In a first aspect, the present invention relates to chimeric antigen receptors (CAR) composed of at least an extracellular domain, a transmembrane domain and an intracellular domain, the extracellular domain comprises a spacer domain located C-terminally to a ligand-binding-domain also present in the extracellular domain, whereby the spacer domain comprises at least part of the CD34 molecule. Further, the present invention relates to the nucleic acid molecule encoding the same as well as vectors and cells containing the same. The cells include engineered T-cells and NK-cells and derivatives thereof. Further, the present invention relates to a pharmaceutical composition comprising the CAR e.g. in form of a vector, a polynucleotide encoding the CAR or the CAR itself and, in addition, cells, cell lines or host cells accordingly. The CAR according to the present invention is useful in adoptive cell therapy. Finally, a method for enrichment or purification of CAR, in particular, of genetically engineered cells, cell lines or host cells expressing the CAR according to the present invention is provided.

PRIOR ART

The idea of exploiting the immune system to target e.g. cancer cells is not new and already well documented in the 19th century, when William Coley repeatedly inoculated live bacteria into malignant skin tumors of patients, with the idea to thereby stimulate the immune system to recognize and kill the tumor cells. Today, immunotherapies against cancers can be divided into three divergent but complementing strategies. Active immunotherapies enhance the patient's immune system to provoke an active immune response, leading to recognition of tumor-associated antigens and elimination of the malignant cells. Passive immunotherapies induce immune responses by administration of exogenously produced components such as lymphocytes and antibodies. Modulatory immunotherapies are thought to amplify endogenous immune responses against cancer cells by general enhancement of the responsiveness of the immune system. Active immunotherapy can involve either polyvalent vaccines that are derived from whole tumor cells and/or dendritic cells or antigen-specific vaccines.

T-cells are the best characterized effector cells mediating specific immunity against malignant or virus-infected cells. T-cells recognize specific antigens on target cells via their T-cell receptor (TCR) however only in association with HLA class I or class II. An important factor to understand the failure of autologous T-cells to recognize and destroy the malignant cells in cancer patients is the process of T-cell development in the thymus. A huge variety of T-cell receptors (TCR) capable of binding to antigens is created by V(D)J recombination of germ-line DNA in T-cell precursors and will be capable of recognizing (almost) any antigen that is present in the body or can be introduced by pathogens. However, natural thymic positive and negative selection processes in humans lead to the loss of >>95% of these T-cells and only permits the survival of autologous T-cells that express TCRs with low or no affinity towards endogenous antigens. This natural selection severely dampens/limits the ability of the patient's immune system to detect the HLA-restricted endogenous antigens that are often expressed on cancer cells. Therefore, genetic engineering of T-cells (and also NK-cells) to stably express activating receptors against selected tumor-associated antigens is an attractive approach to overcome these problems.

Strategies to broaden the adoptive cell therapy (ACT) concentrated on the development of autologous T-cells genetically engineered to express tumor-specific antigen receptors, so these cells can efficiently recognize and kill malignant cells. A summary of adoptive therapy of cancer with CAR redirected T-cells is provided e.g. in Hombach et al., Current Molecular Medicine, 2013, 13(1), 1-10. Therein, the CAR effects are summarized including co-stimulation activity as well as improvement and prolongation of the redirected anti-tumor T-cell response. In addition, the adverse effects of this kind of adoptive therapy are described including “cytokine storm” and “T-cell repression”. These antigen receptors can be classical T-cell receptors (TCRs) or chimeric antigen receptors (CARs) where single-chain antibody fragments (scFv) are fused with the cytoplasmic part of TCR ζ-chain (Dotti G, et al., Immunol Rev. 2014; 257(1):107-26 and Schamel W W, Reth M. Curr Opin Biotechnol 2012; 23(5):780-4.

While transgenic TCRs only recognize antigens/epitopes presented by MHC molecules, the major advantage of CARs is that they recognize the surface antigens on target cells in a non-MHC-restricted manner. The first generation of CARs did not include co-stimulatory signals such as CD28, 4-1BB, OX40 or 2B4 and in the majority of trials demonstrated no clear clinical benefits in patients. However, efficient target recognition and profound activation of the transgenic T-cells with specific and efficient killing of antigen-positive cells was achieved with 2ndgeneration CARs that included one co-stimulatory domain. Third generation CARs carry two co-stimulatory domains, thus providing three signals for the cell after binding of the CAR to the specific antigen peptide, but have not been used widely in research or clinical studies.

Most CARs are composed of the heavy and light chain of a monoclonal antibody scFv and co-stimulatory and different TCR signaling domains (see e.g Dotti G, et al., Immunol Rev. 2014; 257(1):107-26.

