WO2017179015A1 - Compositions for the treatment of cancer - Google Patents

Abstract

The present invention provides chimeric antigen receptors (CARs) comprising: a target binding domain capable of selectively binding placenta-like chondroitin sulfate A (pl-CSA); a spacer region; a transmembrane domain; and at least one costimulatory domain. In one embodiment, methods of treating cancer in a human are provided comprising administering the immunomodulatory cell of the present invention said human. In one embodiment, the methods further comprising expanding a population of said engineered immunomodulatory cell ex vivo prior to administering to said human are provided.

Description

COMPOSITIONS FOR THE TREATMENT OF CANCER

FIELD OF THE INVENTION

The invention relates to engineered chimeric antigen receptors (CARs) or immunomodulatory cells expressing CARs and methods of treating cancer.

BACKGROUND

Adoptive transfer of T cells directed against antigens expressed by neoplastic cells is an immunotherapeutic approach that has proven effective in some patients. Adoptive T cell therapy (ATCT) involves the use of either natural T cells or T cells transduced with chimeric antigen receptors (CARs) or T cell receptors (TCRs) directed against tumor cell associated antigens. CARs are typically engineered to contain three regions. The N-terminal extracellular region dictates the antigen specificity of the CAR and is typically encoded by a single chain fragment variable region (scFv) derived from the linked VH and VL domains of the antigen binding region of a monoclonal antibody (mAb) specific for the intended targeted antigen. This ligand binding component is followed by a flexible hinge sequence, and one or more intracellular signaling domains derived from or associated with immunomodulatory cell signaling. Alternatively, the signaling domain may be directed to another type of immunomodulatory cell such as an NK cell and may comprise, for instance, NKG2D, DAP10, or DAP12 or a functional fragment and/or derivative thereof. The main advantage of CAR technology is that it combines the effector functions of T lymphocytes or other living immune cell carriers with the ability of antibodies to specifically bind antigens with an engineered affinity in a non-MHC restricted fashion.

T cells, especially cytotoxic T cells, play important roles in anti-tumor immunity (Rossing and Brenner (2004) Mol. Ther. 10:5-18). Adoptive transfer of engineered tumor-specific T cells into patients provides a means to treat cancer (Sadelain, et al. (2003) Nat. Rev. Cancer 3 : 35-45). Genetic modification of primary T cells with tumor-specific immunoreceptors, such as full-length T cell receptors or chimeric T cell receptor molecules or CARs can be used for redirecting T cells against tumor cells (Stevens, et al. (1995) J. Immunol. 154:762-771; Oelke, et al. (2003) Nat. Med. 9: 619- 624; Stancovski, et al. (1993) J. Immunol. 151 : 6577-6582; Clay, et al. (1999) J. Immunol. 163: 507- 153). Natural killer (NK) cells are innate effector cells serving as a first line of defense against certain viral infections and tumors (Biron, et al. (1999) Annu. Rev. Immunol. 17: 189-220; Trinchieri (1989) Adv. Immunol. 47: 187-376). NK cells have the ability to "sense" if cells are transformed, infected, or stressed to discriminate between abnormal and healthy tissues. According to the missing self phenomenon (Karre, et al. (1986) Nature (London) 319: 675-678), NK cells accomplish this by looking for and eliminating cells with aberrant major histocompatibility complex (MHC) class I expression; this has been validated by showing that NK cells are responsible for the rejection of the MHC class I-deficient lymphoma cell line RMA-S, but not its parental MHC class I-positive line RMA.

Natural killer (NK) cells can recognize tumor cells as targets and as such may be useful for immunotherapy of cancer (Vivier et al. 2011 , Science 331 : 44-49; Ruggeri et al. 2002, Science 295 : 2097-2100; Cooley et al. 2010, Blood 116: 2411-2419; Miller et al. 2005, Blood 105: 3051-3057; Rubnitz et al. 2010, J Clin Oncol. 28: 955-959). Infusions of NK cells have been used to treat patients with various forms of cancer (Vivier et al. 2011, Science 331 : 44-49; Caligiuri, 2008, Blood 112(3): 461-469; Ruggeri et al. 2002, Science 295: 2097-2100; Miller et al. 2005, Blood 105: 3051-3057). More recently, approaches to engineer NK cells to express immune-receptors have been proposed as a potential means to redirect them directly to tumors (PMID26640245; PMID26155832).

Placental-like chondroitin sulfate (pl-CS) is a differential form of chondroitin sulfate A which in healthy humans is only expressed in placenta. However, it has been found in a wide range of human tumors but not in healthy tissues. These characteristics make it an interesting molecule to target with genetically modified lymphocytes to express chimeric antigen receptors (CARs). The natural binder of pl-CS is VAR2CSA, an anchor protein expressed in Plasmodium falciparum infected erythrocytes. Its minimal binding domain has been described by Salanti, et al. (Cancer Cell. 28(4): 500-14 (2015)). These authors showed specific killing of tumor cells (melanoma, non- Hodgkin's lymphoma & prostate cancer) in mouse xenograft models using a recombinant form of the minimal binding domain (rVAR2) conjugated with diphtheria toxin or hemiasterlin.

Thus, there is a novel opportunity and unmet medical need for new compositions and methods including new CAR constructs for use in immunomodulatory cells including T cell and NK cell subpopulations for the treatment of cancer.

