Chimeric antigen receptors against AXL or ROR2 and methods of using the same

Abstract

The present invention provides a chimeric antigen receptor that binds to Axl and Ror2, and a conditionally active chimeric antigen receptor (CAR) that recognizes Axl and Ror2. [Solution] The present disclosure provides chimeric antigen receptors that bind to Axl and Ror2, and conditionally active chimeric antigen receptors (CARs) that recognize Axl and Ror2. Also provided herein are nucleic acids encoding these CARs, as well as methods for making the CARs and methods of using the CARs, including methods for treating cancer, particularly cancers that express Axl and / or Ror2, such as renal cell carcinoma. The present disclosure also provides cells genetically modified to produce the CARs.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 447,898, filed on January 18, 2017; U.S. Provisional Patent Application No. 62 / 467,059, filed on March 3, 2017; and U.S. Provisional Patent Application No. 62 / 530,193, filed on July 8, 2017. These provisional applications cited in this paragraph are hereby incorporated by reference in their entirety into this specification.

[0002] Sequence Listing The data of the electronic sequence listing submitted simultaneously with this application is hereby incorporated by reference into this specification. The data of the electronic sequence listing was submitted as a text (.txt) file named "F1.002.WO.01_Seqlist.txt" with a size of 126 KB, created on January 17, 2018, and is hereby incorporated by reference in its entirety into this specification.

[0003] Joint Research Agreement F1 Oncology, Inc. and BioAtla, LLC are parties to a joint research agreement regarding the subject matter disclosed herein.

[0004] Field of the Disclosure This disclosure relates to chimeric antigen receptors and the use of chimeric antigen receptors in diagnostic and therapeutic methods.

Background Art

[0005] In cell-based adoptive immunotherapy, immune cells isolated from a patient are modified to express a synthetic protein, which can endow the immune cells with new therapeutic functions when they are then returned to the patient. An example of such a synthetic protein is a chimeric antigen receptor (CAR). An example of a currently used CAR is a fusion of an extracellular recognition domain (e.g., an antigen-specific targeting region or ASTR), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen binding, the intracellular signaling portion of the CAR can initiate activation-related responses in immune cells, such as the release of cytolytic molecules that induce tumor cell death. CARs and CAR-T therapies are highly effective against certain types of blood cancers, but there remains a need for CAR and CAR-T therapies for solid tumors, which have proven far more difficult to achieve to date.

[0006] CARs are an excellent way to treat various diseases, but recently, the safety of CARs has become a concern due to adverse events during clinical trials. One way to reduce these adverse events is by reducing off-tumor ASTR binding. CARs with conditionally active ASTRs bind only to antigens under specific conditions, such as when present in the tumor microenvironment, and do not bind to antigens in normal physiological conditions, reducing off-tumor binding. Thus, the side effects of these CARs are reduced, and the treatment can proceed more safely.

[0007] Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that regulate the range of normal cell processes through ligand-regulated tyrosine kinase activity. Over the past 20 years, deregulation of RTKs has been shown to play an important role in cancer development and progression. Currently, RTKs are recognized as prognostic molecular biomarkers and targets for tumor therapeutics.

[0008] The AXl protein (also known as Ark, UFO, Tyro-7) is a RTK in the Tyro-3 kinase family. The Tyro-3 receptor kinase is characterized by a combination of two immunoglobulin-like domains and double fibronectin type III repeats in the extracellular region and cytoplasmic kinase domain. The ligands of the Tyro-3 receptor kinase are Gas6 (growth-arrest-specific 6) and protein S (two vitamin K-dependent proteins that show 43% amino acid sequence identity and share a similar domain structure). Each protein has an N-terminal Glu domain containing 11g-carboxyglutamic acid residues, followed by four epidermal growth factor (EGF)-like modules, and a C-terminal hormone-binding globulin (SHBG)-like structure consisting of two tandem laminin G domains. The SHBG domain is both necessary and sufficient for binding to the Tyro-3 receptor kinase, while the Glu domain binds to negatively charged membrane phospholipids and plays an important role in the phagocytic action of Tyro-3 kinase apoptotic cells.

[0009] AXl activation leads to signal transduction through other major pathways such as PI-3-kinase / Akt, Ras / Erk, and β-catenin / TCF. AXl is weakly expressed in normal tissues including the organ capsules and connective tissues of the brain, heart, skeletal muscle, and several other organs, and in monocytes, but not in lymphocytes. Akt phosphorylation induced by AXl has been described in the survival of fibroblasts, endothelial cells, vascular smooth muscle cells, and neurons. Furthermore, AXl plays a certain role in cell adhesion and chemotaxis, as AXl knockout animals show impaired platelet aggregation stabilization and thrombosis as a result of reduced activation of platelet integrin IIb3.

