload moiety, the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80
  • any molecule that binds specifically to FolRl may be used as an antigen binding domain.
  • the antigen may be or may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies.
  • the antigen binding domain may be an antibody or antigen binding fragment.
  • said antigen binding domain (such as an antigen binding fragment of an antibody or an scfv) may be fused to an Fragment crystallizable region of an antibody (Fc domain).
  • Said fusion protein may be generated by methods known in the art. Exemplary said fusion protein may be generated by enzymatic or chemical fusion. In another embodiment said fusion protein may be generated by recombinant protein expression.
  • the cytotoxic payload moiety may be a chemotherapeutic agent (e.g. as described by Fu et al 4 ).
  • Said cytotoxic payload moiety is selected from the group of DNA-alkylating agents, Topoisomerase 1 inhibitors, Topoisomerase 2 inhibitors, Transcription inhibitors, Bcl-xL inhibitors, Tyrosine kinase inhibitors, DHFR inhibitors, Microtubule inhibitors or DNA-targeting agents.
  • the cytotoxic domain may be fused to the protein by methods known in the art such as click chemistry. Exemplary the one of the following methods may be used / exemplary methods are disclosed in Fu et al 4 .
  • Said protein drug conjugate as disclosed in this aspect of the invention may be used for immunotherapy
  • This aspect of the invention further provides a method for treating cancer comprising administering to a subject in need thereof said protein drug conjugate disclosed herein (in this aspect of the invention).
  • the treatment of cancer may be in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.
  • This aspect of the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a protein drug conjugate for the antigen FoLRl as disclosed herein (especially in this aspect of the invention) and optional a pharmaceutically acceptable carrier.
  • Pharmaceutical acceptable carriers, diluents or excipients 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., aluminum hydroxide); and preservatives.
  • Bispecific T cell engager Bispecific T cell engager (BiTE)
  • BITE bispecific T cell engager
  • this aspect of the invention provides a protein comprising a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), wherein said first antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20) , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No.
  • HCDR3 comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38) and wherein said second antigen binding domain is specific for an antigen other than FolRl.
  • LCDR1 comprising an amino acid sequence NIGSKS (SEQ ID No. 36)
  • LCDR2 comprising the amino acid sequence DDS
  • LCDR3 having the amino acid sequence QVWDSSSDPNYV
  • said protein which comprises a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), wherein said antigen binding domain comprises wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No.
  • LFR2 comprising an amino acid sequence of SEQ ID No. 40
  • LFR3 comprising an amino acid sequence of SEQ ID No. 41
  • LFR4 comprising an amino acid sequence of SEQ ID No. 42 and wherein said second antigen binding domain is specific for an antigen other than FolRl.
  • said protein may comprise a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35 and wherein said second antigen binding domain is specific for an antigen other than FolRl.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • said protein may comprise a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80 and wherein said second antigen binding domain is specific for an antigen other than FolRl.
  • any molecule or any variants thereof that bind specifically to a target antigen such as FolRl or to an antigen other than FolRl may be used as an antigen binding domain.
  • the antigen binding domain may be or may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies.
  • the antigen binding domain may be an antibody or antigen binding fragment.
  • said antigen binding domains (such as an antigen binding fragment of an antibody or an scfv) may be fused to an Fragment crystallizable region of an antibody (Fc domain).
  • Said additional antigen binding domain (second antigen binding domain) which is specific for an antigen/target other than FolRl may be CD19, CD20, CD33, CD123, FcRH5, FLT3, BCMA, GPRC5D, PSMA, EGFRvIII, DLL3, MUC17 or CLDN18.2.
  • Both antigen binding domains may be fused by methods known in the Art. Exemplary methods are disclosed in Kujawski et al 5 .
  • Said protein drug conjugate may be used for immunotherapy.
  • This aspect of the invention further provides a method for treating cancer comprising administering to a subject in need thereof said protein drug conjugate as disclosed in this aspect of the invention.
  • the treatment of cancer may be in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.
  • This aspect of the invention additionally provides a pharmaceutical composition comprising protein drug conjugate for the antigen FoLRl as disclosed herein and optional a pharmaceutically acceptable carrier.
  • Pharmaceutical acceptable carriers, diluents or excipients 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., aluminum hydroxide); and preservatives.
  • 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
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • the protein comprising the antigen binding domain may comprise a detection moiety.
  • the protein comprises an antigen binding domain (specific for FolRl) and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20)SEQ , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No.
  • said light chain variable region of an antibody comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).
  • LCDR1 comprising an amino acid sequence NIGSKS (SEQ ID No. 36)
  • LCDR2 comprising the amino acid sequence DDS
  • LCDR3 having the amino acid sequence QVWDSSSDPNYV
  • Said protein which comprises an antigen binding domain and a detection moiety, may comprise an antigen binding domain comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL), wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No.