SUMMARY OF THE INVENTION

The present invention provides chimeric antigen receptors (CARs) comprising:

a target binding domain capable of selectively binding placenta-like chondroitin sulfate A (pl-CSA);

a spacer region;

a transmembrane domain; and

at least one costimulatory domain. In one embodiment, methods of treating cancer in a human are provided comprising administering the immunomodulatory cell of the present invention said human. In one embodiment, the methods further comprise expanding a population of said engineered immunomodulatory cell ex vivo prior to administering to said human. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Placental-like CS as a new target candidate for CAR-T cells. FIG. 2: Designing VAR2CSA-CAR Architecture - schematic plan. FIG. 3: Designing VAR2CSA-CAR Architecture - classic co-stimulatory domains. FIG. 4: Designing VAR2CSA-CAR Architecture - unique architecture features. FIG. 5: Co- Stimulatory molecules in CAR-T cells - starting point. FIG. 6: TRAF Family role in signal transduction pathway. FIG. 7: DAP10/DAP12 role in signal transduction pathway. FIG. 8: Expanded CAR co-stimulatory domain tool box. FIG. 9: VAR2CSA Binders.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention chimeric antigen receptors (CARs) are provided comprising: a target binding domain capable of binding placenta-like chondroitin sulfate A (pl-CSA); a spacer region;

a transmembrane domain; and

at least one costimulatory domain.

In one embodiment, the target binding domain comprises a VAR2CS A polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to DBL2X (SEQ ID NO: 7). In another embodiment, the target binding domain comprises a VAR2CSA polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to DBL2Xb (SEQ ID NO: 3). In yet another embodiment, the target binding domain comprises a VAR2CSA polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to ID1 (SEQ ID NO: 2). In one embodiment of the present invention, the target binding domain comprises a VAR2CSA polypeptide comprising an ID2a domain of VAR2CSA. In yet another embodiment, the target binding domain comprises a

VAR2CSA polypeptide comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.

In another aspect of the present invention, the target binding domain comprises a scFv capable of binding to placenta-like chondroitin sulfate A (pl-CSA). In some aspects, the target binding domain may be a Fab or a domain antibody or a combination of any pl-CSA binding fragment.

The amino acid sequence of the portion of VAR2CS A starting with the first amino acid of the ID1 domain and ending with the last amino acid of the ID2a domain and is shown in SEQ ID NO: 1. The amino acid sequence of the ID 1 domain of VAR2CS A is shown in SEQ ID NO: 2, and the amino acid sequence of the DBL2Xb domain of VAR2CSA is shown in SEQ ID NO: 3. The amino acid sequence of the ID2a domain of VAR2CSA is shown in SEQ ID NO: 4

VAR2CSA is a large multi-domain adhesion protein constituted by six Duffy -binding-like domains (DBLX), a cysteine rich inter-domain region (CIDR_PAM) between DBL2X and DBL3X and several inter-domains. The entire FCR3 protein sequence (GU249598) is shown in SEQ ID NO: 5. The amino acid sequences of the six DBLX domains, DBL1X DBL2X, DBL3X, DBL4X, DBL5X, and DBL6X, are shown in SEQ ID NOS: 6 - 11, respectively.

In one aspect, the chimeric antigen receptors of the present invention comprise a spacer region comprising at least one, or multiples of, domains 2, 3 or 4 or a combination thereof of a CD4 molecule; the amino acid sequence of human CD4 is shown in SEQ ID NO: 12 (UniProt, ID NO: P01730). In another aspect, the spacer region comprises CD8 stalk; the amino acid sequence of a CD8 spacer is shown in SEQ ID NO: 20. In another aspect, the spacer region comprises the CH2- CH3 fragment of IgGl; the amino acid sequence of the CH2-CH3 region is presented below as SEQ ID NO:22

In one aspect, the spacer region comprises domain 4 of a CD4 molecule. In one aspect, the spacer region comprises domains 3 and 4 of a CD4 molecule. In one aspect, the spacer region comprises domains 2, 3 and 4 of a CD4 molecule. In another embodiment, the spacer region comprises domains 2 and 3 and two copies of domain 4 of a CD4 molecule. In one embodiment the domain 2 of a CD4 molecule comprises amino acids 126 to 203 of SEQ ID NO: 12. In one embodiment the domain 3 of a CD4 molecule comprises amino acids 204 to 317 of SEQ ID NO: 12. In one embodiment, the domain 4 of a CD4 molecule comprises amino acids 318 to 374 of SEQ ID NO: 12.

In some instances, spacer region comprises an amino acid sequence having at least 90% sequence identity to amino acids 1 to 45 as set forth in SEQ ID NO: 20 (CD8). In some instances, the spacer region comprises an amino acid sequence having at least 90% sequence identity to amino acids 1 to 110 as set forth in SEQ ID NO: 22 (CH2-CH3). In yet another embodiment, the spacer region of the CAR comprises an amino acid sequence having at least 90% sequence identity to amino acids 111 to 217 as set forth in SEQ ID NO: 22 (CH2-CH3). In yet another embodiment, the spacer region of the CAR comprises an amino acid sequence having at least 90% sequence identity to amino acids 1 to 217 as set forth in SEQ ID NO: 22 (CH2-CH3).