[0010] Dysregulation of Axl or its ligand Gas6 has been linked to the etiology of various human cancers. Axl overexpression has been observed in various cancer types, such as breast cancer (Meric et al., Clin. Cancer Res., vol. 8, pp. 361 - 367, 2002; Berclaz et al., Ann. Oncol., vol. 12, pp. 819 - 824, 2001), colon cancer (Chen et al., Int. J. Cancer, vol. 83, pp. 579 - 584, 1999; Craven et al., Int. J. Cancer, vol. 60, pp. 791 - 797, 1995), prostate cancer (Jacob et al., Cancer Detect. Prev., vol. 23, pp. 325 - 332, 1999), lung cancer (Wimmel et al., Eur J Cancer, vol. 37, pp. 2264 - 2274, 2001), gastric cancer (Wu et al., Anticancer Res., vol. 22, pp. 1071 - 1078, 2002), ovarian cancer (Sun et al., Oncology, vol. 66, pp. 450 - 457, 2004), endometrial cancer (Sun et al., Ann. Oncol., vol. 14, pp. 898 - 906, 2003), renal cancer (Chung et al., DNA Cell Biol., vol. 22, pp. 533 - 540, 2003), hepatocellular carcinoma (Tsou et al., Genomics, vol. 50, pp. 331 - 340, 1998), thyroid cancer (Ito et al., Thyroid, vol. 12, pp. 971 - 975, 2002; Ito et al., Thyroid, vol. 9, pp. 563 - 567, 1999), osteosarcoma (Nakano et al., J. Biol. Chem., vol. 270, pp. 5702 - 5705, 2003), melanoma (van Ginkel et al., Cancer Res., vol. 64, pp. 128 - 134, 2004), head and neck squamous cell carcinoma (Green et al., Br J. Cancer., vol. 94, pp.(1446-51, 2006), ovarian cancer, kidney cancer, glioma, endocrine adenocarcinoma, pancreatic cancer, lymphoma, brain cancer, liver cancer, renal cell carcinoma, clear cell renal carcinoma, bladder cancer, rectal cancer, squamous cell carcinoma of the neck, and furthermore, lymphoma and chronic myeloid leukemia (Janssen et al., Oncogene, vol. 6, pp. 2113-2120, 1991; Braunger et al., Oncogene, vol. 14, pp. 2619-2631 1997; O’Bryan et al., Mol. Cell. Biol., vol. 11, pp. 5016-5031, 1991) and various leukemias including acute myeloid leukemia (Rochlitz et al., Leukemia, vol. 13, pp. 1352-1358, 1999).

[0011] AXl expression is induced by targeted chemotherapeutic agents, and drug-induced AXl expression confers resistance to chemotherapeutic agents in acute myeloid leukemia (Hong et al, Cancer Letters, vol. 268, pp. 314-324, 2008), as well as resistance to imatinib and lapatinib / herceptin in gastrointestinal stromal tumors (Mehadevan, et al, Oncogene, vol. 26, pp. 3909-3919, 2007) and breast cancer (Liu et al, Cancer Research, vol. 281, pp. 6871-6878, 2009), respectively.

[0012] Furthermore, since Axl is upregulated in aggressive breast cancer cell lines compared to non-invasive cells, Axl has been identified as being associated with tumor metastasis. In vitro, Axl activity has been found to be required for migration and invasion, and this activity can be inhibited by antibody therapy (WO 04 / 008147). Similarly, in vivo suppression of Axl activity via expression of a dominant negative version of Axl (Vajkoczy, P., et al., Proc. Natl. Acad. Science U.S.A., vol. 103, pp. 5799-5804, 2005), or by siRNA-mediated downregulation of Axl (Holland et al., Cancer Res., vol. 65, pp. 9294-9303, 2005), blocked subcutaneous and orthotopic tumor growth in mouse xenograft experiments.