  • HFR1 comprising an amino acid sequence of SEQ ID No. 23
  • HFR2 comprising an amino acid sequence of SEQ ID No.
  • HFR3 comprising an amino acid sequence of SEQ ID No. 25
  • HFR4 comprising an amino acid sequence of SEQ ID No.
  • said light chain variable region of an antibody comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42
  • said protein may comprise said antigen binding domain and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35.
  • VH heavy chain variable region of an antibody
  • VL light chain variable region of an antibody
  • the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80
  • the protein comprises an antigen binding domain and a detection moiety.
  • Said detection moiety may be a fluorescent moiety.
  • Said detection moiety may be selected from the group of fluorescent dyes, fluorescent proteins or biotin.
  • the protein comprising the antigen binding domain may be fused to an Fc terminus by standard methods known in the art. This Fc terminus may serve as detection moiety.
  • fluorescent dyes are small organic molecule dyes, such as xanthene dyes, like fluorescein, or rhodamine dyes, coumarine dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyl oxazole dyes, pyromethene dyes, acridine dyes, oxadiazole dyes, carbopyronine dyes, benzpyrylium dyes, fluorene dyes, or metallo-organic complexes, such as Ru, Eu, Pt complexes.
  • xanthene dyes like fluorescein, or rhodamine dyes
  • coumarine dyes such as xanthene dyes, like fluorescein, or rhodamine dyes, coumarine dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyl oxazole dyes,
  • fluorescent moi eties might be protein-based, such as phycobiliproteins, nanoparticles, such as quantum dots, upconverting nanoparticles, gold nanoparticles, dyed polymer nanoparticles.
  • fluorescent proteins may be used such as GFP, RFP, YFP and variants thereof.
  • Said detection moiety may be directly covalently coupled or coupled via a linker unit.
  • Any molecule that binds specifically to a target antigen may be used as an antigen binding domain.
  • the antigen may be or may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies.
  • the antigen binding domain may be an antibody or antigen binding fragment.
  • said antigen binding domains (such as an antigen binding fragment of an antibody or an scfv) may be fused to an Fragment crystallizable region of an antibody (Fc domain).
  • Said protein as disclosed in this aspect of the invention comprising said antigen binding domain and a detection moiety, may be used in a method for the detection of cells (preferentially cancer cells) expressing FolRl (figure 6).
  • Said method of detecting FolRl expressing cells (especially cancer cells) in a biological sample may comprise the steps:
  • this aspect of the invention provides a method for detecting FolRl expressing cells (especially cancer cells) in a biological sample, comprising the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No.
  • HCDR1 first complementarity determining region
  • a second complementarity determining region comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).
  • LCDR1 comprising an amino acid sequence NIGSKS
  • LCDR2 comprising the amino acid sequence DDS
  • LCDR3 having the amino acid sequence QVWDSSSDPNYV
  • Said method for detecting FolRl expressing cells (especially cancer cells) in a biological sample may comprise the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, wherein said antigen binding domain comprises wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No.
  • VH heavy chain variable region of an antibody
  • said light chain variable region of an antibody comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42 and a detection moiety;
  • LFR1 first framework region
  • LFR2 comprising an amino acid sequence of SEQ ID No. 40
  • LFR3 comprising an amino acid sequence of SEQ ID No. 41
  • a fourth framework region comprising an amino acid sequence of SEQ ID No. 42 and a detection moiety
  • the invention provides a method detecting FolRl expressing cells (especially cancer cells) in a biological sample, comprising the steps: a) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, said protein, comprising said antigen binding domain and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35; b) Detecting said protein bound to said FolRl expressing cells
  • the invention provides a method for detecting FolRl expressing cells (especially cancer cells) in a biological sample, comprising the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, wherein said, the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80; B) Detecting said protein bound to said FolRl expressing cells.
  • said antigen binding domain is fused to an Fragment crystallizable region of an antibody (Fc domain) and said detection moiety is a fluorescent moiety or biotin, more preferentially a fluorescent moiety.
  • Said method may be a method for immunofluorescence or flow cytometric analysis. Specific protocols and methods are known in the art as disclosed in Kinkhabwala et al 6 and McKinnon 7 .
  • a sample comprising FolRl expressing cells is contacted with said protein comprising said antigen binding domain and a detection moiety.
  • Detection may be done directly, if the detection moiety is e.g. a fluorescent moiety or indirectly using e.g. if the detection moiety is a biotin moiety .
  • one additional protein e.g. an antibody comprising a fluorescent detection moiety and which is specific for biotin may be used.
  • a sample which may be used for the method disclosed in this aspect of the invention may be a tissue sample.
  • This tissue sample may be human.
  • the sample may be or may be derived from a cancer patient. Therefore the sample may comprise folate receptor 1 expressing cancer cells.
