WO2025224123A1 - Chimeric antigen receptors specific for fibroblast activation protein - Google Patents

Abstract

The present dislosure provides a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO:19 (VH) and SEQ ID NO:20 (VL), or SEQ ID NO: 13 (VH) and SEQ ID NO:14 (VL), b) a transmembrane domain, and c) an intracellular signaling domain.

Description

Title

Chimeric antigen receptors specific for fibroblast activation protein

Field of the invention

The present invention generally relates to the field of immunotherapy using immune cells expressing a chimeric antigen receptor, in particular to the field of immunotherapy using immune cells expressing a chimeric antigen receptor specific for the antigen fibroblast activation protein (FAP).

Background of the invention

The use of chimeric antigen receptor (CAR)-expressing immune cells such as T cells re-directed to specifically recognize and eliminate target cells such as malignant cells, greatly increased the scope and potential of adoptive immunotherapy and is being assessed for new standard of care in certain human disorders such as malignancies. CARs are recombinant receptors that typically target surface molecules in a human leukocyte antigen (HLA)-independent manner. Generally, CARs comprise an extracellular antigen recognition moiety, often a single-chain variable fragment (scFv) derived from antibodies or a Fab fragment, linked to an extracellular spacer, a transmembrane domain and intracellular co-stimulatory and signaling domains.

Targeting fibroblast population in solid tumors and fibrotic diseases hold immense potential in developing effective therapeutic solutions. In solid cancer, the cancer associated fibroblasts (CAF) are a major part of the tumor microenvironment (TME) that interferes with T cell activity. The TME is composed of several cell types including fibroblasts, stellate cells, endothelial cells, adipocytes, immune cells, and the extracellular matrix (ECM). The interaction of cancer cells and these surrounding stromal partners modify the microenvironment in a way that supports their proliferation, growth, survival, and metastatic properties.

In the setting of solid tumors, different subtypes of CAFs have been proposed to have disparate effects on tumor establishment, growth and progression, as well as in metastatic capacity (Lo, A. et al. Cancer Res. 75, 2800-2810 (2015); Ozdemir, B. C. et al. Cancer Cell 25, 719-734 (2014)). Therefore, when choosing a CAR-targeted protein, it is important to consider which fibroblast cell subpopulation is going to be depleted (Gascard, P. & Tlsty, T. D. Genes Dev. 30, 1002-1019 (2016)). With this thought in mind, fibroblast activation protein (FAP) has been proposed as a potentially good target. FAP is a surface peptidase that also has gelatinase activity and is widely expressed in a subset of protumoral fibroblasts in many cancer types (Loktev, A. et al. J Nucl Med 59, 1423-1429 (2018); Park, J. E. et al. J. Biol. Chem. 274, 36505-36512 (1999); Scott, A. M. et al. Clin. Cancer Res. 9, 1639-1647 (2003)). FAP expression in pancreatic cancer (Cohen, S. J. et al. Pancreas 37, 154-158 (2008); Lo, A. et al. JCI Insight 2, (2017)) and non-small cell lung cancer (Liao, Y., Ni, Y., He, R., Liu, W. & Du, J. J. Cancer Res. Clin. Oncol. 139, 1523-1528 (2013)) is associated with worse clinical outcome. Depletion of FAP+ cells using genetic depletion strategies appeared to enhance T cell mediated antitumor activity in preclinical models of melanoma and pancreatic ductal adenocarcinoma (Feig, C. et al. Proc. Natl. Acad. Sci. U. S. A. 110, 20212-20217 (2013); Kraman, M. et al. Science 330, 827-830 (2010); Zhang, Y. & Ertl, H. C. J. Oncotarget 7, (2016)).

In addition to solid tumor FAP is an attractive target to treat fibrosis, e.g. cardiac fibrosis. Cardiovascular diseases are the foremost cause for mortality worldwide (Tsao, C. W. et al. Circulation 145, el53-e639 (2022)). Most cardiovascular diseases are accompanied by cardiac fibrosis, i.e. the chronic or acute disease-induced excessive ECM deposition by cardiac fibroblasts (CF) (de Boer, R. A. et al. Eur. J. Heart Fail. 21, 272-285 (2019)). Depending on the underlying disorder, CF and ECM replace whole areas of cardiomyocytes (replacement fibrosis), are diffusely distributed between cardiomyocytes (interstitial fibrosis), or are located around vessels (perivascular fibrosis). The so formed scar tissue is detrimental to heart function by increasing tissue stiffness and pressure, and altering conduction properties potentially leading to arrhythmias (Travers Joshua G. et al. Cardiac Fibrosis. Circ. Res. 118, 1021-1040 (2016)).

Therefore, resolution of excessive ECM and ECM-producing fibroblasts is a promising strategy to alleviate cardiac fibrosis and potentially fibrosis in general. Genetic ablation of activated cardiac fibroblast in mouse models of hypertension or myocardial infarction reduced the fibrotic area in the heart and improved functional parameters, i.e ejection fraction and fractional shortening (Kaur, H. et al. Circ. Res. 118, 1906-1917 (2016)). Recently, Aghajanian et al. (Aghajanian, H. et al. Nature 573, 430-433 (2019)) and Rurik et al. (Rurik, J. G. et al. Science 375. 91-96 (2022)) have provided proof-of-principle studies for the development of immune cell therapy to treat cardiac fibrosis. They have generated CAR T cells directed against fibroblast activation protein (FAP) and assessed their function in an in vivo mouse model of hypertensive cardiac injury and fibrosis. This cell therapeutic treatment resulted in an efficient reduction, not yet complete removal of fibrotic tissue and improved heart function after injury (Aghajanian, H. et al. Nature 573, 430-433 (2019); Rurik, J. G. et al. Science 375. 91-96 (2022)).

WO2014055442A2 discloses CARs targeting stromal cells for the treatment of cancer. W02019067425(A1) discloses CARs targeting FAP expressing fibroblastic cells for the treatment of heart diseases.

There is a need in the art for improved or alternative immunotherapies targeting FAP expressing target cell.

Brief description of the invention

The inventors found novel sequences of antigen binding domains specific for the antigen FAP for use as binders in chimeric antigen receptors.

The invention comprises a CAR comprising an antigen binding domain comprising SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL) that are specific for the antigen fibroblast activation protein (FAP). The use of the CARs as disclosed herein is for directing immune cells such as T cells to target cells expressing FAP in a subject. Said target cells expressing FAP may be (fibroblastic) stromal cells, such as endothelial cells or mesenchymal stromal cells, wherein said target cells expressing FAP is present in (or is associated with) a tumor microenvironment of a subject or is a cancer cell expressing FAP. Therefore, the CARs as disclosed herein are suited for treatment of solid tumors such as pancreatic cancer, non-small cell lung cancer, melanoma, ovarian cancer, breast cancer, or colorectal cancer that are associated with (fibroblastic) stromal cells as part of the TME or that may include cancer cells expressing FAP.

Alternatively, CARs as disclosed herein and expressed in immune cells such as T cells may also be used for treatment of heart diseases. In certain embodiments, the invention provides therapy for treatment or reversal of cardiac fibrosis. The invention involves, in one embodiment, using chimeric antigen receptor T cells specific for fibroblast activation protein (FAP) to reduce pathological cardiac fibrosis and improve cardiac function in a subject in need thereof.

The present invention also comprises nucleic acids encoding the CARs as disclosed herein.

Brief description of the drawings

Figure 1 : Identification of FAP specific scFv binders utilizing CAR pool screening.

(A) CAR pool expression in primary T cells after transduction with CAR pool LV at MOI 10 and 90, compared to untransduced cells (UTD). (B, C) CD4, CD8, and CAR expression at the end of round 1 of co-culture of primary CAR pool T cells with DKMG target cells (B) and HEK 293 cells (C). DKMG and HEK 293 target cells are double negative for CD4 and CD8. (D) CD4, CD8, and CAR expression at end of round 5 of co-culture of primary CAR pool T cells with DKMG target cells. Figure 2: Affinity measurement of FAP scFv binders number 5 and 28.

(A, B) Sensogram plot showing association and dissociation of serial dilutions of FAP5 and FAP28 with human FAP (A) and mouse FAP (B). (C, D) Calculated association constant (Ka), dissociation constant (kdis), and binding affinity (KD) to human FAP (C), and mouse FAP (D). (E) Concentration-dependent binding of FAP scFv binders 5, 14, and 28 to DKMG target cells.

Figure 3. Characterization of FAP targeting CAR T cells.

(A) CAR expression in CAR T cells expressing the FAP scFv clones 1, 3, 5, 7, 10, 14, and 28,

(B) CAR expression of FAP targeting CAR T cells expressing scFv clones 3, 7, 10, and 14 detected with human and mouse FAP recombinant proteins 7 days after transduction (D07) of primary T cells. (C) Cytotoxicity analysis in overnight killing assay of human (hFAP) or mouse FAP (mFAP) expressing HEK 293 cells, and parental HEK 293 cells, at effector to target (E:T) ratios of 20: 1, 10: 1, and 5: 1.