Here, a hinge aka spacer region links the antigen-recognizing domain of the scFv and the transmembrane and signaling cytoplasmic domains in the CAR constructs. Commonly, the constant CH2CH3 domain of IgG1 or IgG4 hinge regions were used due to the flexibility, lack of immunogenicity and easy adaptable modifications of the length (Guest R D, et al, J Immunother. 2005; 28(3):203-11). Also, quick detection of the CARs with these domains on the surface of transduced T-cells in combination with rapid enrichment procedures (MACS) made this spacer region attractive. Other authors successfully employed a hinge region based on human CD8 (e.g. for CD19 CARs). In comparison, the CD8 hinge is a rather short amino acid sequence and the spatial distance between the CAR and the target epitope in relation to the target cell membrane can be an activity-limiting factor. Another disadvantage is that identification and enrichment of transduced T-cells with monoclonal antibodies against the CD8 hinge region is not readily possible, as CD8 is naturally present on T-suppressor cells. In 2010, Hombach et al., Gene Ther. 2010; 17(10):1206-13, demonstrated that the IgG1 CH2CH3 spacer region is activated by cells that express either the FcγRI (CD64) or the FcγRII (CD32) Fc receptors. This unintended but specific activation followed by killing of the Fc receptor-positive cells was independent of the fact whether the targeted antigen structure was or was not present on the target cells. In 2015, Hudecek et al. demonstrated also for the IgG4 CH2CH3 domain that binding to CD32 and CD64 induces activation signals that leads to killing of cells independent from the presence of the CAR target antigen (Hudecek M, et al., Cancer Immunol Res. 2015; 3(2):125-35). Additionally, the authors demonstrated in vivo in mice with human T-cells, that Fc receptor-positive cells in the lungs of the animals activated the CAR transgenic T-cells. These activated T-cells then quickly disappeared in the animals due to activation-induced cell death. Importantly, these CAR-transduced T-cells failed to protect the animals from the CD19+ leukemic cells that were the intended target cells for the CARs. To prevent this loss of anti-tumor activity/specificity of the CAR positive T-cells in vivo in the presence of Fc receptor-positive cells, both groups introduced a few amino acid substitutions that prevent binding of these CH2CH3 domains to Fc receptors.

Recently, chimeric antigen receptors comprising an extracellular spacer which comprises at least part of the extracellular domain of the human low-affinity growth factor (LNGFR also known as NGFR or CD271) or a derivative thereof, are described in WO 2016/042461 A1. It is identified therein, that allegedly CARs containing the LNGFR as a spacer domain thereby substituting the known hinge CH2CH3 Fc domain allows to reduce any off-target immune responses and are not prematurely cleared by the host immune system. In addition, it is identified therein that transduced T-cells can be selected and tracked based on the LNGFR spaced constructs.

Rationale to Use a CD19 CAR for Initial Proof of Principle

In the last 5 years, human studies using CARs against the CD19 antigen which is uniformly present on all B-cell malignancies except plasma cell malignancies, and engineered TCRs against MAGE-A3 have demonstrated the huge therapeutic potential of these re-directed T-cells in patients where all other therapy approaches had ultimately failed. As even a single dose of re-infused T-cells can survive and expand for extended periods of time in the recipients, clinical cure of some malignancies has been achieved for months and years. After the initial proof of concept studies pioneered by Carl June and his colleagues for CD19+ leukemias (Porter D L, et al., N Engl J Med. 2011; 365(8):725-33) and six months event-free survival rate of 67% and an overall survival rate of 78% in these heavily pre-treated patients with refractory ALL, more than 100 investigational new drug applications (INDs) for CAR- and TCR-modified T-cells have been issued in the USA, with approximately ⅓ targeting CD19 on B-cell leukemias and lymphomas. Other important target antigen structures on malignant B-cells that are currently CD20 and CD22, both also (almost) B-cell lineage specific antigens.

However, administering genetically engineered T-cells was also associated with toxic side effects and even mortality (Porter DL, et al., N Engl J Med. 2011; 365(8): 725-33 and Linette GP, et al., Blood 2013; 122(6):863-71). Only second and third generation CAR constructs include co-stimulatory domains that will mediate maximum activation of the T-cell after engagement of the CAR. Therefore, in addition to the complete elimination of all leukemic cells, important on-target/off-tumor toxicities of B-cell lineage-specific CARs e.g. against CD19, CD20 and CD22 are the ablation of normal CD19/CD22/CD20+ B-cells, for as long as the genetically engineered T-cells are present. This side effect of B-cell lineage specific CARs leads to decreased immunoglobulin serum levels that can partially be compensated for by regular infusions of immunoglobulins. Interestingly, CD20 CARs also recognize a small proportion of malignant melanoma cells where CD20 is expressed. Elimination of this subpopulation of primitive cells is sufficient to induce remission in a fraction of these patients. In July 2016, the FDA stopped a Juno Therapeutics CD19 CAR phase I/II trial, after three deaths occurred in patients under 30 years of age with CD19+ malignant cells. Prior to the infusion of transduced autologous T-cells, these three patients had received chemotherapy with cyclophosphamide and fludarabine for mild lymphodepletion and in order to booster the engraftment of transduced cells (www.fiercebiotech.com/biotech/fda-halts-juno-car-t-trial-after-three-patient-deaths).