  • the sample may be a sample of: of triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer. Said sample may be a sample of ovarian cancer.
  • the sample may be a tissue slice (e.g. for imaging applications) or the sample may be present in a dissociated form e.g. as single cell solution (e.g. for detection methods based on flow cytometry).
  • tissue slice may be 5-20pm preferentially 8pm.
  • the tissue may be fixed according to methods known in the art. Standard fixation methods are based on formaldehyde, formalin or acetone.
  • Step b) may be done using a standard fluorescent microscope.
  • standard instruments such as the MACS Quant analyzer may be used.
  • FACS fluorescence activated cell sorting
  • This aspect of the invention further provides a method for detecting FolRl expressing cancer cells and subsequent tumor treatment.
  • the method may comprise the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein according to this aspect of the invention, wherein said protein comprises a detection moiety; B) detecting said protein bound to said FolRl expressing cells and D) Administering to a subject a therapeutically effective amount of a protein such as a CAR, BITE or a protein drug conjugate as disclosed herein.
  • This aspect of the invention further provides a method for detecting FolRl expressing cancer cells and prognosing the probability of a subject to respond to said proteins as disclosed herein.
  • the method may comprise the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein according to this aspect of the invention, wherein said protein comprises a detection moiety; B) Detecting said protein bound to said FolRl expressing cells, Step C) assigning a score to the detection step (B), wherein the score is assigned based on comparison to one or more reference samples.
  • Step D) Comparing the score in step (C) to the score of one or more reference samples wherein a score for the cancer FOLR1 level that is greater than the score for a normal or low FOLR1 expressing reference sample or a score for the cancer FOLR1 level that is equal to or greater than the score for a high FOLR1 expressing reference sample identifies the cancer as likely to respond to the immunotherapeutic treatment based on a CAR, BITE or a protein drug conjugate as disclosed herein.
  • Data obtained from step (b) may be used for scoring and for determining, whether an immunotherapy using this specific conjugate would be successful for treatment. Such a score may be based on the amount and frequency of cells detected as well as the expression profiles and staining intensities.
  • the cancer sample comprises cancer cells.
  • the individual from which the sample was obtained is likely to respond to an immunotherapy based on a CAR, BITE or a protein drug conjugate as disclosed herein.
  • Reference sample may be standard tissue such as Non-neoplastic tissue, e.g. ovary, kidney, lung, cerebellum.
  • folate receptor 1 FolRl, FOLR, FOLR1, and FRl can be used interchangeably. It is understood that this also includes other state of the art synonyms such as folate receptor alpha FRalpha or FRa. Folate-binding protein, FBP or Neurodegeneration due to cerebral folate transport deficiency, NCFTD.
  • An antigen binding domain refers to a region of a protein (generally the variable region of an antibody or fragment thereof) that specifically binds to an antigen. It is composed of one constant and one variable domain of each of the heavy and the light chain. Key elements and most important for specific binding to an antigen are the complementarity-determining regions (CDRs) and framework region (FR) regions.
  • CDRs complementarity-determining regions
  • FR framework region
  • CDRs Complementarity-determining regions
  • a framework region (FR) is a subdivision of the variable region of the antibody.
  • the framework regions are responsible for acting as a scaffold for the complementarity determining regions (CDR).
  • CARs Chimeric antigen receptors
  • T lymphocytes and/or other immune cells are recombinant receptors for antigens which redirect the specificity and function of T lymphocytes and/or other immune cells in a single molecule.
  • the concept of using CARs in immunotherapy is that CARs, which are programmed targeting tumor-associated antigens or disease associated antigens, can be replicated rapidly and homogeneously.
  • a CAR may comprise an extracellular domain (extracellular part) comprising an antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (intracellular signaling domain).
  • the extracellular domain may be linked to the transmembrane domain by a linker or spacer.
  • the extracellular domain may also comprise a signal peptide.
  • signal peptide refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface.
  • an antigen binding domain of a CAR refers to the region that specifically binds to an antigen, e.g. to a tumor associated antigen (TAA), tumor specific antigen (TSA) or a disease associated antigen.
  • the CARs of the invention may comprise one or more antigen binding domains (e.g. a tandem CAR). Generally, the targeting regions of the CAR are localized extracellularly.
  • the antigen binding domain may comprise an antibody, single domain antibody or an antigen binding fragment thereof.
  • the antigen binding domain may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. Often the antigen binding domain is a scFv.
  • variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv.
  • a linker may be for example the “(G4/S)3 -linker” or a “whitlow linker”.
  • Spacer refers to the hydrophilic region which is between the antigen binding domain of the CAR and the transmembrane domain.
  • the spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof.
  • a prominent example of a spacer is the CD8alpha hinge or IgG4 hinge.