Figure 4. Characterization of FAP targeting CAR T cells expressing scFv clones 1, 3, 7, 10, 14, 28.

(A) CAR expression of FAP targeting CAR T cells expressing scFv clones 1, 10, and 28 detected with human FAP recombinant proteins 10 days after transduction (D10) of primary T cells. (B) Cytotoxicity analysis in overnight killing assay of human (hFAP) or mouse FAP (mFAP) over-expressing HEK 293 cells, and parental HEK 293 cells, at effector to target (E:T) ratios of 20: 1, 10: 1, and 5: 1. (C) CAR expression of FAP targeting CAR T cells expressing scFv clones 1, 3, 5, 7, 10, 14, and 28, as detected by anti-G4S antibody (D) Cytotoxicity of FAP targeting CAR T cells against FAP⁺ tumor cell line DKMG at an E:T ratio of 0.3: 1 measured in real time by xCELLigence analysis (E) Time take in hours to kill 50% (KT50) of target cells by different FAP targeting CAR T cells, killing was not detected (ND) for untransduced CAR T cells (UTD).

Figure 5. FAP28 CAR T cells show cytotoxicity, increased activation marker expression and cytokine secretion in co-culture with murine or human FAP expressing cell lines.

(A, B) Flow cytometric analysis of FAP expression in MDA-MB-231 cells transduced with GFP and human FAP (hFAP, A) or murine FAP (mFAP, B) in comparison to the parental cell line expressing GFP. (C-E) Kinetics of killing of MDA-GFP (C), MDA-GFP-hFAP (D) and MDA-GFP-mFAP cells (E) by FAP28 CAR T cells quantified by reduction of green area confluency (GAC). CAR T cells were applied in three different effector to target (E:T) ratios. UTD = untransduced T cells, data normalized to start of the co-culture. (F) Flow cytometric analysis of CAR T cell marker expression after 48h co-culture with target cell lines. (G) Quantification of cytokines in cell culture supernatants collected 48h after start of CAR T cell/target cell line co-culture.

Figure 6. FAP28 CAR T cells show cytotoxicity, increased activation marker expression and cytokine secretion in co-culture with primary, human cardiac fibroblasts.

(A) Flow cytometric analysis of FAP expression in primary, human cardiac fibroblasts (CF) after 4 passages with the respective staining control in comparison to secondary stain control. (B, C) Fluorescence microscopic images of CF in co-culture with untransduced (UTD) T cells

(B) or FAP28 CAR T cells (C). Scale bar= 100 pm. (D) Brightfield microscopic images of CF in co-culture UTD T cells or FAP28 CAR T cells. Scale bar= 100 pm. (E) Flow cytometric analysis of UTD and FAP28 CAR T cell marker expression after 48h co-culture with CF. (F) Quantification of cytokines in cell culture supernatants collected 48h after start of CAR T cell/target cell line co-culture. n = 3 ± SD. (G) Kinetics of killing of CF by FAP28 CAR T cells measured by flow cytometric quantification of CF cell counts over the course of the co-culture.

Detailed description of the invention

In a first aspect the present invention provides a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

Said CAR, wherein the antigen binding domain comprises SEQ ID NO:21 or SEQ ID NO: 15.

Said CAR, wherein said intracellular signaling domain comprises a stimulatory domain comprising one or more immunoreceptor tyrosine-based activation motifs (IT AMs) such as the stimulatory domain of CD3zeta and/or one or more co-stimulatory domain(s) such as CD28 and/or 4- IBB.

Said CAR, wherein CAR comprises SEQ ID NO:24 or SEQ ID NO:25. Said CAR, wherein said antigen FAP is expressed on a target cell. Said antigen FAP may have the sequence set forth in SEQ ID NO:22.

Said target cell expressing FAP may be a (fibroblastic) stromal cell, such as endothelial cell or mesenchymal stromal cell.

Said target cell expressing FAP may be present in a tumor microenvironment or may be a cancer cell expressing FAP.

Said target cell expressing FAP may be a (fibroblastic) stromal cell, such as endothelial cell or mesenchymal stromal cell, wherein said target cell expressing FAP is present in (or is associated with) a tumor microenvironment (in a subject).

Said TME may be a TME of a solid tumor such as pancreatic cancer, non-small cell lung cancer, melanoma, ovarian cancer, breast cancer, or colorectal cancer.

Said solid tumor may be pancreatic cancer, non-small cell lung cancer, melanoma, ovarian cancer, breast cancer, or colorectal cancer.

Said target cell expressing FAP may be a tumor cell such as an epithelial tumor cell.

Said CAR, wherein said target cell expressing FAP is a stromal cell such as a fibroblastic stromal cell.

Said CAR, wherein said target cell expressing FAP is a stromal cell and/or an endothelial cell of the TME, or wherein said target cell that expresses FAP and that is associated with a heart disease is a stromal cell.

Said CAR, wherein said stromal cell is present in a tumor microenvironment or wherein said stromal cell is associated with a heart disease.

Said CAR, wherein the heart disease is selected from the group consisting of cardiac fibrosis, aortic valve stenosis, hypertensive heart disease, diastolic dysfunction, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, myocardial infarction, ischemic cardiomyopathy, hypertrophic cardiomyopathy, arrhythmia, atrial fibrillation, arrhythmogenic right ventricular dysplasia, dilated cardiomyopathy, an inherited form of heart disease, muscular dystrophy, infective cardiomyopathy, transplant cardiomyopathy, radiation induced cardiac fibrosis, an autoimmune related heart condition, sarcoid cardiomyopathy, lupus, a toxin related heart condition, a drug related heart condition, amyloidosis, diabetic cardiomyopathy, reactive interstitial fibrosis, replacement fibrosis, infiltrative interstitial fibrosis, idiopathic dilated cardiomyopathy, aging-related fibrosis, and endomyocardial fibrosis. Preferentially, the heart disease is cardiac fibrosis after myocardial infarction or hypertensive heart disease, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, hypertrophic cardiomyopathy, idiopathic dilated cardiomyopathy, radiation induced cardiac fibrosis, reactive interstitial fibrosis, replacement fibrosis, infiltrative interstitial fibrosis, aging-related fibrosis, or endomyocardial fibrosis.

In another aspect the present invention provides an immune cell expressing a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

In another aspect the present invention provides an immune cell expressing a chimeric antigen receptor (CAR) for use in immunotherapy, the CAR comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

Said immune cell expressing a chimeric antigen receptor (CAR) for use in immunotherapy, wherein the immunotherapy is for treatment of a solid cancer such as pancreatic cancer, nonsmall cell lung cancer, melanoma, ovarian cancer, breast cancer, or colorectal cancer, or wherein the immunotherapy is for the treatment of a heart disease as disclose herein.

Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell. In another aspect the present invention provides an immune cell expressing a chimeric antigen receptor (CAR) for use in treatment of a solid cancer (in a subject) associated with fibroblastic stromal cells that express FAP, the CAR comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

In another aspect the present invention provides an immune cell expressing a chimeric antigen receptor (CAR) for use in treatment of a heart disease (in a subject) associated with fibroblastic stromal cells that express FAP, the CAR comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

In another aspect the present invention provides a (isolated) nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain. In another aspect the present invention provides an immune cell comprising a nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

In a further aspect the present invention provides a vector comprising a nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

A “vector” comprises a (isolated) nucleic acid molecule which can be used to deliver the (isolated) nucleic acid molecule to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. Said vector may be preferentially a retroviral vector such as a lentiviral vector.

In a further aspect the present invention provides a composition comprising (a population of) immune cells expressing a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

Said immune cells may preferentially be T cells, NK cells or a gammadelta T cells.

In a further aspect the present invention provides a pharmaceutical composition comprising i) immune cells expressing a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain, and ii) a pharmaceutically acceptable carrier.

Pharmaceutically 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.

In one embodiment of the invention the immune cells expressing the CAR as disclosed herein are for use in treatment of a disease associated with a target cell of a subject suffering from said disease, wherein said target cell expresses FAP and the disease may be a solid cancer associated with fibroblastic stromal cells that express FAP that are part of the TME of the solid tumor. Immune cells, e.g. T cells or NK cells of a subject may be isolated. The subject may e.g. suffer from said cancer or may be a healthy subject. These cells are genetically modified in vitro to express the CAR as disclosed herein. These engineered cells may be activated and expanded in vitro. In a cellular therapy these engineered cells are infused to a recipient in need thereof. These cells may be a pharmaceutical composition (said cell plus pharmaceutical acceptable carrier). The infused cells may be e.g. able to kill (or at least stop growth of) cancerous cells in the recipient. The recipient may be the same subject from which the cells was obtained (autologous cell therapy) or may be from another subject of the same species (allogeneic cell therapy).