In one embodiment of the present invention, the target binding domain of the CAR has a binding affinity of less than about 500 nanomolar (nM) for pl-CSA. In one embodiment, the chimeric antigen receptor of the present invention comprises a transmembrane (TM) domain comprising the transmembrane domain of CD4. In one aspect, the chimeric antigen receptor comprises a spacer region comprising a domain of CD8. In one aspect, the chimeric antigen receptor of the present invention comprises a transmembrane domain comprising the transmembrane domain of CH2-CH3 of IgGl. In one embodiment, the transmembrane domains of the CARs of the present invention comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25 (TM region and spacer of CD4). In one embodiment, the transmembrane domains of the CARs of the present invention comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 (TM region of CD8). In one embodiment, the transmembrane domains of the CARs of the present invention comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23 (TM region of CH2-CH3). In one embodiment, the costimulatory domain of the CARs of the present invention comprise a stimulatory molecule. Suitably, the stimulatory molecule is a CD3 zeta (CD3z or CO3Q signaling domain or functional fragment or variant thereof. In one embodiment, the costimulatory domain comprises at least one first intracellular effector domain or a functional variant thereof. Suitably, the first costimulatory domain comprises at least one intracellular effector domain or a functional variant thereof, selected from CD28, CD27, 4-1BB, OX40, ICOS, CD30, CD40, PD-1, CD2, CD7, LIGHT, NKG2C, NKG2D, CD150, NTBA, B7-H3, SLAM1, SLAM 6, TRAF2, TRAF3, TRAF5, TRAF6, TRAF7, 2B4, DAPIO, DAP12, lymphocyte function-associated antigen- 1(LF A- 1) or any combination thereof. Suitably, the costimulatory domain of the CARs of the present invention comprise a Οϋ3ζ signaling domain and at least one first intracellular effector domain (or co-stimulatory signaling domain) or functional variant thereof selected from CD28, CD27, 4-1BB, OX40, ICOS, CD30, CD40, PD-1, CD2, CD7, LIGHT, NKG2C, NKG2D, CD150, NTBA, B7-H3, SLAM1, SLAM 6, TRAF2, TRAF3, TRAF5, TRAF6, TRAF7, 2B4, DAP10, DAP 12, lymphocyte function-associated antigen- l(LFA-l) or any combination thereof. In some instances, the costimulatory domain comprises at least one additional intracellular effector domain (or co-stimulatory signaling domain) or functional variant thereof. Suitably, the at least one additional co-stimulatory intracellular effector domain may be selected from CD28, CD27, 4-lBB, OX40, ICOS, CD30, CD40, PD-1, CD2, CD7, LIGHT, NKG2C, NKG2D, CD150, NTBA, B7-H3, SLAM1, SLAM 6, TRAF2, TRAF3, TRAF5, TRAF6, TRAF7, 2B4, DAP10, DAP12, lymphocyte function-associated antigen-1 (LFA-1). As is understood in the art, the intracellular effector domain or signaling domain of a CAR can be selected to activate the immunomodulatory cell carrying said CAR. For example, an intracellular effector domain that is known to activate a T-cell such as, but not limited to, ICOS, OX40, 4-lBB, or CD28 could be selected if the immunomodulatory cell is derived from a T-cell. While an intracellular effector domain such as, but not limited to, 4-lBB, NKG2D, DAP10 or DAP12 could be selected if the immunomodulatory cell is derived from an NK cell. The amino acid sequence of Οϋ3ζ (NCBI Reference Sequence: NP 000725.1; amino acids

52-163) is shown in SEQ ID NO: 13. The amino acid sequence of CD28 (NCBI Reference Sequence: XP 011510498.1; amino acids 96-137) is shown in SEQ ID NO: 14. The amino acid sequence of 4- 1BB (NCBI Reference Sequence: NP 001552.2; amino acids 187-255) is shown in SEQ ID NO: 15. The amino acid sequence of OX-40 (NCBI Reference Sequence: NP 003318.1; amino acids 215-277) is shown in SEQ ID NO: 16. The amino acid sequence of 2B4 (NCBI Reference Sequence:

NP 057466.1; amino acids 225-365) is shown in SEQ ID NO: 17. The amino acid sequence of DAP10 (NCBI Reference Sequence: NP 055081.1; amino acids 7-51) is shown in SEQ ID NO: 18. The amino acid sequence of DAP12 (NCBI Reference Sequence: NP 003323.1; amino acids 12-113) is shown in SEQ ID NO: 19. The amino acid sequence of SLAM1 (NCBI Reference Sequence: NP 003028.1; amino acids 238-335) is shown in SEQ ID NO: 26. The amino acid sequence of

CD84 (NCBI Reference Sequence: NP 003865.1; amino acids 226-345) is shown in SEQ ID NO: 27. The amino acid sequence of SLAM6 (NCBI Reference Sequence: NP 443163.1; amino acids 227- 331) is shown in SEQ ID NO: 28.

In one embodiment, the present invention provides polynucleotide encoding the chimeric antigen receptors of the present invention. In another embodiment, expression vectors are provided comprising the polynucleotide of the present invention.

In yet another embodiment, immunomodulatory cells comprising the chimeric antigen receptors and/or vectors expressing the CARs of the present invention are provided. The immunomodulatory cells of the present invention may be derived from an inflammatory T- lymphocyte, cytotoxic T-lymphocyte, helper T-lymphocyte, naive or stem-cell like cell, central memory type T cell, or a Natural Killer Cell.

Also provided herein are immunomodulatory cells for use in therapy. Also provided herein are methods of engineering an immunomodulatory cell, comprising:

(a) providing an immunomodulatory cell;

(b) introducing an expression vector comprising a polynucleotide encoding a CAR of the present invention into said immunomodulatory cell; and

(c) expressing said expression vector in the immunomodulatory cell. In one embodiment, methods of treating cancer in a human are provided comprising administering the immunomodulatory cell of the present invention said human. In one embodiment, the methods further comprising expanding a population of said engineered immunomodulatory cell ex vivo prior to administering to said human.