  • the “transmembrane domain” of the CAR may be from any desired natural or synthetic source for such domain. When the source is natural the domain may be from any membranebound or transmembrane protein. The transmembrane domain may be for example from CD8alpha or CD28.
  • the cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR).
  • "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.
  • the intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function.
  • the intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions.
  • Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and coreceptors that initiate signal transduction following antigen receptor engagement.
  • T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co- stimulatory signaling domain).
  • primary cytoplasmic signaling sequences primary cytoplasmic signaling domain
  • secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal secondary cytoplasmic signaling sequences, co- stimulatory signaling domain.
  • an intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and/or one or more secondary cytoplasmic signaling domains.
  • Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs).
  • ITAMs immunoglobulin-based activation motifs
  • Examples of ITAM containing primary cytoplasmic signaling domains often used in CARs are that those from TCRzeta (CD3zeta), FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence from CD3zeta.
  • the cytoplasmic domain of the CAR may be designed to comprise the CD3zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s).
  • the cytoplasmic domain of the CAR can comprise a CD3zeta chain portion and a co-stimulatory signaling region (domain).
  • the co-stimulatory signaling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule.
  • a co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
  • Examples for a co-stimulatory molecule are CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3.
  • LFA- 1 lymphocyte function-associated antigen- 1
  • the cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order.
  • a short oligo- or polypeptide linker which is preferably between 2 and 10 amino acids in length, may form the linkage.
  • a prominent linker is the glycine-serine doublet.
  • the antigen binding domain of a CAR binds a tag or hapten that is coupled to a protein or polypeptide (“haptenylated” or “tagged” polypeptide/ Protein), wherein the polypeptide may bind to a disease-associated antigen such as a tumor associated antigen (TAA) that may be expressed on the surface of a cancer cell, herein FolRl.
  • TAA tumor associated antigen
  • a CAR may be referred to as “anti -tag” CAR or “adapterCAR” or “universal CAR” as disclosed e.g. in US9233125B2.
  • the haptens or tags may be coupled directly or indirectly to a polypeptide (the tagged polypeptide), wherein the polypeptide may bind to said disease associated antigen expressed on the (cell) surface of a target, e.g. FolRl.
  • the tag may be e.g. dextran or a hapten such as biotin or fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or thiamin, but the tag may also be a peptide sequence e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide.
  • the tag may also be streptavidin.
  • the tag portion of the tagged polypeptide is only constrained by being a molecule that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR.
  • the tag when the tag is FITC (Fluorescein isothiocyanate), the tag-binding domain may constitute an anti-FITC scFv.
  • the tag when the tag is biotin or PE (phycoerythrin), the tag-binding domain may constitute an anti-biotin scFv or an anti-PE scFv, respectively.
  • tagged protein or “tagged polypeptide” as used herein refers to a polypeptide that has bound thereto directly or indirectly at least one additional component, i.e. the tag.
  • the tagged polypeptide as used herein is able to bind an antigen expressed on a target cell.
  • the polypeptide may be an antibody or antigen binding fragment thereof that binds to an antigen expressed on the surface of a target cell such as a tumor associated antigen (TAA) on a cancer cell.
  • TAA tumor associated antigen
  • the polypeptide of the tagged polypeptide alternatively may be a cytokine or a growth factor or another soluble polypeptide that is capable of binding to an antigen of a target cell.
  • the terms “adapter” or “adapter molecule” or “tagged polypeptide” as used herein may be used interchangeably.
  • the tag may be e.g. a hapten or dextran and the hapten or dextran may be bound by the antigen binding domain a CAR comprising an antigen binding domain specific for the tag.
  • Haptens such as e.g. FITC, biotin, PE, thiamin, streptavidin or dextran are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.
  • the tag may also be a peptide sequence e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide.
  • the peptide may be selected from the group consisting of c-Myc-tag, Strep-Tag, Flag-Tag, and Polyhistidine-tag.
  • the tag may also be streptavidin.
  • the tag portion of the tagged polypeptide is only constrained by being a molecular that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR.
  • the tag is FITC (Fluorescein isothiocyanate)
  • the tagbinding domain may constitute an anti-FITC scFv.
  • the tag-binding domain may constitute an anti-biotin scFv or an anti-PE scFv.
  • engineered can refer to one or more human-designed alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome.
  • An “engineered cell” or “genetically modified cell” can refer to a cell with an added, deleted and/or altered gene.
  • the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state.
  • T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.
  • the engineered cell expressing a CAR may be further modified by genetic engineering using methods well known in the art e.g. Meganucleases, TALEN, CrisprCas, zink finger nucleases, shRNA and /or miRNA.
  • Said cells may be modified to reduce or lack expression of a specific gene, which is normally expressed in the cell e.g. T cell receptor (TCR), MHC, co- inhibitory molecules like PD-1, CTLA-4, BTLA, TIGIT, Tim-3, CD244, LAIR, Lag-3, CD160, HVEM.