The immune cells, preferentially T cells or NK cells engineered to express the CAR as disclosed herein may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention may comprise a cell population of genetically modified cells (a plurality of immune cells) as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions 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.

Preferentially, the compositions of the present invention are formulated for intravenous administration. The administration of cell compositions to the subject may be carried out in any convenient manner known in the art.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated. Appropriate dosages may be determined by clinical trials. But the quantity and frequency of administration will also be determined and influenced by such factors as the condition of the patient, and the type and severity of the patient's disease.

A pharmaceutical composition comprising the immune cells, preferentially T cells or NK cells as disclosed herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight. The cell compositions may also be administered several times at these dosages. The compositions of cells may be injected e.g. directly into a tumor, lymph node, or site of infection.

The genetically engineered immune cells may be activated and expanded to therapeutic effective amounts using methods known in the art.

The immune cells of the invention may be used in combination with e.g. chemotherapy, radiation, immunosuppressive agents, antibodies or antibody therapies.

In a further aspect the present invention provides in in-vivo method for treatment of a subject suffering from a solid cancer associated with fibroblastic stromal cells that express FAP or from a heart disease associated with fibroblastic stromal cells that express FAP, the method comprising administering to said subject an immune cell expressing a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), preferentially in the order of sequence from N to C-terminus VL-VH, b) a transmembrane domain, and c) an intracellular signaling domain.

Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

Generation of of CAR T cells

Processes of generation of CAR immune cells such as CAR T cells are well known in the art. Exemplarily in the following methods of generation of immune cells expressing a CAR are disclosed.

The genetically modified immune cells expressing the CAR as disclosed herein, preferentially T cells, may be generated preferentially in an automated process in a closed system. A process for the generation of genetically modified cells, preferentially T cells, is disclosed e.g. in WO2015162211A1 and may comprise the e.g. steps: a) providing a cell sample comprising immune cells (e.g. from a PBMC) b) preparation of the cell sample by centrifugation c) magnetic separation of the immune cells, preferentially T cells, d) activation of the enriched immune cells, preferentially T cells, using modulatory agents e) genetically modifying the immune cells, preferentially T cells, to express the CAR as disclosed herein f) expansion of the genetically modified immune cells, preferentially T cells, in a cultivation chamber g) washing of the cultured immune cells, preferentially T cells.

All these steps may be performed automatically in a closed system, preferentially in a closed and sterile system.

The process is especially suited for preparing gene modified cells such as immune cells, preferentially T cells, wherein the enriched immune cells, preferentially T cells, are gene- modified by using viral and/or non-viral vectors, preferentially using a lentiviral vector.

In case of magnetically enrichment of T cells from PBMC or leukapheresis anti-CD4 and/or anti-CD8 antibodies or antigen binding fragments coupled to beads may be used. The modulatory agents may be selected from agonistic antibodies such as anti-CD3 and/or anti- CD28 antibodies or antigen binding fragments thereof (especially in case of modifying T cells), and/or cytokines.

The gene-modified immune cells, preferentially T cells, may be enriched by magnetic labelling of immune cells and magnetic separation before or after cultivation to obtain higher frequency of gene-modified immune cells, preferentially T cells, in the final cellular product.

The cultivation (expansion) may be over several day such as 8 to 12 days, or may be a shorter cultivation process without or with less cultivation/expansion as disclosed e.g. in WO2020239866A1. In case of a shorter in-vitro process of generation of immune cells such as T cells, the generated immune cells such as T cells may expand in-vivo after administration to a subject in need thereof to therapeutically effect amounts of immune cells expression the CAR as disclosed herein (see e.g. WO2020239866A1) . Such a short ex-vivo process may comprise e.g. (in a closed and sterile system for cell modification) the steps a) providing a sample (e.g. from PBMC) comprising immune cells such as T cells b) preparation of said sample by centrifugation c) enrichment of the immune cells such as T cells of step b d) activation of the enriched immune cells such as T cells using modulatory agents e) genetic modification of the activated immune cells such as T cells by transduction e.g. with lentiviral vector particles f) removal of said modulatory agents, thereby generating a sample of genetically modified immune cells such as T cells, wherein said method is performed e.g. in equal or less than 3 days (72h).

As a closed and sterile system for cell modification, the fully automated cell processing device CliniMACS Prodigy® and associated tubing sets (Miltenyi Biotec GmbH, Germany) may be used (W02009/072003). This closed system meets the requirements of GMP -grade processing of almost any kind of cellular products and may allow reducing clean room requirements, improve technology transfer and harmonization of cell manufacturing processes.

Nucleotides, Expression, Vectors, and Host Cells

The nucleic acids encoding a CAR as used herein may comprise a nucleotide sequence encoding any of the leader sequences, antigen binding domains, transmembrane domains, and/or intracellular T cell signaling domains described herein.

In some embodiments, the nucleotide sequence may be codon-modified. Without being bound to a particular theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

"Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide", "nucleic acid molecule" and “nucleic acid sequence” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.

A recombinant nucleic acid may be one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).

The nucleic acid can comprise any isolated or purified nucleotide sequence which encodes any of the CARs or functional portions or functional variants thereof. Alternatively, the nucleotide sequence can comprise a nucleotide sequence which is degenerate to any of the sequences or a combination of degenerate sequences.

Also provided is a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.

In an embodiment, the nucleic acids can be incorporated into a recombinant expression vector. In this regard, an embodiment provides recombinant expression vectors comprising any of the nucleic acids. For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors are not naturally-occurring as a whole.

However, parts of the vectors can be naturally-occurring. The recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double- stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.

In an embodiment, the recombinant expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.

The recombinant expression vector may be a viral vector, e.g., a retroviral vector or a lentiviral vector. A lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector. Other examples of lentivirus vectors that may be used in the clinic, include, for example, and not by way of limitation, the LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica pic, the LENTIMAX.TM. vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

A number of transfection techniques are generally known in the art. Transfection methods include e.g. calcium phosphate co-precipitation, direct micro injection into cultured cells, electroporation, liposome mediated gene transfer, and lipid mediated transduction. If DNA or RNA is introduced into cells by using viral vector carriers, then the technique is called transduction.

Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.

The recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based. The recombinant expression vector may comprise restriction sites to facilitate cloning.

The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the CAR (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the CAR. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a nonviral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.

The recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

Further, the recombinant expression vectors can be made to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

An embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5a cell. For purposes of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell. For purposes herein, the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Thl and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells, i.e. Tscm, naive T cells, and the like. The T cell may be a CD8+ T cell or a CD4+ T cell.

In an embodiment, the CARs as described herein can be used in suitable non-T cells. Such cells are those with an immune-effector function, such as, for example, NK cells, and T-like cells generated from pluripotent stem cells.

General methods of treatment

It is contemplated that the CARs disclosed herein can be used in methods of treating or preventing a disease in a mammal. In this regard, an embodiment provides a method of treating cancer or of treating a heart disease as disclosed herein in a mammal, comprising administering to the mammal (the subject) the CARs, the nucleic acids encoding the CARs, the recombinant expression vectors encoding the CARs, the immune cells expressing the CARs disclosed herein in an amount effective to treat cancer or a heart disease in the mammal.

An embodiment, especially for the treatment of cancer, further comprises lymphodepleting the mammal prior to administering the CARs disclosed herein. Examples of lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

For purposes of the methods, wherein immune cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal. As used herein, allogeneic means any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically. As used herein, “autologous” means any material derived from the same individual to whom it is later to be re-introduced into the individual.

The mammal referred to herein can be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the mammal is a human.

With respect to the methods, the cancer can be any cancer in that FAP expressing target cells are involved, e.g. in a TME of a solid tumor The cancers may include pancreatic cancer, nonsmall cell lung cancer, melanoma, ovarian cancer, breast cancer, or colorectal cancer.

With respect to the methods, the heart diseases to be treated include but are not limited to cardiac fibrosis, aortic valve stenosis, hypertensive heart disease, diastolic dysfunction, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, myocardial infarction, ischemic cardiomyopathy, hypertrophic cardiomyopathy, arrhythmias including atrial fibrillation, arrhythmogenic right ventricular dysplasia, dilated cardiomyopathy (including idiopathic and familial forms), hypertensive heart disease, inherited forms including muscular dystrophy, infective cardiomyopathy (e.g. Chagas disease, rheumatic fever), transplant cardiomyopathy, radiation induced cardiac fibrosis, autoimmune (Sarcoid cardiomyopathy, lupus), toxin or drug related, amyloidosis, diabetic cardiomyopathy, and other types of cardiac fibrosis including but not limited to reactive interstitial fibrosis, replacement fibrosis, infiltrative interstitial fibrosis, idiopathic dilated cardiomyopathy, aging-related fibrosis, and endomyocardial fibrosis.