[0013] Accordingly, anti-Axl monoclonal antibodies have been suggested for use in the treatment of cancer. For example, documents related to anti-Axl antibodies include WO 2009 / 06395, WO 2009 / 062690, WO 2011 / 014457, US 2014 / 0227283, and US 8,853,369. US 2014 / 0227283 discloses monoclonal anti-Axl antibodies and their use in diagnostic and therapeutic methods. WO 2009 / 062690 discloses antibodies that bind to the extracellular domain of the Axl protein and at least partially inhibit Axl activity. However, there are still many challenges that make it extremely difficult to generate conditional active CARs against targets such as Axl for which monoclonal antibodies have been identified and are available as reagents.

[0014] Ror2, also known as receptor tyrosine kinase-like orphan receptor 2, another RTK, is a membrane-bound receptor that is activated by non-canonical Wnt signaling through binding to the Wnt5A glycoprotein during normal bone and cartilage development. Ror2 has a single transmembrane domain that separates its extracellular and intracellular domains. Ror2 is known to play an important role in the normal development of various organs and tissues. In mammals, Ror2- and Wnt5A-deficient mice show similar abnormalities during morphogenesis in the developmental process, reflecting their impairments in convergent extension movements and planar cell polarity. Furthermore, mutations in the human Ror2 gene are involved in the autosomal dominant skeletal disorder brachydactyly type B and the autosomal recessive Robinow syndrome. Ror2 has been found to mediate polarized cell migration, and dysfunction of Ror2 results in hereditary skeletal disorders and tumor invasion (Minami et al., “Ror-family receptor tyrosine kinases in noncanonical Wnt signaling: their implications in developmental morphogenesis and human diseases,” Dev Dyn., vol. 239, pp. 1-15, 2010). Furthermore, Debebe et al. (“Ror2 as a therapeutic target in cancer,” Pharmacol. Ther., vol. 50, pp. 143-148, 2015) have disclosed that Ror2 mediates both canonical and noncanonical signaling pathways.

[0015] Ror2 has also been reported to have a tumor-promoting effect. U.S. Patent Application Publication No. 2014 / 0322234 discloses that the expression and activity of Ror2 in various cancers are different from those in normal tissues. Therefore, it is suggested that deregulation of Ror2 plays a certain role in the etiology of various human cancers. U.S. Patent Application Publication No. 2014 / 0322234 also contemplates that antibodies against Ror2 can be used for the diagnosis of cancer and the suppression of cancer cell proliferation. For example, such antibodies can be conjugated with cytotoxic drugs that have a high cytotoxicity against cancer cells expressing Ror2, whereby the cytotoxic drugs can effectively kill cancer cells. The Ror2 gene can also be used for the classification of cancers based on the Ror2 expression pattern in cancers.

[0016] Ror2 is involved in the occurrence and progression of cancer (“The dual role of the novel Wnt receptor tyrosine kinase, Ror2, in human carcinogenesis,” International Journal of Cancer, vol. 133, pp. 779-787, 2013). Specifically, Ror2 has been found to play an important role in the carcinogenesis of a number of cancers, including colon cancer, hepatocellular carcinoma, metastatic melanoma, and renal cell carcinoma. For example, Ror2 is overexpressed in osteosarcoma, melanoma, renal cell carcinoma, prostate cancer, squamous cell carcinoma of the head and neck, colon cancer, breast cancer, lymphoma, leukemia, thyroid cancer, pancreatic endocrine gland, brain cancer, ovarian cancer, renal papillary carcinoma, lung cancer, pancreatic cancer, liver cancer, renal clear cell carcinoma, bladder cancer, endometrial cancer, rectal cancer, squamous cell carcinoma of the cervix, and stromal tumors. In most of these cancer types, Ror2 expression is associated with a more invasive cancer state. Therefore, Ror2 has the potential to be a drug target for cancer treatment by inhibiting the Wnt signaling pathway.

[0017] There remains a need for effective therapies that harness the power of the immune system to fight cancer while reducing or eliminating off-target off-tumor as well as off-target effects. Monoclonal antibodies against Ror2 and Axl are commercially available, but there is a need for chimeric antigen receptors (CARs) that contain antibody fragments that are conditionally active in specific environments such as the cancer microenvironment and that effectively target cells that express only Ror2 or Axl. There are numerous challenges in generating such conditionally active CARs. For example, when the antibody fragment is expressed on the surface of T cells or NK cells as part of the CAR, an antibody fragment must be generated and identified that not only binds to Axl or Ror2 but also has the ability to recognize epitopes exposed on cancer cells. Further, such CARs bind predominantly to their targets in a conditionally active manner, particularly under the acidic pH of tumors as compared to normal physiological pH. Further, such candidate CARs need to activate T cells or NK cells expressing the CAR to express cytotoxic functions when they bind to their targets. Thus, there are many requirements for such CARs that contain antibody fragments to help solve the problems arising from current CAR-T methods. Against Axl or Ror2, such conditionally active CARs hold great promise for the treatment of solid cancers using CAR-T therapy and thus will overcome most of the limitations of current CAR-T therapy. SUMMARY OF THE INVENTION