  • Said cells may be modified to express additional transgenes such as therapeutic controls, cytokines and/or fragments, cytokine receptors and/or fragments, cytokine receptor fusion proteins, costimulatory receptors or armoring molecules.
  • immune cell or “immune effector cell” may be used interchangeably and refer to a cell that may be part of the immune system and executes a particular effector function such as T cells, alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, monocytes or macrophages.
  • ILC innate lymphoid cells
  • CIK cytokine induced killer
  • LAK lymphokine activated killer
  • gamma-delta T cells monocytes or macrophages.
  • monocytes or macrophages gamma-delta T cells
  • Preferred immune cells are cells with cytotoxic effector function such as T cells, alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells, macrophages or gamma-delta T cells. Most preferred immune effector cells are T cells and NK cells.
  • Effective function means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.
  • antibody as used herein is used in the broadest sense to cover the various forms of antibody structures including but not being limited to monoclonal and polyclonal antibodies (including full length antibodies), single domain antibodies, multispecific antibodies (e.g. bispecific antibodies), antibody fragments, i.e. antigen binding fragments of an antibody, immunoadhesins and antibody-immunoadhesin chimeras, that specifically recognize (i.e. bind) an antigen.
  • Antigen binding fragments comprise a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof (“an antigen binding fragment of an antibody”).
  • antigen binding fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), VHH fragments, diabodies, dsFv, Fab’, diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • the terms “having specificity for”, “specifically binds” or “specific for” with respect to an antigen-binding domain of an antibody, of a fragment thereof or of a CAR refer to an antigenbinding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific.
  • An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.
  • the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, haptens and combinations thereof, for example a glycosylated protein or a glycolipid.
  • antigen refers to a molecular entity that may be expressed on the surface of a target cell and that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.
  • epitope means the part of an antigen, e.g. a soluble antigen, that may be recognized and specifically bound by antibodies or antigen bindings fragments thereof (antigen binding domains).
  • the tumor associated antigen refers to an antigenic substance produced by tumor cells. Tumor associated antigens are useful tumor or cancer markers in identifying tumor/cancer cells with diagnostic tests and are potential candidates for use in cancer therapy. Preferentially, the TAA may be expressed on the cell surface of the tumor/cancer cell, so that it may be recognized by the antigen binding receptor as disclosed herein.
  • target cell refers to cell which expresses an antigen on its cell surface that should be recognized (bound) by the protein according to the current invention.
  • Said target cell may be e.g. a cancerous cell or a cell associated with an autoimmune disease or a cell associated with an infectious disease
  • Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies.
  • treatment means to reduce the frequency or severity of at least one sign or symptom of a disease.
  • terapéuticaally effective amount or “therapeutically effective population” mean an amount of a cell population which provides a therapeutic benefit in a subject.
  • the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the individual is a human. The subject may be a subject suffering from a disease such as cancer.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter in a cell.
  • isolated is used herein to indicate that the polypeptide, nucleic acid or host cell exist in a physical milieu distinct from that in which it occurs in nature.
  • the isolated polypeptide may be substantially isolated (for example enriched or purified) with respect to the complex cellular milieu in which it naturally occurs, such as in a crude extract.
  • cancer is known medically as a malignant neoplasm. Cancer is a broad group of diseases involving unregulated cell growth and includes all kinds of leukemia. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans.
  • ADC Antibody-drug conjugate
  • a drug such as a cytotoxic agent.
  • ADCs can be used to specifically target a drug to cancer cells through specific binding of the antibody to a tumor antigen expressed on the cell surface.
  • Exemplary drugs for use with ADCs include of DNA-alkylating agents, Topoisomerase 1 inhibitors, Topoisomerase 2 inhibitors, Transcription inhibitors, Bcl-xL inhibitors, Tyrosine kinase inhibitors, DHFR inhibitors, Microtubule inhibitors or DNA-targeting agents.
  • Bispecific T cell engager or BiTE is an antigen binding molecule. It comprises a single chain polypeptide and can bind to two different antigens.
  • Autoimmune diseases are a condition arising from autoimmunity or disbalance in the immune homeostasis resulting in pathologies that can affect multiple different organ systems.
  • Behcet’s disease Juvenile idiopathic arthritis, Type 1 diabetes, Rheumatoid arthritis, Wegener Granulomatosis, Systemic lupus erythematosus, Systemic sclerosis, Crohn's disease, Graves' disease, Hashimoto thyroiditis, Goodpasture syndrome, Primary biliary cholangitis, Myasthenia gravis, Dermato polymyositis, Vasculitis, Mixed connective tissue disease, Scleroderma, Multiple sclerosis, Psoriasis, Ulcerative colitis and Uvetis.