The term "chimeric antigen receptors" ("CARs") as used herein, refers to an engineered receptor which comprises an extracellular target binding domain, a spacer region, a transmembrane region, and one or more intracellular effector domains or signaling domains. CARs have also been referred to as chimeric T cell receptors or chimeric immunoreceptors (CIRs). CARs are genetically introduced into immunomodulatory cells such as hematopoietic cells, including but not limited to T cells and NK cells, to redirect specificity for a desired cell-surface antigen. As used herein "target binding domain" refers to an oligo- or polypeptide that is capable of binding a specific target, such as an antigen or ligand. In particular, the target may be a cell surface molecule. For example, the target binding domain may be chosen to recognize a target that acts as a cell surface marker on pathogenic cells, including pathogenic human cells, associated with a particular disease state. In some instances, the target may be specific to a tumor cell. In some embodiments, the target binding protein can be a single chain Fv (scFv). In some aspects the scFv may be designed to bind to pl-CS A. In some embodiments the target binding protein can be a VAR2CSA polypeptide capable of binding to pl-CSA, in particular, to a sulfated sugar moiety on pl-CSA.

As used herein the term "VAR2CSA polypeptide" refers to the extracellular part of a specific Erythrocyte Membrane Protein 1 (PfEMPl) protein expressed by Plasmodium falciparum interacting with chondroitin sulfate proteoglycans (CSPG) and fragments and/or variants thereof with the ability to bind placenta-like chondroitin sulfate A (pl-CSA) that could be presented on a proteoglycans (CSPG). In some embodiments, the VAR2CSA polypeptide according to the present invention at least comprises the protein fragment of VAR2CSA, which fragment comprises the ID1 domain or a functional fragment and/or variant thereof. In some embodiments, the VAR2CS A polypeptide according to the present invention at least comprises the protein fragment of VAR2CSA, which fragment comprises the DBL2Xb domain of VAR2CS A or functional fragments and/or variants thereof. In some embodiments, the VAR2CSA polypeptide according to the present invention at least comprises the protein fragment of VAR2CSA, which fragment comprises one or more of a) ID 1 (SEQ ID NO: 2), and b) DBL2Xb (SEQ ID NO: 3), and c) ID2a (SEQ ID NO: 4).

The term "ID 1" as used herein refers to a domain of VAR2CSA characterized by having an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence identified by 1-152 of SEQ ID NO: 1 or SEQ ID NO: 2.

The term "DBL2Xb" as used herein refers to a domain of VAR2CSA characterized by having an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with to amino acid sequence identified by 153-577 of SEQ ID NO: 1 or SEQ ID NO: 3. The term "ID2a" as used herein refers to a domain of VAR2CSA characterized by having an amino acid sequence of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, or at least 62, such as the 63 consecutive amino acids from the N-terminal of amino acids 578-640 of SEQ ID NO: 1 or SEQ ID NO: 4 and with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to such a sequence of consecutive amino acids.

The term "spacer region" as used herein, refers to an oligo- or polypeptide that functions to link the transmembrane domain to the target binding domain in a CAR. This region may also be referred to as a "hinge region" or "stalk region." The spacer region may be derived from, but is not limited to, CD8, CD4, and/or CH2/CH3 amino acid sequences. The length and flexibility of the spacer region plays an important role in the functionality of the CAR. The length of the spacer can be varied depending on the position of the target epitope in order to maintain a set distance (e.g. , about 14 nanometers) upon CAR:target binding. Immune synapses are described to be about 4 nanometers. With respect to T cells and NK cells the immune synapse is typically about 15 nanometers to about 20 nanometers. This distance between immunomodulatory cell (for instance a T cell or NK cell) and target cell is important for the correct initiation and performance of the signaling pathway downstream of the membrane interaction in the immunomodulatory cell. The term "domain" refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. The term "transmembrane domain" as used herein refers to the part of the CAR molecule which traverses the cell membrane of an immunomodulatory cell.

As used herein the term "immunomodulatory cell" refers to any cell capable of being transduced, transformed or transfected to carry a CAR or a modified TCR and/or a polynucleotide encoding a CAR or modified TCR. The immunomodulatory cells of the present invention may be or may be derived from an inflammatory T-lymphocyte, cytotoxic T-lymphocyte, regulatory T- lymphocyte, helper T-lymphocyte or a Natural Killer Cell. Additionally, immunomodulatory cells of the present invention may be a naive or a stem-cell. Immunomodulatory cells also include stem cells or other pluripotent precursors that are differentiated to the T cell lineage and then transducer.

Immunomodulatory cells may include autologous as well as allogeneic immune cell subsets that could be used from cross donors.

The term "intracellular effector domain" (also referred to as the "signaling domain") as used herein refers to the domain in the CAR which is responsible for intracellular signaling following the binding of the target binding domain to the target. The intracellular effector domain is responsible for the activation of at least one of the normal effector functions of the immunomodulatory cell in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines. Alternatively, the intracellular effector domain may be directed to another type of immunomodulatory cell such as an NK cell and may comprise NKG2D, DAP10, or DAP12 or a functional fragment and/or derivative thereof. As is understood in the art the signaling domain of a CAR can be selected to activate the immunomodulatory cell carrying said CAR.

The term "single variable domain" refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain. As used herein a single variable domain also includes a "single chain variable fragment" or "scFv." A single variable domain is capable of binding an antigen or epitope independently of a different variable region or domain. A "domain antibody" or "dAb™" may be considered the same as a "single variable domain". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid VHH dAbs™. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from camelid species including bactrian and dromedary camels, llamas, vicunas, alpacas, and guanacos, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art, and such domains are considered to be "single variable domains". As used herein VH includes camelid VHH domains.

As used herein, a "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for preparing scFvs are known, and include methods described in U.S. Pat. No. 4,694,778, Science, vol. 242, pp. 423-442 (1988), Nature, vol. 334, p. 54454 (1989), and Science, vol. 242, pp. 1038-1041 (1988).

"Affinity" is the strength of binding of one molecule, e.g., the target binding protein of the CAR molecule of the invention, to another, e.g., its target antigen, at a single binding site. The binding affinity of an antigen binding protein to its target may be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE™ analysis).