[0018] The present disclosure provides chimeric antigen receptors (CARs) that bind to AXl and / or Ror2, nucleic acids comprising nucleotide sequences encoding the CARs, and conditionally active biological (CAB) CARs that bind to AXl and Ror2. The present disclosure provides cells genetically modified to produce the CARs, and methods of making such cells. The CARs of the present disclosure can be used in various methods, and these methods are also provided, and these methods include methods of activating immune cells under specific conditions such as a pH below a threshold value, CAR therapy, for example, methods of performing adoptive immune cell therapy such as CAR therapy for cancer, for example, renal cell carcinoma.

[0019] Details of the aspects and embodiments provided herein are provided throughout the present disclosure. For clarity, this summary section is not intended to be construed as limiting the scope of the present disclosure provided herein, nor should it be so construed.

Brief Description of the Drawings

[0020]

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[0021] Definitions As used herein, the terms "chimeric antigen receptor" or "CAR" or "CAR(s)" mean a genetically modified receptor that confers antigen specificity to a cell, such as a T cell, NK cell, macrophage, and stem cell. The CARs of the present invention can include at least one antigen-specific targeting region (ASTR), a hinge or stalk domain (i.e., extracellular stalk domain (ESD)), a transmembrane domain (TM), one or more co-stimulatory domains (CSD), and an intracellular activation domain (IAD). In certain embodiments, the ESD and / or CSD are optional. In some embodiments, the CARs provided herein have a particularly described domain (e.g., the CD3Z intracellular activation domain). For such particularly described domains, the domain retains the activity of the wild-type domain and can thus be effectively employed in a CAR (i.e., the CAR retains the ability to bind to the target and, in response, transmits a signal through the intracellular activation domain recognized within the CAR), and it is intended that such a particular domain have at least 80% sequence identity to a known human sequence as compared to that portion of the domain that confers such activity. For example, the CD3Z intracellular domain has at least 80% sequence identity at the amino acid level to a known human CD3Z sequence and, when found on a CAR, retains the ability to transmit a signal to its target upon binding of the ASTR of the CAR. In another embodiment, the CAR is a bispecific CAR, which is specific for two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the IAD activates intracellular signaling. For example, the IAD can redirect T cell specificity and reactivity towards a selected target in a non-MHC-restricted manner and can utilize the antigen-binding properties of an antibody. Non-MHC-restricted antigen recognition confers on T cells expressing the CAR the ability to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor escape. Further, when expressed in T cells, the CAR conveniently does not dimerize with the endogenous T cell receptor (TCR) alpha and beta chains.

[0022] As used herein in connection with CAR or ASTR, the term "conditionally active" means a CAR or ASTR that has a lower binding affinity for one or more target antigens under conditions in a target microenvironment than under conditions in a normal physiological environment, or, in an exemplary embodiment, a higher binding affinity. In an exemplary embodiment, the conditionally active CAR provided herein is more active under conditions in a tumor microenvironment or in an in vitro tumor surrogate assay than under non-tumor microenvironment or normal conditions. Conditions in a tumor microenvironment include a lower pH, higher concentrations of lactic acid and pyruvic acid, hypoxia, lower concentrations of glucose, and a slightly higher temperature compared to a non-tumor microenvironment. For example, a conditionally active CAR is, in certain embodiments, substantially inactive at normal body temperature but active at the higher temperature in a tumor microenvironment. In yet another embodiment, a conditionally active CAR has low activity in normal oxygenated blood but higher activity under the lower oxygenation environment in a tumor. In some exemplary embodiments provided herein, a conditionally active CAR has low activity in a normal physiological pH of 7.2 to 7.8 but higher activity under the acidic pH of 6.0 to 6.8 present in a tumor microenvironment. There are other conditions in a tumor microenvironment known to those of skill in the art where conditionally active CARs and ASTRs have different binding affinities and can be used as well in the context of the present invention. As used herein, a conditionally active CAR may also be referred to as a "conditionally active biological CAR" or "CAB-CAR" and a "microenvironment-restricted biological CAR" or "MRB-CAR".