  • Infection infectious disease is the invasion of an organism's body tissues by diseasecausing agents, their multiplication, and the reaction of host tissues to the infectious agents and the toxins they produce. Infections are caused by infectious agents (pathogens) including: viruses, bacteria, fungi and parasites. Said infection may be an acute or a chronic infection.
  • administering refers to local and systemic administration, e.g., including enteral, parenteral, pulmonary, and topical/transdermal administration.
  • the administration may be directly intratumoral.
  • Routes of administration for pharmaceutical ingredients include, e.g., oral administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery, intrathecal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, or subcutaneous administration to a subject.
  • Administration can be by any route including parenteral and transmucosal (e.g, oral, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic and intracranial.
  • the anti-FOLRl CAR T candidate selection process consisted of a an initial phage display selection, followed by a flow cytometric screening, to ensure specificity of the candidates to FOLR1 compared to other FOLR variants. Following, a CAR T screening approach and advanced in vitro co-culture assays allowed to identify a novel, functional and specific anti-FOLRl CAR candidate (Fig 1).
  • Example 1 Identification of antibody fragments
  • Two human naive scFv phage display libraries constructed at Miltenyi Biotec were used for the identification of novel binders against FOLR1. These libraries differed in the variable domains of the light chain, they contained either the K or the X isotype.
  • mouse FOLR1 mFOLRl
  • human FOLRl-hlgGlFc hFOLRl-Fc
  • human FOLR2 hFOLR2
  • Human FOLR3 was purchased from R&D systems
  • human IgGl-Fc (hlgGlFc) was purchased from amsbio.
  • hFOLR2 Five different panning strategies were used to identify binder candidates using hFOLRl and mFOLRl antigen for positive selection, respectively.
  • hFOLR2, hFOLR3, and hlgGl-Fc antigens were used for competition (Fig. 2 A).
  • each library was incubated on antigens immobilized on Maxi Sorb ELISA Plates (Nunc). After washing, scFv phage particles were eluted using trypsin. Eluted phage particles were used to infect E. coli TGI and phage production was facilitated using M13K07 helper phage. After the third panning round eluted phage particles were used to infect A. coli TGI and single cell colonies were inoculated the next day for expression of scFvs.
  • scFvs were analyzed for their capability to bind hFOLRl-Fc, mFOLRl, hFOLR2 or hlgGlFc in ELISA. This lead to the identification of 2266 first hits that bind either hFOLRl and/or mFOLRl and do not bind hFOLR2 or hlgGlFc (Fig. 2B). Separating the ELISA hits by panning strategy it became obvious, that binder selected in strategies without mFOLRl in the selection process indeed recognized preferably hFOLRl over mFOLRl. On the other hand panning strategy 3 with selection on mFOLRl revealed binder binding preferably to mFOLRl over hFOLRl as expected. Panning strategies using both hFOLRl and mFOLRl lead to the selection of scFvs that are cross-reactive against both targets (Fig. 2C).
  • scFv genes were sequenced for 817 clones. Alignment of the scFv genes revealed that 349 out of 817 clones contain unique scFv sequences. From these unique sequences, 189 clones were cloned as scFv-Fc fusion proteins into mammalian expression vectors (Fig. 2D). After expression and purification, 94 of these scFv-Fc proteins were used for analysis by flow cytometry.
  • [00182]Potential anti-FOLRl scFv-Fc candidates were manufactured via transient transfection of HEK293 cells. On the day of transfection, HEK293 cells were seeded at a density of 2xl0 6 cells per well in a total volume of 800 pl expression medium in a 96 deep well plate. Next, transfection reagent was mixed with diluted plasmid DNA, encoding for the respective anti- FOLRl scFv-Fc candidate and added to the cells after an incubation period of 10 minutes. Cells were incubated at 37°C and 8% CO2 on an orbital shaker.
  • Example 3 Flow cytometric screening of anti-FOLRl scFv-Fc candidates
  • Anti-FOLRl scFv-Fc candidates were manufactured as described in example 2 and protein concentrations were normalized.
  • non-target cells were labeled with CellTraceTM Violet or Cell TraceTM FarRed (ThermoFisher) and IxlO 5 cells per cell line were seeded into a 96-well plate.
  • Cells were pelleted at 1200 g for 2 min at 4°C and stained with 50 pL staining mixture containing 1 pg scFv-Fc.
  • Staining was performed for 30 min at 4°C followed by two successive washing steps with 150 pL PBS / 2 mM EDTA / 0.5% BSA (referred to as PEB, 1200 g, 2 min).
  • PEB 150 pL PBS / 2 mM EDTA / 0.5% BSA
  • 50 pl of secondary staining mixture comprising the AffinitPure F(ab')2 Fragment Goat Anti-Human IgG-PE (Jackson ImmunoResearch) as well as 7-AAD as dead cell marker (Miltenyi Biotec) was added to the cells.