Avidity is the sum total of the strength of binding of two molecules to one another at multiple sites, e.g. , taking into account the valency of the interaction.

The term "antigen" as used herein refers to a structure of a macromolecule which is selectively recognized by an antigen binding protein. Antigens include but are not limited to protein (with or without polysaccharides) or proteic composition comprising one or more T cell epitopes. As is contemplated herein, the target binding domains of the CAR molecules of the present invention may recognize a sugar side chain of a glycoprotein rather than a specific amino acid sequence or of a macromolecule. Thus, the sugar moiety or sulfated sugar moiety serves as an antigen.

The term "epitope" as used herein refers to that portion of the antigen that makes contact with a particular binding domain, e.g., the target binding domain of the CAR molecule. An epitope may be linear or conformational/discontinuous. A conformational or discontinuous epitope comprises amino acid residues that are separated by other sequences, i.e., not in a continuous sequence in the antigen's primary sequence. Although the residues may be from different regions of the peptide chain, they are in close proximity in the three dimensional structure of the antigen. In the case of multimeric antigens, a conformational or discontinuous epitope may include residues from different peptide chains. Particular residues comprised within an epitope can be determined through computer modeling programs or via three-dimensional structures obtained through methods known in the art, such as X-ray crystallography. As is contemplated herein the term epitope includes post-translational modification to a polypeptide that can be recognized by an antigen binding protein or domain, such as sugar moiety of a glycosylated protein.

Sequence identity as used herein is the degree of relatedness between two or more amino acid sequences, or two or more nucleic acid sequences, as determined by comparing the sequences. The comparison of sequences and determination of sequence identity may be accomplished using a mathematical algorithm; those skilled in the art will be aware of computer programs available to align two sequences and determine the percent identity between them. The skilled person will appreciate that different algorithms may yield slightly different results.

"Percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the "Identities" value, expressed as a percentage, that is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed. Such pair-wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein.

"Percent identity" between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair- wise BLASTP alignment is performed. Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein.

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

As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g. , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms "individual," "subject," and "patient" are used herein interchangeably. In one embodiment, the subject is a mammal, such as a primate, for example a marmoset or monkey, or a human. In a further embodiment, the subject is a human.

The CAR described herein may also be used in methods of treatment of a subject in need thereof. Treatment can be therapeutic, prophylactic or preventative. Treatment encompasses alleviation, reduction, or prevention of at least one aspect or symptom of a disease and encompasses prevention or cure of the diseases described herein.

The CAR described herein is used in an effective amount for therapeutic, prophylactic or preventative treatment. A therapeutically effective amount of the antigen binding protein described herein is an amount effective to ameliorate or reduce one or more symptoms of, or to prevent or cure, the disease.

As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and, in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as "liquid tumors." Specific examples of clinical conditions based on hematologic tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS) and Waldenstrom's

macroglobulinemia (WM); lymphomas such as non-Hodgkin's lymphoma, Hodgkin's lymphoma; and the like.

The cancer may be any cancer in which an abnormal number of blast cells or unwanted cell proliferation is present or that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute

myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocyte (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.

Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and/or extranodal sites. Lymphoid cancers include B- cell malignancies, which include, but are not limited to, B-cell non-Hodgkin's lymphomas (B-NHLs). B-NHLs may be indolent (or low-grade), intermediate-grade (or aggressive) or high-grade (very aggressive). Indolent B cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa- associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B- NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B- NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman's disease. NHL may also include T-cell non-Hodgkin's lymphomas (T-NHLs), which include, but are not limited to T-cell non-Hodgkin's lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.

Hematopoietic cancers also include Hodgkin's lymphoma (or disease) including classical Hodgkin's lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma, lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocyte depleted Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, MGUS, plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues which include hematopoietic cells referred herein to as "hematopoietic cell tissues" include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings. The term "anti-tumor effect" as used herein, refers to a biological effect which can be manifested by various means, including but not limited to, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in tumor cell proliferation, a decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

By the term "stimulation," used in the context of immune-receptor engineered TCR/CAR T/CAR NK cells, is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 or CAR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the CAR/CD3 or TCR/CD3 complex.

Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like.

A "stimulatory molecule," used in the context of CAR T cells or CAR NK cells, means a molecule expressed by a T cell that provide the primary cytoplasmic signaling sequence(s) that regulate primary activation of the immune-receptor engineered cell in a stimulatory way for at least some aspect of the T cell signaling and/or NK cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of IT AM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcRy, FcR , CD3y, CD3A, CD3e, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS") and CD66d. In a specific CAR of the invention, the cytoplasmic signaling molecule in any one or more CARS of the invention, including CARs comprises a cytoplasmic signaling sequence derived from CD3C,. In a specific CAR of the invention, the cytoplasmic signaling sequence derived from CD3C, is the sequence provided as SEQ ID NO: 13 or the equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, ape and the like. The CD3 (cluster of differentiation 3) T-cell co-receptor helps to activate the cytotoxic T-

Cell. It consists of a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3e chains. These chains associate with a molecule known as the T-cell receptor (TCR) and the ζ-chain(zeta-chain) to generate an activation signal in T lymphocytes. The TCR, ζ-chain, and CD3 molecules together comprise the TCR complex.

As used herein "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein provided as GenBank Acc. No. BAG36664.1, or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like, and a "zeta stimulatory domain" or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof. In one aspect, the "zeta stimulatory domain" or a "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO 13.

The CD3y, CD35, and CD3e chains are highly related cell-surface proteins of

the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. Phosphorylation of the IT AM on CD3 renders the CD3 chain capable of binding an enzyme called ZAP70 (zeta associated protein), a kinase that is important in the signaling cascade of the T cell.