The CD34 antibody QBend10 is utilized in the CliniMACS device from Miltenyi Biotec GmbH, Germany. This antibody binds to the class II epitope in CD34, which is resistant to neuraminidase but sensitive to glycoprotease. The antibody binding site in CD34 was mapped as being present within a region of 16 amino acids by Philip et al., Blood 2014; 124(8):1277-87. Specifically, this 16 amino acid region containing the epitope of the QBend10 antibody is located between positions 42 to 57 in human CD34, see also SEQ ID. No. 2 corresponding to the CD34 protein encoded by the sequence of NM 001773.2 and the codon optimized nucleic acid sequence of Seq. ID No. 1.

In WO 2013/153391 A1 a polypeptide useful in adoptive cell therapy has been described. The epitope of the QBend10 antibody was inserted in a polypeptide framed by rituximab binding epitopes whereby these rituximab binding epitopes represent a suicide epitope. Therein, the polypeptide with the QBend10 binding epitope framed by the rituximab binding epitopes have been added to a transfer vector which co-expresses e.g. a CAR molecule via a T2A site. Thus, this polypeptide should allow on the one hand selecting cells which are identified as expressing the QBend10 binding epitope while on the other side, these cells allow for suicide due to the rituximab binding epitopes.

Miltenyi Biotec GmbH, Germany, provides clinically approved magnetic beads based systems allowing enrichment or purification of specific cell types based on antigen antibody binding whereby the antibody is bound to the magnetic or parametric particles accordingly. Thus, automated cell labelling and enrichment is possible.

Production of genetically engineered cells for adaptive cell therapy, typically, T-cells or NK-cells, are performed by transfecting or transducing said cells with appropriate means including plasmids, vectors like viral vectors etc. After transfection or transduction, these cells are cultured for selection of the desired cells accordingly. Typically, the selection is based on known selection systems including usage of resistance genes (e.g. neomycin or puromycin, see Fehse B. et al. Blood. 2004; 104(10):3408-9.) or enrichment by expression of MACS-selectable special proteins on the cell surface e.g. truncated CD34, truncated CD19, truncated EGFR or similar, see e.g Philip et al., see above, Di Stasi et al., N Engl J Med. 2011; 365(18):1673-83. (CD19), and Wang X, et al. 2011; 118(5):1255-63 EGFR.

However, these techniques require some time to generate/grow the respective cells including long-term cultivation. That is, the transduction of human blood lymphocytes is effected by standard techniques including cultivation and selection based on e.g. antibiotic resistance accordingly. However, there is still a need for improved CAR molecules and cells expressing the same, in particular, in view of immunogenicity and fast availability.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

In a first aspect, the present invention relates to a chimeric antigen receptor (CAR) composed of at least the following domains:

an extracellular domain, a transmembrane domain and an intracellular domain whereby the extracellular domain comprises a ligand-binding-domain, like an antigen-binding-domain, and a spacer domain C-terminal to the ligand-binding-domain and whereby the spacer domain comprises at least part of the CD34 molecule. In particular, the CAR is a CAR molecule wherein the spacer domain comprises at least one of the sequences of SEQ ID Nos. 5, 6, 7, 8 or a sequence at least 90% identical thereto. In an embodiment, the spacer domain consists of the above sequences. Further the present invention relates to a polynucleotide encoding the CAR according to the present invention, a vector comprising said polynucleotide as well as a cell, cell line or host cell containing the vector or containing a nucleic acid molecule according to the present invention or expressing the CAR molecule according to the present invention accordingly.

In addition, an embodiment of the present invention relates to a pharmaceutical composition comprising the CAR as defined herein as well as a polynucleotide encoding the same or a vector containing said polynucleotide.

In addition, a CAR is provided suitable in adoptive cell therapy. Further, it has been recognized that using at least part of the CD34 molecule as a hinge region in the production of a CAR molecule has beneficial effects.

Further, a therapeutic method, namely adoptive transfer of genetically modified cells is provided using the cells or CAR molecule according to the present invention. Of course, the method for treating and/or preventing a disease in a subject may also be conducted with the vector containing the nucleotide sequence encoding said CAR molecule accordingly. That is, a method for treating e.g. cancer or other diseases involving an immune response against cells, like cancer cells, based on the CAR molecule according to the present invention is provided.