  • Staining was performed for 10 min at 4°C followed by two successive washing steps with 150 pL PEB.
  • Example 4 Identification of chimeric antigen receptors (CARs) for FOLR1 -expressing target cell recognition by CAR-transgenic T cells.
  • CARs chimeric antigen receptors
  • scFv Human naive single chain variable fragment
  • scFv Human naive single chain variable fragment
  • the scFv contained a (648)3 linker domain (SEQ ID 9, 10).
  • Lentiviral vectors contained a truncated human low-affinity nerve growth factor receptor (ALNGFR) expression cassette separated by a 2A element for co-expression as transduction marker.
  • ANGFR truncated human low-affinity nerve growth factor receptor
  • Lentiviral particles were used to transduce primary human T cells of three independent donors for CAR expression. Frequency and absolute numbers of CAR T cells were determined by quantification of CD3 + LNGFR + events using flow cytometry.
  • CAR T cells were cultured at an effector-to-target ratio of 2.5 to 1 with FOLR1 -expressing or FOLR1 KO OV-90 cells, which were transduced to express GFP.
  • CAR T cells were cultured in absence of target cells, and OV-90 or OV-90 FOLR1 KO cells were cultured in absence of CAR T cells. Additionally, OV-90 and OV-90 FOLR1 KO cells were cultured with untransduced T cells or with T cells expressing one of two positive control CARs, respectively.
  • CAR T cells CD3 + LNGFR + .
  • the total duration of three rounds of co-culture was 11 days, and cytotoxicity towards OV-90 and OV-90 FOLR1 KO cells, respectively, was quantified every 2h by acquisition of the GFP signal over time by measuring the Green Calibrated Units per pm 2 /image.
  • candidates FOLR1 CAR 6, 9, 10, 11, 13, 15, 17, 20, 21 were either least or not functional after three rounds of co-culture from at least one donor.
  • Co-cultures of CAR T cells with OV-90 FOLR1 KO cells allowed to identify CAR T candidates, which mediated cytotoxicity in the first and up to the third round of co-culture (Fig. 4D-F), i.e. antigen-independent cytolysis.
  • Candidates FOLR1 CAR 16 lysed tumor cells unspecifically in the absence of FOLR1.
  • CAR T cell expansion was observed for four CAR constructs, i.e. FOLR1 CAR 5, 7, 12, 18, for all three donors in co-cultures with OV-90 cells at the end of the first and the third round, i.e. numbers were exceeding the initial amount ofCD3+LNGFR+T cells plated at day 0 (Fig. 4 G-I). This was also true for T cells expressing the CAR, i.e. FOLR1 CAR 16 fate which mediated cytotoxicity towards OV-90 FOLR1 KO cells.
  • Proliferation was not measurable in at least one donor for candidates FOLR1 CAR 1, 2, 3, 4, 6, 9, 10, 11, 13, 14, 15, 17, 20, and 21 (SEQ ID NOs: 81, 82, 83, 84, 86, 89, 90, 91, 92, 93, 94, 96, 99 and 100).
  • Example 5 Generation and identification of a lead chimeric antigen receptor (CAR) for FOLR1 -expressing target cell recognition.
  • CAR lead chimeric antigen receptor
  • CAR constructs were generated lacking the LNGFR reporter by cloning human naive FOLR1 directed scFvs into lentiviral vector constructs together with a CD8alpha hinge (SEQ ID 1,2), a CD8alpha transmembrane domain (SEQ ID 3,4), a human 41BB costimulatory domain (SEQ ID 5,6) and a human CD3 zeta domain linker (SEQ SEQ ID 7,8) (Fig. 5A).
  • scFv Heavy and light chain of the scFv were linked via a (G4S)s linker (SEQ ID 9,10).
  • Lentiviral particles were produced via transfection of HEK293T cells and were harvested from cell culture supernatants.
  • SUP-T1 cells were transduced with varying amounts of lentiviral vector and transduction efficiency was determined via flow cytometry by detection of the CAR using biotinylated FOLRl-Fc protein. All four candidate CARs were expressed at high and comparable levels.
  • CAR T cells of three different donors were co-cultured with repeatedly added GFP-expressing target cells (OV-90 wt) or non-target cells (OV-90 FOLR1 KO) at an initial effector-to-target ratio of 0.5 to 1.
  • OV-90 cells were cultured without T cells as well as with untransduced T cells.
  • positive control CAR T cells were co-cultured with OV-90 cells, known to specifically and efficiently induce lysis of FOLR1 -expressing cells. After 48h, fresh OV-90 cells were added to the co-culture. OV-90 cell lysis causes a decrease in GFP signal, whereas inefficient or no lysis increases GFP signal over time.