A "costimulatory molecule" refers to the cognate binding partner on an immunomodulatory cell such as a T cell or NK cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell or NK cell, such as, but not limited to, activation and/or proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la/CD18) and 4-lBB (CD 137). Examples of the intracellular effector domain comprising a secondary cytoplasmic signaling sequence that can be used in the present invention include sequences derived from CD2, CD4, CD5, CD8a, CD8 , CD28, CD134, CD 137, ICOS, and CD154. Specific examples thereof include peptides having sequences of amino acid numbers 236 to 351 of CD2 (NCBI Ref Seq: NP 001758.2), amino acid numbers 421 to 458 of CD4 (NCBI Ref Seq: NP_000607.1), amino acid numbers 402 to 495 of CD5 (NCBI Ref Seq: NP__055022.2), amino acid numbers 207 to 235 of CD8.alpha. (NCBI Ref Seq: NP_001759.3), amino acid numbers 196 to 210 of CD83 (Gen Bank: AAA35664.1), amino acid numbers 181 to 220 of CD28 (NCBI Ref Seq: NP_006130.1), amino acid numbers 214 to 255 of CD137 (4-1BB, NCBI Ref Seq: NP_001552.2), amino acid numbers 241 to 277 of CD134 (OX40, NCBI Ref Seq: NP_003318.1), and amino acid numbers 166 to 199 of ICOS (NCBI Ref Seq: NP 036224.1), and their variants having the same function as these peptides have.

Many NK cell activating receptors belong to the Ig superfamily (IgSF) (such receptors also may be referred to as Ig-like receptors or "ILRs" herein). Activating ILR NK receptors (AILRs) include, e.g. , CD2, CD16, CD69, DNAX accessory molecule-1 (DNAM-1), 2B4, NK1.1; killer immunoglobulin (Ig)-like activating receptors (KARs); ILTs/LIRs; and natural cytotoxicity receptors (NCRs), such as NKp44, NKp46, and NKp30. Several other activating receptors belong to the CLTR superfamily {e.g., NKRP-1, CD69; CD94/NKG2C and CD94/NKG2E heterodimers, NKG2D homodimer, and in mice, activating isoforms of Ly49, such as Ly49A-D). Still other activating receptors (e.g. , LFA-1 and VLA-4) belong to the integrin protein superfamily and other activating receptors may have even other distinguishable structures. Many activating receptors possess extracellular domains that bind to MHC-I molecules, and cytoplasmic domains that are relatively short and lack the immunoreceptor tyrosine-based inhibition motif (ITIM) signaling motifs characteristic of inhibitory NK receptors. The transmembrane domains of these receptors typically include a charged amino acid residue that facilitates their association with signal transduction- associated molecules, e.g., CD3ζ, FceRIy, DAP12, and DAP10 (2B4, however, appears to be an exception to this general rule), which contain short amino acid sequences termed an "immunoreceptor tyrosine-based activating motif' (ITAMs) that propagate NK cell-activating signals. Receptor 2B4 contains 4 Immunoreceptor Tyrosine-based Switch Motifs (ITSMs) in its cytoplasmic tail. ITSM motifs can also be found in NKCARs CS1/CRACC and NTB-A. The cytoplasmic domains of 2B4 and SLAM contain two or more unique tyrosine-based motifs that resemble motifs presents in activating and inhibitory receptors and can recruit the SH2-domain containing proteins SHP-2 and SLAM-associated protein (SAP).

As used herein "4- IBB" is defined as member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, ape and the like; and a "4- IBB costimulatory domain" are defined amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. In one aspect, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 15 or the equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, ape and the like. An "effective amount" as used herein, means an amount which provides a therapeutic or prophylactic benefit.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

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

A "transfer vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid 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 "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to further 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 transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, gamma retroviral vectors, lentiviral vectors, and the like. "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g. , lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

A "lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

A "lentiviral vector" is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al. Mol. Ther. 17(8): 1453-1464 (2009). Other Examples or lentivirus vectors that may be used in the clinic as an alternative to the pELPS vector, include but not limited to, e.g. , the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ 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

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al. J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al. Mol. Cell. Probes 8: 91-98 (1994)). The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

By the term "specifically binds," and grammatical variations thereof as used herein with respect to an antibody, is meant an antibody or antibody fragment which recognizes and binds with a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody. The term "therapeutic" as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

The term "therapeutically effective amount" refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

By the term "treating" and grammatical variations thereof as used herein, is meant therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate or prevent the condition of one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition, (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, (4) to slow the progression of the condition or one or more of the biological manifestations of the condition and/or (5) to cure said condition or one or more of the biological manifestations of the condition by eliminating or reducing to undetectable levels one or more of the biological manifestations of the condition for a period of time considered to be a state of remission for that manifestation without additional treatment over the period of remission. One skilled in the art will understand the duration of time considered to be remission for a particular disease or condition. Prophylactic therapy is also contemplated thereby.

The skilled artisan will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing cancer, such as when a subject has a strong family history of cancer or when a subject has been exposed to a carcinogen.

The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The phrase "under transcriptional control" or "operatively linked" as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid 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 or a virus. 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.

Examples

The following examples illustrate various non-limiting aspects of this invention.

Example 1: Target Binding Domain

Considering the complexity and the large size of VAR2CSA, it is essential to define smaller regions that can be included in a CAR. The obvious approach has been to define the glycan-binding site, however this has not been straightforward and the molecular mechanism underlying the interaction between VAR2CS A and pl-CSA remains unresolved. This is in part due to the difficulties in analyzing protein-glycan binding in vitro, which easily gives rise to false positive identification of nonspecific glycan-binding proteins because of the large impact of low affinity charge-charge interactions (Dahlback et al. J Biol Chem. 286(18): 15908-17 (2011).