Preferentially, the heart disease is cardiac fibrosis after myocardial infarction or hypertensive heart disease, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, hypertrophic cardiomyopathy, idiopathic dilated cardiomyopathy, radiation induced cardiac fibrosis, reactive interstitial fibrosis, replacement fibrosis, infiltrative interstitial fibrosis, aging-related fibrosis, or endomyocardial fibrosis. The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods can provide any amount or any level of treatment of cancer or heart diseases in a mammal.

Furthermore, the treatment or prevention provided by the method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

Another embodiment provides for the use of the CARs, nucleic acids, recombinant expression Vectors and immune cells for the treatment or prevention of disorder, e.g., cancer or heart disease, in a mammal.

Any method of administration can be used for the disclosed therapeutic agents, including local and systemic administration. For example topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration can be used. The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic). In cases in which more than one agent or composition is being administered, one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally and a composition of immune cells expressing the CARs as disclosed herein may be administered intravenously.

Methods of administration include injection for which the CAR, CAR T cell or the compositions are provided in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds or compositions (e.g. the cells expressing the CARs as disclosed herein) can be used, for instance by applying the compounds or compositions to a region of tissue from which a tumor has been removed, or a region suspected of being prone to tumor development. In some embodiments, sustained intra-tumoral (or near-tumoral) release of the pharmaceutical preparation that includes a therapeutically effective amount of the compounds or compositions may be beneficial. In other examples, the conjugate is applied as an eye drop topically to the cornea, or intravitreally into the eye.

The disclosed therapeutic agents can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the disclosed therapeutic agents may be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1- 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions.

Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner.

Treatment of cancer

In some embodiments, the disclosed methods include providing surgery, radiation therapy, and/or chemotherapeutics to the subject in combination with a disclosed CAR or T cell expressing a CAR (for example, sequentially, substantially simultaneously, or simultaneously). Methods and therapeutic dosages of such agents and treatments are known to those skilled in the art, and can be determined by a skilled clinician. Preparation and dosing schedules for the additional agent may be used according to manufacturer's instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described elsewhere.

In some embodiments, the combination therapy can include administration of a therapeutically effective amount of an additional cancer inhibitor to a subject. Non-limiting examples of additional therapeutic agents that can be used with the combination therapy include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent can be administered in combination with the CARs or CAR- T cells disclosed herein. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.

Additional chemotherapeutic agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example, capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfm; and other agents , such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib, and tretinoin. Selection and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.

The combination therapy may provide synergy and prove synergistic, that is, the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.

Treatment of heart diseases

Heart disease or cardiovascular disease generally refers to conditions that involve narrowed or blocked blood vessels that can lead to a heart attack, chest pain (angina) or stroke. Other heart conditions, such as those that affect the heart's muscle, valves or rhythm, also are considered forms of heart disease. Fibroblasts comprise the largest cell population in the myocardium. In heart disease, the number of fibroblasts is increased either by replication of the resident myocardial fibroblasts, migration and transformation of circulating bone marrow cells, or by transformation of endothelial/epithelial/epicardial cells into fibroblasts and myofibroblasts. The primary function of fibroblasts is to produce structural proteins that comprise the extracellular matrix (ECM). This can be a constructive process; however, hyperactivity of cardiac fibroblasts can result in excess production and deposition of ECM proteins in the myocardium, known as fibrosis, with adverse effects on cardiac structure and function. Cardiac fibrosis may refer to an abnormal thickening of the heart valves due to inappropriate proliferation of cardiac fibroblasts, but more commonly refers to the excess deposition of extracellular matrix in the cardiac muscle. Cardiac fibrosis can also contribute to pathology in some forms of genetic cardiac diseases including muscular dystrophies.

Nearly all forms of heart failure are associated with cardiac fibrosis, including those with reduced ejection fraction or preserved ejection fraction (heart failure with preserved ejection fraction, HFpEF). Myocardial fibrosis and associated poor diastolic relaxation are thought to be the central drivers of symptoms in patients with HFpEF. Many forms of cardiomyopathy not associated with coronary artery disease also display excessive fibrosis, including ischemic cardiomyopathy, sarcoid cardiomyopathy, hypertrophic cardiomyopathy, hypertensive heart disease, and inherited forms of muscular dystrophy and dilated cardiomyopathy. Although quiescent fibroblasts are an important component of the normal structure of the myocardium, activated, pathological fibroblasts induced by injury or disease negatively impact compliance and stiffness and signal to cardiac myocytes to further negatively impact function. WO2019067425A1 discloses the use of CAR T cells expressing anti-FAP CAR to treat heart diseases associated with FAP expressing cells.

The kind of treatment of a heart disease by using an immune cell expressing an anti-FAP CAR as disclosed herein may be as disclosed herein (especially in section “General methods of treatment”). In certain embodiments, the modified cells expressing the anti-FAP CARs disclosed herein are administered in such a way as to focus their delivery to the heart. For example, the cell or population of cells can be injected intravenously into the coronary arteries. In certain embodiments a cardiac catheter can be used to deliver the cell or population of cells. In certain embodiments, a focused delivery of the cell or population of cells to the myocardium is administered.

All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.

Definitions

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

As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not. As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1% from the specified value

In general, a CAR may comprise an extracellular domain (extracellular part) comprising the 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 or hinge region). The extracellular domain may also comprise a signal peptide.

A "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.

Generally, an “antigen binding domain” refers to the region of the CAR that specifically binds to an antigen, e.g. to a tumor associated antigen (TAA) or tumor specific antigen (TSA). The CARs may comprise one or more antigen binding domains (e.g. a tandem CAR). Generally, the targeting regions on the CAR are extracellular. The antigen binding domain may comprise an antibody or an antigen binding fragment thereof. The antigen binding domain may comprise, for example, full length heavy chain, Fab fragments (Fab), single chain Fv (scFv) fragments, divalent single chain antibodies, nanobodies, single domain antibodies, VHH 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 or nanobody. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4/S)3-linker”.

In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will be used in. For example, when it is planned to use it therapeutically in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human or humanized antibody or antigen binding fragment thereof. Human or humanized antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art. “Spacer” or “hinge” as used herein refers to the hydrophilic region which is between the antigen binding domain and the transmembrane domain. The CARs may comprise an extracellular spacer domain but is it also possible to leave out such a spacer. 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.

The transmembrane domain of the CAR may be derived from any desired natural or synthetic source for such domain. When the source is natural the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in the CARs described herein may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8alpha, CD9, CD 16, CD22, CD28, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19. The transmembrane domain preferentially may be derived from CD8alpha or CD28. When the key signaling and antigen recognition modules (domains) are on two (or even more) polypeptides then the CAR may have two (or more) transmembrane domains. The splitting key signaling and antigen recognition modules enable for a small molecule-dependent, titratable and reversible control over CAR cell expression (e.g. WO2014127261A1) due to small molecule-dependent heterodimerizing domains in each polypeptide of the CAR.

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, otherwise it is indicated explicitly as an inhibitory CAR (iCAR)). "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. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement.

Generally, 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). Therefore, 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).

Examples of IT AM containing primary cytoplasmic signaling domains often used in CARs are that those derived from TCR^ (CD3Q, FcRgamma, FcRbeta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence derived from CD3^.

The cytoplasmic domain of the CAR may be designed to comprise the CD3^ signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3^ 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, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3.

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.

As an example, the cytoplasmic domain may comprise the signaling domain of CD3^ and the signaling domain of CD28. In another example the cytoplasmic domain may comprise the signaling domain of CD3^ and the signaling domain of CD137. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3^, the signaling domain of CD28, and the signaling domain of CD137.

As aforementioned either the extracellular part or the transmembrane domain or the cytoplasmic domain of a CAR may also comprise a heterodimerizing domain for the aim of splitting key signaling and antigen recognition modules of the CAR.

The CAR may be further modified to include on the level of the nucleic acid encoding the CAR one or more operative elements to eliminate CAR expressing immune 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. In one embodiment, the nucleic acid expressing and encoding the CAR can be further modified to express an enzyme such thymidine kinase (TK) or cytosine deaminase (CD). The CAR may also be part of a gene expression system that allows controlled expression of the CAR in the immune cell. Such a gene expression system may be an inducible gene expression system and wherein when an induction agent is administered to a cell being transduced with said inducible gene expression system, the gene expression system is induced and said CAR is expressed on the surface of said transduced cell.

In some embodiments the CAR may be a “SUPRA” (split, universal, and programmable) CAR, where a “zipCAR” domain may link an intra-cellular costimulatory domain and an extracellular leucine zipper (WO2017/091546). This zipper may be targeted with a complementary zipper fused e.g. to an scFv region to render the SUPRA CAR T cell tumor specific. This approach would be particularly useful for generating universal CAR T cells for various tumors; adapter molecules could be designed for tumor specificity and would provide options for altering specificity post-adoptive transfer, key for situations of selection pressure and antigen escape.