[0023] As used herein, the term "microenvironment" means any part or region of a body or tissue that has a physical or chemical difference, either constantly or temporarily, from other regions of the body or tissue. With respect to a tumor, as used herein, the term "tumor microenvironment" means the environment in which the tumor exists, which is the non-cellular site within the tumor and the region immediately outside the tumor tissue, but does not belong to the intracellular compartments of the cancer cells themselves. The tumor and the tumor microenvironment are closely related and constantly interact. The tumor can change its microenvironment, and the microenvironment can affect how the tumor grows and spreads. Typically, the tumor microenvironment has a low pH in the range of 5.8 to 7.0, more commonly in the range of 6.0 to 6.8, and in the range of 6.2 to 6.8. On the other hand, normal physiological pH is in the range of 7.2 to 7.8. The tumor microenvironment is also known to have lower concentrations of glucose and other nutrients compared to plasma, but is also known to have higher concentrations of lactate. Additionally, the tumor microenvironment may have a temperature that is 0.3°C to 1°C higher than normal physiological temperature. The tumor microenvironment is discussed in Gilles et al., "MRI of the Tumor Microenvironment," Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002. This document is hereby incorporated by reference in its entirety. The term "non-tumor microenvironment" means the microenvironment at sites other than tumors.

[0024] As used herein in the same sense, the terms "polynucleotide" and "nucleic acid" mean polymeric nucleotides of any length, ribonucleotides or deoxynucleotides. Thus, the term includes, but is not limited to, short-chain, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural nucleotide bases, chemically or biochemically modified, non-natural, derivatized nucleotide bases.

[0025] As used herein, the terms "antibody" and "immunoglobulin" include any isotype of an antibody or immunoglobulin, as well as epitopes, and fragments of antibodies that retain specific binding to the same epitope as a typically full-length antibody, including, but not limited to, fragments that contain both the heavy and light chains of the antibody and are present in the assay, such as Fab, Fab’, Fab’-SH, (Fab’)2, Fv, scFv, diabody, Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins that contain antigen-specific targeting regions of antibodies and non-antibody proteins.

[0026] An "antibody fragment" contains a portion of a full antibody, for example, the antigen-binding or variable region of a full antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-binding sites and can still cross-link antigens.

[0027] A "single-chain Fv" "scFv" or "sFv" antibody fragment contains the V H and V L domains of an antibody, which domains are present in a polypeptide single chain. In some embodiments, the Fv polypeptide contains the V H and V LIt further contains a polypeptide linker between domains, which enables the sFv to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269 - 315 (1994).

[0028] As used herein, the term "affinity" means the equilibrium constant for the reversible binding of two reagents and is expressed as the dissociation constant (Kd). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody for an irrelevant amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or greater. As used herein, the term "binding strength" means the resistance to dissociation after dilution of a complex consisting of two or more reagents. The terms "immunoreactive" and "preferentially binds" are used herein in the same sense with respect to antibodies and / or antigen-binding fragments.

[0029] The term "binding" means a direct binding between two molecules by hydrogen bond interactions including, for example, covalent bonds, electrostatic bonds, hydrophobic bonds, and hydrogen bond interactions such as ionic and / or salt bridges and water bridges. Nonspecific binding is binding at an affinity of less than about 10 -7 M, for example, 10 -6 M, 10-5 M, 10 -4 It means a bond such as M.

[0030] As used herein, the term "hinge region" refers to a flexible polypeptide connector region (also referred to herein as "hinge" or "spacer") that confers structural flexibility and spacing to adjacent polypeptide regions and can be composed of natural or synthetic polypeptides. The "hinge region" derived from immunoglobulin (e.g., IgG1) is typically defined as the stretch from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22:161 - 206). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the inter - heavy - chain disulfide (S - S) bonds at the same positions. The hinge region may be natural or non - natural, including, but not limited to, the modified hinge regions described in U.S. Patent No. 5,677,425. The hinge region may include the complete hinge region from the hinge region of the CH1 domain derived from antibodies of different classes or subclasses. The term "hinge region" may also include regions derived from other receptors such as CD8, CD28, or that provide a similar function of conferring flexibility and spacing to adjacent regions.