DBL2X, where the actual binding domain lies in VAR2CSA, will be combined with partial or complete adjacent domains to find the most suitable structure to permit the binding and establish a functional immunological synapse.

Several soluble recombinant fragments have already been tested for targeting tumors in vitro and in vivo. All of them contain DBL2X, but these fragments were chosen not only for their small size but for their high affinity for the target molecule. Using a CAR approach the recombinant fragments need to be tested for optimal functionality when they are expressed with this new format the T-cell surface. Not always do the highest affinity binders confer a better killing capacity to the CAR T-cell.

Recombinant VAR2CSA constructs will be derived from the protein sequence of the FCR3 variant of Plasmidium falciparum (Gene Bank™, GU249598). Several codon optimized sequences for expression in mammal, insect and bacterial cells will be design to produce either soluble protein or complete CAR constructs expressed in human primary cells and cells lines.

The DBL2X domain of VAR2CSA has been shown to bind to placenta-like chondroitin sulfate A. As shown in FIG. 1 a functional binding fragment of this domain will bind to placental-like chondroitin sulfate A (Bentley & Gamain. Nature Structural & Molecular Biology 15, 895 - 897 (2008)). The polypeptide sequence of this domain as set forth in SEQ ID NO: 7 or a functional fragment can be used to direct a chimeric antigen receptor or an immunomodulatory cell comprising a CAR to a tumor cell expressing placenta-like chondroitin sulfate A. It is contemplated that variants of the domain will be tested for optimal binding to placenta-like chondroitin sulfate A. As is also contemplated, truncated version or a fragment of SEQ ID NO: 7 including truncated variants of SEQ ID NO: 7 having at least 70% sequence identity over the truncated domain may be tested for target binding. Additionally, the target binding domain of the CAR may comprise a DBL2X or a fragment and/or a variant thereof with additional amino acid sequences at either the N-terminus or C-terminus. In some instance, the target binding domain will be flanked by interdomain amino acids sequences from VAR2CSA. In some instances, the target binding domain will comprise the DBL2Xb (SEQ ID NO: 3) domain of VAR2CSA or a fragment and or variant thereof. In some instances, the target binding domain will comprise the ID1 (SEQ ID NO: 2) domain of VAR2CSA or a fragment and/or variant thereof. In some instances, the target binding domain will comprise a ID2a domain of VAR2CSA. VAR2CSA proteins or variants thereof to be used as the target binding domain for CARs of the present invention can be determined according to the scheme presented in FIG. 9.

SPACER:

The spacer plays an important role in the functionality of the CAR. Immunosynapses are described to be about 15 nanometers to about 20 nanometers and this distance between T cell and target cell is important for the correct initiation and performance of the signaling pathway downstream the membrane interaction in the effector cell.

The well-established spacers such as the CD8 stalk and the CH2-CH3 fragment of IgGl will be compared with a CD4 spacer recently in development. CD4 spacer is a modular molecule and the D2-D4 domains will be combined in different ways to attain the best combination with the binders to have a functional VAR2CSA-CAR T-cell able to target and eliminate specifically several major tumor types.

Also, CH3 fragment alone as a shorter spacer and a CAR structure without a spacer will be studied.

CO-STIMULATORY DOMAINS:

Physiologically, optimal activation requires CD28 engagement to be followed by co- stimulation through other T cell signaling molecules.

Other co-stimulatory domains include, but are not limited to, OX40 (CD134), which is expressed on T cells 24 hour after antigen and CD28 stimulation. The ligand for OX40 (OX40L) is expressed on antigen-presenting cells (APCs) several hours to days after their own activation. In vitro and in vivo studies have detailed how OX40 signaling can further augment CD28-activated T cell responses, enhancing proliferation, cytokine secretion, and survival. Hence, CD28 signaling provides the initial costimulus for proliferation, while subsequent OX40 signaling allows effector T cells to survive and continue proliferating

The intracellular signaling modules are derived from lymphocyte signal-initiating molecules. CAR designs of the present invention include the ζ-chain of the TCR/CD3 complex alone or in combination with one or two co-stimulatory domains. The 0)3ζ stimulatory domain initiate the phosphatidylinositol and tyrosine kinase cascade leading to cell activation and cellular responses to tumor cells. CARs designed including co-stimulatory molecules enhance cytokine production and enable sequential rounds of T-cell proliferation in response to tumor-associated antigens compared with CARs that only contain Οϋ3ζ (Maher et al. Nat Biotechnol. 2002 Jan; 20(1): 70-5). Improved responses will be examined, including different combinations of the co-stimulatory signals as CD28, ICOS, OX40 (CD 134), 4-1BB (CD137), 2B4 (CD244), DAP10 and DAP12 or other co-stimulatory domains belonging to CD28-superfamily, TNFR family, Immunoglobulin family, SLAM family and their adaptor proteins alone and in tandem with CD3C,.

Although CD28 is the most used and studied co-stimulatory molecule some other properties and advantages as augment T-cell effector function, and/or extend T-cell survival can be find utilising other domains. It has been shown that 4-1BB in combination with CO3^ can ameliorate CAR T-cell exhaustion induced by persistent CAR signaling (Long et al. Nat Med. 2015 Jun;21(6): 581-90). OX40 incorporation shown to abrogate IL-10 secretion but not impact in T-cell mediated cytolysis. OX40 signaling also enhance T-cell amplification, pro-inflammatory cytokines secretion and survival comparing with CD28 effect (Pule et al. Mol Ther. 2005 Nov; 12(5): 933-41). Second generation CARs seem to have superior signaling properties compared with the first generation only carrying 033ζ (Sadelain. Cancer J. 2009 Nov-Dec; 15(6): 451-5) but in occasions they may not initiate the full signaling capabilities of T-cells. Consequently, third-generation CARs have been developed including two costimulatory signals and CD3ζ in the CAR gene constructs (Dai et al. J Natl Cancer Inst. 2016 Jan 27; 108(7)). These are promising receptors but more studies are needed, including in vivo studies, to assess their therapeutic potential and safety profile.