The CARs as disclosed herein may be designed to comprise any portion or part of the above- mentioned domains as described herein in any order and/or combination resulting in a functional CAR, i.e. a CAR that mediated an immune effector response of the immune effector cell that expresses the CAR as disclosed herein.

The engineered cell expressing a CAR as disclosed herein 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 e.g. 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- 1

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 such as, but not limited to, metalloproteases/ECM-degrading enzymes.

The term “FAP” refers to fibroblast activation protein. The term should be construed to include not only fibroblast activation protein, but variants, homologs, fragments and portions thereof to the extent that such variants, homologs, fragments and portions thereof retain the activity of FAP as disclosed herein. The FAP may be a human FAP as set forth in SEQ ID NO:22 or may be a murine FAP as set forth in SEQ ID NO:23.

The term "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), 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”). Examples of antigen binding fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), single domain antibodies (VHH and nanobodies), diabodies, dsFv, Fab’, F(ab')2, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

A “fully human antibody” or “human antibody” is an antibody or antigen binding fragment thereof which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some embodiments, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals.

The term “CDR” denotes a complementarity determining region as defined by at least one manner of identification to one of skill in the art. The precise amino acid sequence boundaries of a given CDR or framework region (FR) can be readily determined using any of a number of well-known schemes, including the numbering system of Kabat.

As used herein, 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, and combinations thereof, for example a glycosylated protein or a glycolipid. The term “antigen” as used herein 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.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter in a cell.

As used herein, 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.

The terms “nucleic acid”, “nucleic acid sequence”, “nucleic acid molecule” or “polynucleotide” may be used interchangeably herein and refer to polymers of nucleotides. Polynucleotides, which can be hydrolyzed into monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, the term “polynucleotides” encompasses, but is not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means. A recombinant nucleic acid may be one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques well-known in the art.

In some embodiments, the nucleic acid sequence may be codon-modified. Without being bound to a particular theory, it is believed that codon optimization of the nucleic acid sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleic acid sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleic acid sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

A recombinant protein is a biotechnologically generated protein that does not occur naturally in a eukaryotic and/or prokaryotic cell. Often it is composed of different domains from different proteins, e.g. as used herein, a viral envelope protein is fused (at its ectodomain) to a polypeptide that comprises an antigen binding domain specific for an antigen.

The term “transduction” means the transfer of genetic material from a viral agent such as a lentiviral vector particle into a eukaryotic cell such as a T cell.

The terms “having specificity for”, “specifically binds” or “specific for” with respect to an antigen-binding domain of an antibody or a fragment thereof refer to an antigen-binding 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.

Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". 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. Cancer immunotherapy as an activating immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses cell-based, preferentially T cell-based cytotoxic responses to attack cancer cells. T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient or are directly generated in-vivo. Then the immunotherapy is referred to as “CAR T cell immunotherapy”.

The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

The term “(therapeutically) effective amount” as used herein means an amount of a pharmaceutical composition which is sufficient to significantly and positively modify the symptoms and/or conditions to be treated. The effective amount of an active ingredient such a a pseudotyped retroviral vector particle or a genetically modified immune cell for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable carrier(s) utilized.

The terms “engineered cell” and “(genetically) modified cell” as used herein can be used interchangeably. The terms mean containing and/or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. Especially, 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. For example, T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.

For enrichment, isolation or selection of specific immune cells, e.g. T cells such as CD4+ and/or CD8+ T cells, in principle any sorting technology can be used. This includes for example affinity chromatography or any other antibody-dependent separation technique known in the art. Any ligand-dependent separation technique known in the art may be used in conjunction with both positive and negative separation techniques that rely on the physical properties of the cells. An especially potent sorting technology is magnetic cell sorting. Methods to separate cells magnetically are commercially available e.g. from Invitrogen, Stem cell Technologies, in Cellpro, Seattle or Advanced Magnetics, Boston. For example, monoclonal antibodies can be directly coupled to magnetic polystyrene particles like Dynal M 450 or similar magnetic particles and used e.g. for cell separation. The Dynabeads technology is not column based, instead these magnetic beads with attached cells enjoy liquid phase kinetics in a sample tube, and the cells are isolated by placing the tube on a magnetic rack. However, in a preferred embodiment for enriching e.g. CD4+ and/or CD8+ T cells from a sample comprising T cells monoclonal antibodies or antigen binding fragments thereof are used in conjunction with colloidal superparamagnetic microparticles having an organic coating by e.g. polysaccharides (Magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec B.V. & Co. KG, Germany)). These particles (nanobeads or MicroBeads) can be either directly conjugated to monoclonal antibodies or used in combination with anti-immunoglobulin, avidin or anti- hapten-specific MicroBeads. The MACS technology allows cells to be separated by incubating them with magnetic nanoparticles coated with antibodies directed against a particular surface antigen. This causes the cells expressing this antigen to attach to the magnetic nanoparticles. Afterwards the cell solution is transferred on a column placed in a strong magnetic field. In this step, the cells attach to the nanoparticles (expressing the antigen) and stay on the column, while other cells (not expressing the antigen) flow through. With this method, the cells can be separated positively or negatively with respect to the particular antigen(s)/marker(s).

In case of a positive selection the cells expressing the antigen(s) of interest, which attached to the magnetic column, are washed out to a separate vessel, after removing the column from the magnetic field.

In case of a negative selection the antibody used is directed against surface antigen(s) which are known to be present on cells that are not of interest. After application of the cells/magnetic nanoparticles solution onto the column the cells expressing these antigens bind to the column and the fraction that goes through is collected, as it contains the cells of interest. As these cells are non-labelled by an antibody coupled to nanoparticels, they are “untouched”.

As used herein “autologous” means that cells, a cell line, or population of cells used for treating subjects are originating from said subject.

As used herein “allogeneic” means that cells or population of cells used for treating subjects are not originating from said subject but from a donor.

The terms “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, regulatory T cells (Treg), monocytes or macrophages. Preferentially these immune cells are human immune cells. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells or gamma-delta T cells. Most preferred immune effector cells are T cells and/or NK cells. Tumor infiltrating lymphocytes (TILs) are T cells that have moved from the blood of a subject into a tumor. These TILs may be removed from a patient' s tumor by methods well known in the art, e.g. enzymatic and mechanic tumor disruption followed by density centrifugation and/or cell marker specific enrichment. TILs are genetically engineered as disclosed herein, and then given back to the patient. "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.

T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. There are several subsets of T cells, each with a distinct function.

T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen- presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate a different type of immune response. Signaling from the APC directs T cells into particular subtypes.

Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells.

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

Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described — Foxp3+ Treg cells and Foxp3- Treg cells.

Natural killer T cells (NKT cells - not to be confused with natural killer cells of the innate immune system) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CDld. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic/cell killing molecules).

Natural killer cells (NK cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor-generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter the circulation. NK cells differ from natural killer T cells (NKTs) phenotypically, by origin and by respective effector functions; often, NKT cell activity promotes NK cell activity by secreting ZFNy. In contrast to NKT cells, NK cells do not express T cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD 16 (FcyRIII) and CD56 in humans, NK1.1 or NK1.2 in C57BL/6 mice. Up to 80% of human NK cells also express CD8. Continuously growing NK cell lines can be established from cancer patients and common NK cell lines are for instance NK-92, NKL and YTS.

The term "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. For example, 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.

A transgene may be a gene that has been transferred by genetic engineering techniques into a host cell that normally does not bear this gene. The gene may be a naturally gene that occurs in other cells or may be a recombinant gene. Normally the transgenes used in the present invention may be the chimeric antigen receptor specific for the antigen FAP as disclosed herein. The expressed transgene may also be referred to a heterologous protein or transgenic polypeptide. Examples

The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.

Example 1 : Binder discovery for FAP using CAR pool method and binding characterization of FAP binders

Derivation of human FAP binders from a fully human yeast library

A large yeast display human naive single chain variable fragment antibody library was used to isolate anti-human FAP antibodies described herein. The library was constructed using a collection of human antibody gene repertoires from 30 individuals. Three rounds of magnetic- activated cell sorting (MACS) were performed to enrich human binders to the recombinant human FAP (ectodomain)-his and biotin tagged (Aero Biosystems). For the first round of yeast library panning, the yeast display library (5xlO¹⁰ cells) was incubated with 5 pg/ml FAP-His- biotin in 15 ml PBSA (consisting of 0.1% Bovine Serum Albumin (BSA) in Dulbecco's phosphate-buffered saline (PBS) buffer), at room temperature on a rotator for 1.5 hours. After two times washing with 25 ml PBSA, the yeast library mix was incubated with 100 pL anti -his microbeads (Miltenyi Biotec) at room temperature on a rotator for 30 minutes. After one time washing, the library mix was resuspended in 50 ml of PBSA and loaded onto the MACS cell separation column (LS column). After three times washing with 10 ml PBSA. The yeast displayed binders to the column were then eluted two times with 2 ml PBSA. These eluted yeast cells were combined and then resuspended into 50 ml SDCAA medium (20 g D-glucose, 6.7 g BD Difco™ Yeast Nitrogen Base without Amino Acids, 5 g Bacto™ Casamino Acids, 5.4 g Na2.HPC>4, and 8.56 g NaEEPCU.EEO in 1 L water) and amplified with shaking at 225 rpm at 30°C for 20 hours. The amplified pool was then induced in SGCAA medium (consisting of the same composition of SDCAA medium, but containing galactose instead of glucose), with shaking at 225 rpm at 30°C for another 16 hours and used for next round of panning. The same process was repeated two more times to enrich the FAP specific binders.