[0031] An "isolated" polypeptide is a polypeptide that has been identified and separated and / or recovered from the components of its natural environment. Contaminant components of its natural environment are substances that may interfere with the diagnostic or therapeutic uses for the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified to (1) greater than 90%, greater than 95%, or greater than 98% by weight of the antibody, e.g., greater than 99% by weight as measured by the Lowry method, (2) at least 15 residues of the N-terminus or to an extent sufficient to obtain an internal amino acid sequence using a spinning cup sequenator, or (3) homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie blue or silver staining under reducing or non-reducing conditions. Isolated polypeptides include in situ polypeptides within recombinant cells, as these are thought to lack at least one component of the polypeptide's natural environment. In some instances, the isolated polypeptide is prepared by at least one purification step.

[0032] As used herein, the term "immune cell" generally includes white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) generated in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells (i.e., T lymphocytes), B cells, natural killer (NK) (CD3−CD56+) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of immune cells expressing CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and γδ T cells, as well as NK T cells (CD3+ and CD56+). One of ordinary skill in the art will understand that the T cells and / or NK cells used throughout the present disclosure can include T cells only, NK cells only, or both T cells and NK cells. In certain exemplary embodiments and aspects provided herein, the T cells are activated and transduced. Further, in certain exemplary composition embodiments and aspects provided herein, T cells are provided. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, NK-T cells, γδ T cells, and neutrophils, which are cells that can mediate a cytotoxic response.

[0033] As used herein, the term "stem cell" generally includes pluripotent or multipotent stem cells. "Stem cells" include, for example, embryonic stem cells (ES); mesenchymal stem cells (MSC); induced pluripotent stem cells (iPS); and directed progenitor cells (hematopoietic stem cells (HSC); bone marrow-derived cells, etc.).

[0034] As used herein, unless otherwise indicated, the term "Axl" refers to any native Axl from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" unprocessed Axl, as well as any form of Axl resulting from processing in cells. This term also encompasses native Axl variants, such as splice variants or allelic variants. The amino acid sequence of human Axl is well-known in the art and is available from public databases such as GenBank.

[0035] As used herein, the term "Axl activation" refers to the activation or phosphorylation of the Axl receptor. Generally, Axl activation results in signal transduction (e.g., signal transduction caused by the intracellular kinase domain of the Axl receptor that phosphorylates tyrosine residues in Axl or a substrate polypeptide). Axl activation can be mediated by an Axl ligand (Gas6) that binds to the Axl receptor of interest. The binding of Gas6 to Axl activates the kinase domain of Axl, thereby resulting in phosphorylation of tyrosine residues in Axl and / or further phosphorylation of tyrosine residues in a substrate polypeptide.

[0036] As used herein, the term "Axl-mediated anti-apoptosis" refers to all Axl-related processes that prevent programmed cell death (apoptosis) in human cells, preferably, but not limited to, human cancer cells. In particular, Axl-mediated anti-apoptosis refers to the process of preventing apoptosis induction in human cells, preferably, but not limited to, human cancer cells, by growth factor withdrawal, hypoxia, exposure to chemotherapeutic agents or radiation, or initiation of Fas / Apo-1 receptor-mediated signal transduction, and is preferably stimulated or mediated by the non-catalytic or catalytic activity of Axl, including Axl phosphorylation and / or Axl-mediated signal transduction.

[0037] As used herein, the term "Ror2" refers to receptor tyrosine kinase-like orphan receptor 2, which is predicted to be a 943 amino acid protein having in vitro protein kinase activity and is shown in GenBank accession number AAI30523. Many lineage-restricted receptor tyrosine kinases were initially regarded as "orphan" receptors homologous to known receptors and were only later used to identify their unknown growth factors. DeChiara et al. (2000) identified one such orphan encoded by Ror2.

[0038] As used herein, the terms "treatment", "treating", etc. mean obtaining a desired pharmacological and / or physiological effect. The effect may be a prevention from the perspective of completely or partially preventing a disease or its symptoms, and / or a treatment from the perspective of partial or complete cure of the disease and / or the deleterious effects caused by the disease. As used herein, "treatment" includes any treatment of a disease in a mammal such as a human, including (a) preventing the onset of a disease in a subject who is susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its onset; and (c) alleviating the disease, i.e., causing the disease to regress.

[0039] As used herein in the same sense, the terms "individual", "subject", "host", and "patient" mean mammals including, but not limited to, humans, mice (e.g., rats, mice), rabbits (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), etc.

[0040] "Therapeutically effective amount" or "effective amount" means an amount of a drug, or a combination of two drugs, that is sufficient to affect such treatment of a disease when administered to a mammal or other subject for the treatment of the disease. The "therapeutically effective amount" varies depending on the drug, the disease, and its severity, as well as the age, weight, etc. of the subject being treated.