Sequence Concordance Table

Claims

1. A chimeric antigen receptor (CAR) comprising:

a target binding domain capable of selectively binding placenta-like chondroitin sulfate A (pl-CSA);

a spacer region;

a transmembrane domain; and

at least one costimulatory domain.

2. The chimeric antigen receptor of claim 1 wherein the target binding domain comprises a VAR2CSA polypeptide comprising an amino acid sequence having at least 70% sequence identity to DBL2X (SEQ ID NO: 7) domain of VAR2CSA, and/or a VAR2CSA polypeptide comprising an amino acid sequence having at least 70% sequence identity to DBL2Xb (SEQ ID NO: 3) domain of VAR2CSA, and/or a VAR2CSA polypeptide comprising an amino acid sequence having at least 70% sequence identity to IDl (SEQ ID NO: 2) domain of VAR2CSA.

3. The chimeric antigen receptor of claim 1 or claim 2 wherein the target binding domain comprises a VAR2CSA polypeptide comprising an ID2a (SEQ ID NO: 4) domain of VAR2CSA.

4. The chimeric antigen receptor of any one of claims 1 to 3 wherein the target binding

domain comprises a VAR2CSA polypeptide comprising an amino acid sequence having 70% sequence identity to SEQ ID NO: 1.

5. The chimeric antigen receptor of claim 1, wherein the target binding domain comprises a scFv capable of binding to placenta-like chondroitin sulfate A (pl-CSA).

6. The chimeric antigen receptor of any one of claims 1 to 5, wherein the spacer region

comprises at least one, or multiples of, domains 2, 3 or 4 or a combination thereof of a CD4 molecule.

7. The chimeric antigen receptor of any one of claims 6, wherein domain 2 of a CD4 molecule comprises amino acids 126 to 203 of SEQ ID NO: 12.

8. The chimeric antigen receptor of any one of claims 6 to 7, wherein domain 3 of a CD4 molecule comprises amino acids 204 to 317 of SEQ ID NO: 12.

9. The chimeric antigen receptor of any one of claims 6 to 8, wherein domain 4 of a CD4 molecule comprises amino acids 318 to 374 of SEQ ID NO: 12.

10. The chimeric antigen receptor of any one of claims 1 to 5 wherein the spacer region

comprises an amino acid sequence having at least 90% sequence identity to amino acids 1 to 45 as set forth in SEQ ID NO: 20.

11. The chimeric antigen receptor of any one of claims 1 to 5 wherein the spacer region

comprises an amino acid sequence having at least 90% sequence identity to: amino acids 1 to 110, or amino acids 111 to 217, or amino acids 1 to 217, as set forth in SEQ ID NO: 22.

12. The chimeric antigen receptor of any one of claims 1 to 11 wherein the transmembrane domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25.

13. The chimeric antigen receptor of any one of claims 1 to 12, wherein the costimulatory domain comprises a Οϋ3ζ signaling domain.

14. The chimeric antigen receptor of any one of claims 1 to 13, wherein the costimulatory domain comprises at least one first intracellular effector domain or a functional variant thereof selected from CD28, CD27, 4-lBB, OX40, ICOS, CD30, CD40, PD-l, CD2, CD7, LIGHT, NKG2C, NKG2D, CD150, NTBA, B7-H3, SLAM1, SLAM 6, TRAF2, TRAF3, TRAF5, TRAF6, TRAF7, 2B4, DAP10, DAP12, lymphocyte function-associated antigen- l(LFA-l) or any combination thereof, and optionally, at least one additional intracellular effector domain or functional variant thereof selected from CD28, CD27, 4-lBB, OX40, ICOS, CD30, CD40, PD-l, CD2, CD7, LIGHT, NKG2C, NKG2D, CD 150, NTBA, B7-H3, SLAM1, SLAM 6, TRAF2, TRAF3, TRAF5, TRAF6, TRAF7, 2B4, DAP10, DAP 12, lymphocyte function-associated antigen- 1(LF A- 1).

15. The chimeric antigen receptor of any one of claims 1 to 14, wherein the target binding domain has a binding affinity of less than about 500 nanomolar.

16. A polynucleotide encoding the chimeric antigen receptor of any one of claims 1 to 15.

17. An expression vector comprising the polynucleotide of claim 16.

18. The expression vector of claim 17, wherein the expression vector is a lentiviral vector.

19. An immunomodulatory cell comprising a polynucleotide of claim 16 or an expression vector of claim 17 or claim 18.

20. An immunomodulatory cell comprising the chimeric antigen receptor of any one of claims 1 to 15.

21. The immunomodulatory cell of claim 19 or 20, which is derived from an inflammatory T- lymphocyte, cytotoxic T-lymphocyte, helper T-lymphocyte, naive or stem-cell like cell, central memory type T cell, or a Natural Killer Cell.

22. The immunomodulatory cell of any one of claims 19 to 21 for use in therapy.

23. A method of engineering an immunomodulatory cell, comprising: providing an immunomodulatory cell; introducing the expression vector of claim 17 or 18 into said immunomodulatory cell; and expressing said expression vector in the immunomodulatory cell.

24. A method of treating cancer in a human comprising administering the immunomodulatory cell of any one of claims 19 to 22 to said human.

25. The method of claim 24 further comprising expanding a population of said engineered immunomodulatory cell ex vivo prior to administering to said human.