To further enrich the binders with higher affinity and better specificity, FACS based sorting was employed to isolate the strongest binders from the pool. The induced pool was incubated with 0.1 pg/ml of biotinylated FAP (Aero biosystems) at room temperature for 1 hour and then stained with Anti-c-Myc-FITC and Streptavidin-APC conjugates, the top 1% of the pool with the highest APC versus FITC signal was gated and sorted. The sorted pool of approximately 1000 binders was amplified in SDCAA medium and yeast plasmid DNA was extracted and transformed into bacteria for cloning into CAR construct.

Generation of CAR T constructs incorporating fully human FAP-targeting scFv sequences Fully human scFv binders targeting FAP were PCR amplified and cloned into the CAR construct. The scFv sequences were linked in frame to CD8 hinge and transmembrane domain, 4-1BB costimulatory domain and CD3 zeta activation domain. CAR sequences were incorporated into third-generation lentiviral vectors and which were used in transduction of human primary T cells to generate the FAP CAR T cells.

Identification of FAP binding scFv clones by CAR-Pool method

The enriched binder pool cloned into CAR construct was used to produce a lentiviral pool that was used to transduce primary T cells. CAR expression was measured using flow cytometry by incubating with histidine-tagged FAP (Aero Biosystems) and detection by anti-His-PE (Miltenyi Biotec). The T cells were analyzed for CAR expression before and after enrichment that showed a significant increase in CAR expression after the enrichment process. These T cells expressing the “CAR-Pool” were used for co-culture with FAP-positive tumor cell line DKMG at an E:T ratio of 0.3 : 1. Upon completion of 5 rounds of co-culture, RNA was extracted from “CAR-Pool” CAR T cells, and cDNA was synthesized from the co-cultures and the scFv fragments were PCR amplified to confirm an approximately 800 base pair PCR product. The individual clones were transformed into bacteria and single clones were sequenced using sanger sequencing, and the top seven clones were identified for further analysis.

FAP-binder affinity measurements

Quantitative affinity measurement of binders to FAP antigen was performed using biolayer interferometry in OctetR8 (Sartorius) instrument and anti -human Fc capture (AHC) biosensors (Sartorius). AHC biosensors were loaded with Fc-tagged FAP binders at 10, 5, and 2.5 pg/ml in PBS containing 0.1% BSA, 0.02% Tween 20. Recombinant FAP (Aero Biosystems) were diluted in the same buffer at 200 nM concentration to measure association and dissociation of the FAP analyte to AHC biosensors loaded with FAP scFv binders. Association and dissociation curves were then exported to Octet Analysis studio software and analyzed using a 1 : 1 binding model analysis. Results

Binder discovery for FAP using CAR Pool method:

The CAR pool method was used to identify the binders for FAP. Primary T cells were transduced at MOI 10 and 90 to produce CAR T cells, that did not show a large difference in CAR pool expression (Figure 1A). The CAR pool cells were co-cultured with DKMG glioblastoma target cell line and negative control HEK293 cells. At the end of round 1, the CAR pool cells showed effective killing of DKMG cells and CAR expression (Figure IB), whereas HEK293 cells did not show any clearance by CAR pool T cells (Figure 1C). Rechallenge with DKMG cells were continued until round 5, and target cell killing and CAR expression was continued at this stage (Figure ID). At this stage of serial re-challenge assay, RNA extraction was carried out followed by cDNA synthesis, cloning, and sequencing, to identify 7 scFv binders that were further tested for functional characterization. The binders were numbered as clones 1, 3, 5, 7, 10, 14, and 28 (FAP#1, FAP#3, FAP#5, FAP#7, FAP#10, FAP#14 and FAP#28).

Binding characterization of FAP binders

Fc tagged scFv fusion protein was produced to characterize the binding affinity to using biolayer interferometry and cell based binding assays. The Fc tag enabled the binding of the scFv binders to biosensors and also detection in cell based assays using anti-Fc antibody. To characterize the scFv formatted FAP binders, affinity measurements were carried out for FAP5 and FAP28 against human FAP (Figures 2A) and mouse FAP (Figures 2B). The FAP5 and FAP28 scFv binders showed affinity constant (KD) values of 4.11 nM and 4.76 nM, respectively to human FAP (Figure 2C). To mouse FAP, the corresponding affinity constant values (KD) were 2.73 pM and 7.08 nM, respectively (Figure 2D). The binding was also tested in target cell line DKMG and showed dose-dependent binding over the serial dilutions tested (Figure 2E). Binder scFv #28 showed the best binding ability followed by #14, and #5 showed the weakest binding to FAP expressing DKMG cells.

Example 2: Functional characterization of FAP targeting CAR T cells

Lentiviral particle production

VSV-G pseudotype lentiviral particles encoding the FAP28 CAR (SEQ ID NO:24), murine FAP (SEQ ID NO:23) or human FAP (SEQ ID NO:22) were generated by transfection of HEK293 cells using a four plasmid system. 24h after transfection, 10 mM Sodium Butyrate (Sigma Aldrich) was added to the culture medium. Lentiviral particles were harvested 48h and 72h after transfection by passing the cell culture supernatant through a 0.45 pm filter and centrifugation over night at 4°C. Lentiviral particles were resuspended in TexMACS Medium (Miltenyi Biotec) and stored at -70 °C until transduction.

Cell lines and cell culture

For in vitro experiments, MDA-MB-231 breast cancer cell line, and DKMG glioblastoma cell line were purchased from American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. Ectopic expression of mouse and human version of FAP in HEK293 cells were also performed to test functional analysis against those cell lines.

Flow cytometry analysis

Flow cytometry analysis was carried out in MACSQuant 10 analyzer. For cell staining, 0.5 million cells were harvested from culture and washed two times in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). Non-transduced cells were used as negative controls. Dead cells in all studies were excluded by 7AAD staining (BD Biosciences, San Jose, CA) or ViobilityTM fixable dye (Miltenyi Biotec). Cells were washed twice and resuspended in 200 ul Staining Buffer before quantitative analysis by flow cytometry. Flow cytometric analysis and data plots were generated using MACSQuant software or FlowJo software.

CAR T cell generation and cultivation

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from whole blood of healthy donors. Pan T cells were isolated from PBMC using the Pan T Cell Isolation Kit, human (Miltenyi Biotec). T cells were cultured at a density of 1E+06 cells per ml in TexMACS Medium (Miltenyi Biotec) supplement with 12.5 ng/ml IL-7 and IL-15 (Miltenyi Biotec) and activated with CD3 and CD28 agonists (T cell TransAct, Miltenyi Biotec). 24h after activation, T cells were transduced with VSV-G pseudotype lentiviral particles. Three days after isolation, the majority of TransAct was removed by replacing % of the media volume with fresh medium supplemented with IL-7 and IL-15. Transduction efficiency was determined via flow cytometry by staining with PE-labeled Human FAP protein (Aero Biosystems). CAR T cells were used for assays 10-14 days after isolation. Cytotoxicity assay in HEK293 cell lines

HEK293 cells were stably transduced to express human or mouse FAP and firefly luciferase (ffLuc) and used as target cell. HEK293 cells expressing ffLuc only were used as negative control. Target cells were cocultured with FAP targeting CAR T cells at various effector to target (E:T) ratios and incubated overnight. SteadyGlo reagent (Promega, Madison, WI) was added to each well and the resulting luminescence was quantified as counts per second (sample CPS). Target only wells (max CPS) and target only wells plus 1% Tween-20 (min CPS) were used to determine assay range. Percent specific lysis was calculated as: (l-(sample CPS-min CPS)/(max CPS-min CPS)).

Cytotoxicity assay in DKMG cell line

FAP targeting CAR T cells were analysed for cytotoxicity using xCELLigence RTCA MP analyser (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions. Briefly, 40,000 DKMG target cells were co-cultured with effector FAP CAR T cells at an E:T ratio of 0.3: 1, and cytolysis was measured for 36 hours. Data were analysed by RTCA Software Pro (Agilent Technologies, Santa Clara, CA, US).