[0041] As used herein, the terms "evolution" or "evolving" mean generating different polynucleotides encoding different polypeptides that are self-improved biomolecules and / or contribute to the generation of another improved biomolecule using one or more mutagenesis methods.

[0042] "Physiological" or "normal" or "normal physiological" conditions are conditions that are considered to be within the normal range for a subject at the site of administration or in the tissue or organ of the site of action, including but not limited to temperature, pH, osmotic pressure, osmolality, oxidative stress and electrolyte concentration, and other parameters.

[0043] It should be understood that the present disclosure and the aspects and embodiments provided herein are, of course, modifiable and thus are not limited to the specific examples disclosed. Also, since the scope of the present disclosure is limited only by the appended claims, it should be understood that the terms used herein are for the purpose of disclosing only specific examples and embodiments and are not intended to be limiting.

[0044] Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range and any other prescribed value or intervening value in the prescribed range is to be understood as being included in the invention, unless there is a clear contrary indication in the context. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and also, subject to the existence of any specifically excluded range within the indicated range, be included within the scope of the invention. Where the indicated range includes one or both of its limit values, ranges excluding either or both of those included limit values are also included in the invention.

[0045] Unless otherwise indicated, all 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications.

[0046] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a chimeric antigen receptor" includes a plurality of such chimeric antigen receptors and equivalents thereof known to those of ordinary skill in the art, etc. Further, it should be noted that claims may be drafted to exclude any optional element. Therefore, this description is intended to serve as a basis for using limiting terms such as "solely", "only", etc. or for using "negative" limitations in connection with the recitation of elements of the claims.

[0047] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment may be provided separately or in any suitable sub-combination. All combinations of embodiments associated with the invention are specifically embraced by the invention and are disclosed herein as if each and every combination was individually and explicitly disclosed. Further, all sub-combinations of the various embodiments and their elements are also specifically embraced by the invention and are disclosed herein as if each and every sub-combination was individually and explicitly disclosed herein.

[0048] Detailed Description Aspects and embodiments disclosed herein overcome problems of the off-tumor effects of current therapies, in one aspect, by providing a conditionally active chimeric antigen receptor (CAR) for binding to Axl and / or Ror2. The CAR for binding to Axl and / or Ror2 is active in the tumor environment but not in normal physiological tissues / organs. In addition to various embodiments of the CAR for binding to Axl and / or Ror2, provided herein are embodiments of nucleic acids comprising nucleotide sequences encoding any of the CARs provided herein, as well as viral constructs for expressing any of the CARs, cells infected with at least one viral construct, and recombinant cells expressing the CAR. The CARs of the disclosure can be used in a variety of methods, which are also provided, along with methods of infecting T cells and other cytotoxic cells with expression vectors such as recombinant viral vectors encoding the CARs of the disclosure.

[0049] Chimeric Antigen Receptor The present disclosure provides a chimeric antigen receptor, which is referred to herein as "CAR" for simplicity. In an exemplary embodiment, the CAR of the present disclosure binds to Axl or Ror2, and in a further exemplary embodiment, the CAR binds to Axl or Ror2 in a conditionally active manner. In certain exemplary embodiments, the CAR provided herein comprises: a) at least one conditionally active antigen-specific targeting region (ASTR) that exhibits increased binding at pH 6.7 compared to pH 7.4; b) a transmembrane domain; and c) an intracellular activation domain. In an exemplary embodiment, the antigen-specific targeting region of the CAR is a conditionally active scFv portion of an anti-AXl or anti-Ror2 antibody. Further, in an exemplary embodiment, the ASTR exhibits increased activity in a tumor environment or in in vitro tumor surrogate assay conditions compared to a normal physiological environment.

[0050] The CAR of the present disclosure can be present in the cell membrane of eukaryotic cells, such as mammalian cells. Suitable mammalian cells include, but are not limited to, cytotoxic cells, T lymphocytes, stem cells, progeny of stem cells, progenitor cells, progeny of progenitor cells, and NK cells, NK-T cells, and macrophages. When present in the cell membrane of a eukaryotic cell, the CAR of the present disclosure is active in the presence of Axl and / or Ror2, which binds to the ASTR under specific conditions. Axl and Ror2 are the second members of a specific binding pair. The Axl and / or Ror2 of the specific binding pair can be a soluble (e.g., non-cell-binding) factor; a factor present on the surface of a cell such as a target cell; a factor presented on a solid surface; a factor present in a lipid bilayer; and the like. When the ASTR is an antibody, the second member of the specific binding pair is an antigen, and the antigen can be a soluble (e.g., non-cell-binding) antigen; an antigen present on the surface of a cell such as a target cell; an antigen presented on a solid surface; an antigen present in a lipid bilayer; and the like.