  • OV-90 cell lysis was measured for 120h and is displayed as green area confluency [%]. All CAR T candidates from all three donors efficiently eradicate OV-90 cells expressing FOLR1 after two additions of OV-90 cells, slightly outperforming the positive control (Fig. 5B-D). Additionally, all CAR T candidates from all three donors do not eradicate OV-90 FOLR1 KO cells (Fig. 5E-G).
  • CD69 expression of FOLR1 CAR 18 and FOLR1 CAR 7 is elevated compared to FOLR1 CAR 5, FOLR1 CAR 12 and the positive control CAR T cells after co-culture with OV-90 FOLR1 KO cells (Fig. 5 H-J).
  • anti-FOLRl CAR T candidates were co-cultured with FOLRl low (OVCAR-3) expressing cells and compared to OV-90 wt (FOLRl hlgh ) and OV-90 FOLR1 KO cells.
  • CD69 and CD137 activation marker expression of CAR T candidates was increased after 48h of co-culture with OV-90 cells expressing FOLR1, while minimal activation of all CAR T candidates was observed after co-culture with OV-90 FOLR1 KO cells.
  • OVCAR-3 cells only displayed 15% FOLR1 expression
  • the positive control CAR T cells and FOLR1 CAR 12 show higher expression of the activation markers CD69 and CD137, compared to untransduced T cells and other candidates (Fig. 5N, O).
  • FOLR1 CAR 12 was activated by FOLRl low expressing target cells and showed superior activation after co-culture with FOLRl hlgh expressing cells compared to other candidates, FOLR1 CAR 12 was nominated as lead candidate (SEQ ID 11-44).
  • Example 6 Immunofluorescence detection of FOLR1 expression in human cell lines and tissue using anti-FOLRl scFv-Fc candidates.
  • the four lead CAR T candidates (FOLR1 CAR5, FOLR1 CAR7, FOLR1 CAR12 and FOLR1 CAR18) that were identified in the previous CAR T screening approach were further characterized for their capability to detect FOLR1 expressing cells via immunofluorescence analysis.
  • the scFv-Fc fusion proteins as described in example 3 were used.
  • the candidates were directly labeled with a fluorescent dye namely PE.
  • OV-90 cells expressing FOLR1 were analyzed with the PE-labeled scFv-Fc fusion proteins (Fig. 6E).
  • specificity of the anti-FOLRl scFv-Fc fusion proteins was assessed and detect elevated signals of the candidates relative to the negative control antibody (Fig. 6F).
  • OV-90 cells were modified via CRISPR/CAS9 to ablate endogenous FOLR1 expression.
  • these OV-90 FOLR1 KO cells were analyzed with the PE-labeled scFv-Fc fusion proteins (Fig. 6G).
  • specificity of the anti-FOLRl scFv-Fc fusion proteins was assessed and detect no significant signal of the candidates relative to the positive and negative control antibodies.
  • the signal of the scFv-Fc candidates co-localizes with the positive control.
  • SEQ ID NO 1 Human CD8 alpha hinge nucleotide sequence
  • SEQ ID NO 2 Human CD8 alpha hinge amino acid sequence
  • SEQ ID NO 3 Human CD8alpha transmembrane domain nucleotide sequence
  • SEQ ID NO 4 Human CD8alpha transmembrane domain amino acid sequence
  • SEQ ID NO 5 Human 4 IBB costimulatory domain nucleotide sequence
  • SEQ ID NO 6 Human 41BB costimulatory domain amino acid sequence
  • SEQ ID NO 7 Human CD3 zeta domain nucleotide sequence
  • SEQ ID NO 8 Human CD3 zeta domain amino acid sequence
  • SEQ ID NO 10 (G4S)3 linker amino acid sequence
  • SEQ ID NO 11 FOLR1 CAR 12 scFv Vh nucleotide sequence
  • SEQ ID NO 12 FOLR1 CAR 12 scFv Vh CDR1 nucleotide sequence
  • SEQ ID NO: 56 recombinant hFOLR3 amino acid sequence including 3x HA tag and hFOLRl GPI-anchor domain

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Abstract

L'invention concerne une protéine ayant un domaine de liaison à l'antigène spécifique du FolRl. La liaison spécifique de la cible a été représentée dans plusieurs applications différentes. Selon l'application, la protéine de la présente invention peut comprendre des caractéristiques et/ou domaines structurels supplémentaires. En fonction des caractéristiques structurales, la protéine ayant un domaine de liaison à l'antigène spécifique du FolRl peut être, par exemple, un récepteur antigénique chimérique, un conjugué protéine-médicament, un anticorps bispécifique engageant les cellules T (BITE) ou un réactif de détection.
PCT/EP2024/079876 2023-10-26 2024-10-23 Récepteur antigénique chimérique spécifique du récepteur 1 du folate Pending WO2025087935A1 (fr)

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