Results

To characterize the anti-FAP CAR T cells, primary T cells were transduced to produce CAR T cells expressing anti-FAP scFv binders 1,3, 5, 7, 10, 14, and 28, together with CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3zeta activation signal. All CAR T cells showed comparable CAR expression at day 7 except #5, which had the lowest expression of CAR+ T cells. Subsequently the FAP#5 clone was not tested in further analysis.

Primary T cells were transduced to produce CAR T expressing FAP CARs. All the CAR T cells showed good recognition of both human and mouse FAP antigen (Figure 3 A). To assess the function of FAP targeting CAR T cells, a target cell line was produced by over expressing either human or mouse FAP in HEK293 cells. The target cells were used to test overnight cytotoxicity of FAP 3, 7, 10, and 14 CAR T cells (Figure 3B). All the CAR T cells were effective in clearing human FAP over expressing HEK293 cells. Interestingly, only FAP 10 CAR T cells were able to show cytotoxicity against mouse FAP expressing HEK293 cells (Figure 3B), although all 4 CAR T cells were reactive to mouse FAP (Figure 3 A). To further test FAP1, 10, and 28 CAR T cells, CAR expression was detected using human FAP antigen (Figure 4A). The CAR T cells were effective in killing both human and mouse FAP over expressing HEK293 cells (Figure 4B).

To finalize testing the FAP targeting CAR T cells against tumor cells, FAP CAR T cells 1, 3, 5, 7, 10, 14, and 28 were analyzed alongside each other. CAR expression for FAP5 was the lowest, whereas FAP28 showed the highest. In impedance-based cytotoxicity assay, all CAR T cells were able to complete DKMG target cell cytolysis by 18 hours (Figure 4D), but killing time for 50% cytotoxicity was shortest for FAP28 CAR T cell (Figure 4E). FAP 28 binder is cross-reactive to both human and mouse FAP, showed low non-specific cytotoxicity, and showed most effective CAR expression and target cell cytotoxicity and was used in subsequent functional test in cardiac fibrosis model.

Example 3: FAP28 CAR T cells show cytotoxicity, increased activation marker expression and cytokine secretion in co-culture with murine or human FAP expressing cell lines and primary human cardiac fibroblasts

Cytotoxicity assay MDA-MB-231 cell lines

MDA-MB-231 cells expressing firefly luciferase and green fluorescent protein (GFP) were transduced to express human FAP or murine FAP. Target cells were seeded in a density of 2E+04 cells per well in 96-well plates the day before the cytotoxicity assay. CAR transduction efficiency was determined via flow cytometry. On the day of the cytotoxicity assay, medium was changed to TexMACS (Miltenyi Biotec) and CAR T cells were added to target cells in three effector to target (E:T) ratios (0.5: 1, 1 : 1, 2: 1). Total T cell numbers were equalized by adding untransduced T cells to lower E:T ratios. Cytotoxicity was assessed by measuring confluency of target cells (green area confluence, GAC) with Incucyte®S3 Live-Cell Analysis Instrument (Sartorius) and analyzing with the IncuCyte® S3 2019A software over the course of the co-culture. Data was normalized to GAC at start of the co-culture. T cell activation marker expression and cytokine secretion were assessed via flow cytometry at 48h of cocultivation. For cytokine measurements 50 pl of supernatant were taken and stored at -20°C until analysis. Quantification of cytokines was performed using MACSplex Cytokine 12 Kit, human (Miltenyi Biotec) according to manufacturers instruction. Cytotoxicity assay primary cardiac fibroblasts

Human primary ventricular cardiac fibroblasts (CF) obtained from Cell Applications were cultured in HCF Growth Medium (Cell Applications). For co-culture with FAP28 CAR T cells, CF were seeded in a density of 5E+05 cells/well in a 96-well plate. On the day of cytotoxicity assay, medium was changed to Cardiac Cultivation Medium (Miltenyi Biotec). For cell count determination of CF in co-cultures with T cells, CF were labeled with CellTrace™ CFSE dye (Invitrogen) according to manufacturer’s instruction prior to seeding. At the indicated timepoints, T cells were removed by pipetting up and down and removing the cell culture medium and CF were harvested using Multi Tissue Dissociation Kit 3 (Miltenyi Biotec). Cells were washed and resuspended in PBS/EDTA/BSA buffer (PEB). Cell counts were determined via flow cytometry. T cell activation marker expression and cytokine secretion were assessed via flow cytometry at 48h of co-cultivation.

Immunofluorescence staining

At 48h of co-culture with FAP28 CAR T cells, CF were washed with PBS and fixed with 4% PFA for 20 min. Cells were washed again twice with PBS and permeabilized for 10 min with Permeabilization Solution (MACS Clearing Kit, Miltenyi Biotec). Staining with anti-FAP antibody (clone: Fl 1-24, Santa Cruz, 1 :200 in Antibody Staining Solution, MACS Clearing Kit, Miltenyi Biotec) was performed at 4 °C overnight. Cells were washed again twice with PBS and stained with secondary antibody for FAP (Goat anti-Mouse IgG Alexa Fluor 647, Thermo Fisher, 1 : 1000) and anti-CD3 (clone: REA613, Miltenyi Biotec, 1 :500) in Antibody Staining Solution for 1 h at RT. Cells were washed twice and images were acquired with Zeiss LSM 710 confocal laser scanning microscope.

Results

To evaluate FAP28 CAR T cell functionality, we assessed their cytotoxicity, activation marker expression and cytokine secretion in co-culture with target cell lines. In a first step, target cell lines were generated from GFP expressing MDA-MB-231 cells by lentiviral transduction with murine or human FAP, respectively. Transduction resulted in 94.9% and 70.0% expression of human or murine FAP, respectively (Fig. 5A, B). Transduced human T cells with the FAP28 CAR were co-cultured with target cell lines. FAP28 CAR T cells did not kill the parental FAP negative cell line (Fig. 5C). However, FAP28 CAR T cells specifically killed human (Fig. 5D) and murine FAP (Fig. 5E) expressing cell lines. Additionally, activation marker expression (Fig. 5F) and pro-inflammatory cytokine secretion (Fig. 5G) of FAP28 CAR T cells were increased in co-culture with FAP positive cell lines .

Next, the capability of FAP28 CAR T cells to eliminate primary, human cardiac fibroblasts (CF) was tested. FAP was expressed in 69.2% of in vitro cultured human CF (Fig. 6A). FAP28 CAR T cells effectively killed CF in co-culture indicated by the absence of FAP expressing cells in the culture (Fig. 6B, C) and a reduction in total CF confluency (Fig. 6D). To quantify the killing we labeled CF with a fluorescent cell dye and observed a decrease in cell counts over time in co-culture with FAP28 CAR T cells compared to CF cultured without T cells (Fig. 6E). In addition, we observed upregulation of activation markers (CD69, CD25 and 4- IBB) and increased secretion of pro-inflammatory cytokines (GM-CSF, IFN-y, TNF-a) by FAP28 CAR T cells (Fig. 6F, G). Of note, CF alone secreted high levels of IL-6, further confirming their activated phenotype.

Sequences of the disclosure (in standard one letter code for amino acids)

SEP ID NO: 1 (FAP#1 VH)

EVQLVQSGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGS

TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSRYAPEGMDVWG QGTLVTVSS

SEP ID NO:2 (FAP#1 VL)

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPS

GVSNRFSGSKSGNT ASLTISGLQAEDEAD YYC S S YTS S SGVFGGGTQLTVLG

SEP ID NO:3 (FAP scFv binder 1 (FAP#1))

EVQLVQSGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGS

TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSRYAPEGMDVWG

QGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNY

VSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCS

S YT S S SGVFGGGTQLTVLG

SEP ID NO:4 (FAP#3 VH)

EVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG

STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLYPYGMDVWGQG TLVTVSS

SEQ ID N0:5 (FAP#3 VL)

DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPPTFGQGTKLEIKR

SEP ID NO:6 (FAP scFv binder 3 (FAP#3))

EVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG

STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLYPYGMDVWGQG

TLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYP PTFGQGTKLEIKR

SEP ID NO:7 (FAP#5 VH)

QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTN TLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVT vss

SEQ ID NO:8 (FAP#5 VL)

QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGV

PARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIK

SEP ID NOV (FAP scFv binder 5 (FAP#5))

QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTN

TLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVT

VSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQK SGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPT FGGGTKLEIK

SEP ID NO: 10 (FAP#7 VH)

EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYYMSWIRQAPGRGLEWVASISTRTRS STIYYADSVKGRVTISRDDAKNSLSLQMNNLRAEDTAIYYCARGGLSRFDSWGQGTL VTVSS

SEP ID NO: 11 (FAP#7 VL)

QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRP SGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLEVFGTGTKLTVLG

SEP ID NO: 12 (FAP scFv binder (FAP#7))

EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYYMSWIRQAPGRGLEWVASISTRTRS STIYYADSVKGRVTISRDDAKNSLSLQMNNLRAEDTAIYYCARGGLSRFDSWGQGTL VTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSW YQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTS SSTLEVFGTGTKLTVLG

SEP ID NO: 13 (FAP# 10 VH)

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGT ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATARSGPGEYFQHWGQG TL VTVSS

SEP ID NO: 14 (FAP# 10 VL)

SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGI PERF SGS S SGNTVSLTITGAQ AADEAD YYCLSRDS SGNVVFGGGTK VTVLG

SEP ID NO: 15 (FAP scFv binder 10 (FAP# 10))

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGT ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATARSGPGEYFQHWGQG TL VT VS SGGGGSGGGGSGGGGS S SELTQDP AVS VALGQTVRITCQGDSLRS YYASWY QQKPGQAPVLVIYGKNNRPSGIPERFSGSSSGNTVSLTITGAQAADEADYYCLSRDSS GN VVFGGGTK VTVLG SEP ID NO: 16 (FAP# 14 VH)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGGILPIFGT ANYAQKFQGRVTITADESTST AYMELS SLRSEDT AVYYCAREGPDF S S SFFNWFDPW GQGTLVTVSS

SEP ID NO: 17 (FAP# 14 VL)

DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGGAPKILIYGAANLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPRTFGQGTKLEIKR

SEP ID NO: 18 (FAP scFv binder 14 (FAP# 14))

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGGILPIFGT ANYAQKFQGRVTITADESTST AYMELS SLRSEDT AVYYCAREGPDF S S SFFNWFDPW GQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLN WYQQKPGGAPKILIYGAANLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY TTPRTFGQGTKLEIKR

SEP ID NO: 19 (FAP#28 VH)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAISGSGD STYYADSVKGRFTISRDTSKNTLSLQMNSLRAEDTAVYYCAKDRWYGGNSVFDYW GQGTLVTVSS

SEP ID NQ:20 (FAP#28 VL)

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPS GVSNRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGGWVFGGGTKLTVLG

SEQ ID NO:21 (FAP scFv binder 28 (FAP#28))

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAISGSGD STYYADSVKGRFTISRDTSKNTLSLQMNSLRAEDTAVYYCAKDRWYGGNSVFDYW GQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYN YVSWYQQHPGKAPKLMIYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC CSYAGGWVFGGGTKLTVLG

SEQ ID NO:22 (Amino acid sequence of human FAP)

MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTF FPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLE SDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYL KQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDT DIP VIAYS YYGDEQ YPRTINIPYPKAGAKNPVVRIFIIDTTYP AYVGPQEVP VP AMIAS S DYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTG WAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVT QDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYA LVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMI LPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQ GDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASG

TGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVD YLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHM THFLKQCFSLSD

SEQ ID NO:23 (Amino acid sequence of mouse FAP) MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTY

FPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLES

DYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLK

QRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSD

IPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASS

DYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRT

GWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRV

TQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYY

ALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWY

KMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTA

FQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALA

SGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNV

DYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTH

MTHFLKQCFSLSD (Amino acid sequence of complete anti ■FAP(#28) CAR)

MLLLVTSLLLCELPHPAFLLIPASVLAQAAEVQLVESGGGLVQPGRSLRLSCAASGFT

FDDYAMHWVRQAPGKGLEWVSAISGSGDSTYYADSVKGRFTISRDTSKNTLSLQMN

SLRAEDTAVYYCAKDRWYGGNSVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQS ALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGV

SNRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGGWVFGGGTKLTVLGGQAGPTSG GSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG TCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP

R (Amino acid sequence of complete anti FAP(#10) CAR)

MLLLVTSLLLCELPHPAFLLIPASVLAQAAEVQLVESGGGLVQPGRSLRLSCAASGFT

FDDYAMHWVRQAPGKGLEWVSAISGSGDSTYYADSVKGRFTISRDTSKNTLSLQMN

SLRAEDTAVYYCAKDRWYGGNSVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQS

ALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGV

SNRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGGWVFGGGTKLTVLGGQAGPTSG

GSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG

TCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR

VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP

R

ID NO 26 (Amino acid sequence of complete anti FAP(#1) CAR)

EVQLVQSGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGS

TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSRYAPEGMDVWG

QGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNY

VSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCS

SYTSSSGVFGGGTQLTVLGGQAGPTSGGSAAATTTPAPRPPTPAPTIASQPLSLRPEAC

RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF MRP VQTTQEEDGC SCRFPEEEEGGCELRVKF SRS ADAP AYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR

ID NO (Amino acid sequence of complete anti FAP(#3) CAR) EVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLYPYGMDVWGQG TLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWY

QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYP PTFGQGTKLEIKRGQAGPTSGGSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTT QEEDGC SCRFPEEEEGGCELRVKF SRS ADAP AYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR

ID NO:28 (Amino acid sequence of complete anti ■FAP(#5) CAR)

QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTN

TLYNEKFKGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVT

VSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQK

SGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSFNPPT

FGGGTKLEIKGGQAGPTSGGSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQE

EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR

RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR

29 (Amino acid sequence of complete anti ■FAP(#7) CAR)

EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYYMSWIRQAPGRGLEWVASISTRTRS

STIYYADSVKGRVTISRDDAKNSLSLQMNNLRAEDTAIYYCARGGLSRFDSWGQGTL

VTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSW

YQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTS

SSTLEVFGTGTKLTVLGGQAGPTSGGSAAATTTPAPRPPTPAPTIASQPLSLRPEACRP

AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR

PVQTTQEEDGC SCRFPEEEEGGCELRVKF SRS ADAP AYQQGQNQLYNELNLGRREEY

DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD

GLYQGLSTATKDTYDALHMQALPPR (Amino acid sequence of complete anti ■FAP(#14) CAR)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGGILPIFGT

ANYAQKFQGRVTIT ADESTST AYMELS SLRSEDT AVYYCAREGPDF S S SFFNWFDPW

GQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLN

WYQQKPGGAPKILIYGAANLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY

TTPRTFGQGTKLEIKRGQAGPTSGGSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPA

AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP

VQTTQEEDGC SCRFPEEEEGGCELRVKF SRS ADAP AYQQGQNQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG

LYQGLSTATKDTYDALHMQALPPR

Claims

Claims

1) A chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), b) a transmembrane domain, and c) an intracellular signaling domain.

2) The CAR of claim 1, wherein said intracellular signaling domain comprises a stimulatory domain comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs) such as the stimulatory domain of CD3zeta and/or one or more co-stimulatory domain(s) such as CD28 and/or 4- IBB.

3) The CAR of claim 1 or 2, wherein said antigen FAP is expressed on a target cell.

4) The CAR of claim 3, wherein said target cell expressing FAP is present in a tumor microenvironment (TME) of a solid tumor or is a cancer cell expressing FAP, or wherein said target cell expressing FAP is associated with a heart disease.

5) The CAR of claim 4, wherein said target cell expressing FAP is a stromal cell and/or an endothelial cell of the TME of the solid tumor, or wherein said target cell that expresses FAP and that is associated with a heart disease is a stromal cell.

6) The CAR of claim 5, wherein said solid tumor is pancreatic cancer, non-small cell lung cancer, melanoma, ovarian cancer, breast cancer, or colorectal cancer , or wherein the heart disease is selected from the group consisting of cardiac fibrosis, hypertensive heart disease, diastolic dysfunction, heart failure with preserved ejection fraction, myocardial infarction, ischemic cardiomyopathy, hypertrophic cardiomyopathy, arrhythmia, atrial fibrillation, arrhythmogenic right ventricular dysplasia, dilated cardiomyopathy, an inherited form of heart disease, muscular dystrophy, infective cardiomyopathy, transplant cardiomyopathy, radiation induced cardiac fibrosis, an autoimmune related heart condition, sarcoid cardiomyopathy, lupus, a toxin related heart condition, a drug related heart condition, amyloidosis, diabetic cardiomyopathy, reactive interstitial fibrosis, replacement fibrosis, infiltrative interstitial fibrosis, and endomyocardial fibrosis. 7) An immune cell expressing a CAR according to any one of claims 1 to 6.

8) The immune cell according to claim 7 for use in immunotherapy.

9) An isolated nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), b) a transmembrane domain, and c) an intracellular signaling domain.

10) A vector comprising a nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen fibroblast activation protein (FAP), wherein the antigen binding domain comprises SEQ ID NO: 19 (VH) and SEQ ID NO:20 (VL), or SEQ ID NO: 13 (VH) and SEQ ID NO: 14 (VL), b) a transmembrane domain, and c) an intracellular signaling domain.