[0051] In some cases, the CARs of the present disclosure are present in the cell membrane of eukaryotic cells and, when activated by Axl and / or Ror2, increase the expression of at least one nucleic acid in the cell. For example, in some cases, when present in the cell membrane of eukaryotic cells, the CARs of the present disclosure, when activated by Axl and / or Ror2, increase the expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the level of transcription of the nucleic acid in the absence of Axl and / or Ror2.

[0052] As an example, the CARs of the present disclosure can include an immunoreceptor tyrosine activation motif (ITAM)-containing intracellular signaling polypeptide; in such cases, the CARs of the present disclosure, when present in the cell membrane of eukaryotic cells and activated by Axl and / or Ror2, increase nuclear factor of activated T cells (NFAT)-dependent transcription. NFAT-dependent transcription includes transcription induced by any member of the NFAT family, such as NFATel, NFATc2, NFATc3, NFATc4, NFAT5; AP-1; Spl; NKKB; and the like.

[0053] The CARs of the present disclosure are present in the cell membranes of eukaryotic cells and, when activated by Axl or Ror2, in some cases result in an increase in the production of one or more cytokines in the cell. For example, when present in the cell membrane of a eukaryotic cell, the CARs of the present disclosure, when activated by Axl or Ror2, increase cytokine production by the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold compared to the amount of cytokine produced by the cell in the absence of Axl and / or Ror2. In some embodiments, when present in the cell membrane of a eukaryotic cell, the CARs of the present disclosure, when activated by Axl or Ror2, increase cytokine secretion by the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold compared to the amount of cytokine secreted by the cell in the absence of Axl and / or Ror2. Cytokines whose production can be increased include, but are not limited to, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; chemokines; growth factors; and the like.

[0054] In some cases, the CARs of the present disclosure, when present in the cell membrane of a eukaryotic cell and activated by Axl and / or Ror2, can result in an increase in nucleic acid transcription in the cell, an increase in cytokine production by the cell, and an increase in cytokine secretion by the cell.

[0055] In some cases, the CARs of the present disclosure are present in the cell membrane of eukaryotic cells and, when activated by Axl and / or Ror2, result in a cell-mediated cytotoxic activity against target cells that express an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds on its cell surface. For example, when the eukaryotic cell is a cytotoxic cell (e.g., an NK cell or a cytotoxic T lymphocyte), the CARs of the present disclosure are present in the cell membrane of the eukaryotic cell and, when activated by Axl and / or Ror2, increase the cell-mediated cytotoxic activity of the cell against target cells that express Axl and / or Ror2 on its cell surface. For example, when the eukaryotic cell is an NK cell or a T lymphocyte, the CARs of the present disclosure, when present in the cell membrane of the cell and when activated by Axl and / or Ror2, increase the cell-mediated cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the cell-mediated cytotoxic activity in the absence of Axl and / or Ror2.

[0056] In some cases, the CARs of the present disclosure are present in the cell membrane of eukaryotic cells and, when activated by Axl and / or Ror2, can result in other CAR activation-related events such as proliferation and expansion (due to increased cell division or anti-apoptotic responses).

[0057] In some cases, the CARs of the present disclosure are present in the cell membrane of eukaryotic cells and, when activated by Axl and / or Ror2, can result in other CAR activation-related events such as intracellular signal transduction regulation, cell differentiation, or cell death.

[0058] The CARs of the present disclosure can be present in the eukaryotic cell membrane, where the first and second polypeptides of the CAR are not covalently linked to each other. The CARs of the present disclosure can be present in the eukaryotic cell membrane as a single heterodimer that is not covalently linked to any other polypeptide in the membrane. Alternatively, the first CAR of the present disclosure can be present in the eukaryotic cell membrane as a heterodimer that is covalently or non-covalently linked to the second CAR of the present disclosure. In some cases, the first and second CARs are covalently linked via disulfide bonds formed between cysteines present in a hinge region that is present in both the first polypeptide of the first CAR and the first polypeptide of the second CAR.

[0059] In some cases, the CARs of the present disclosure can be present in the eukaryotic cell membrane, where the first polypeptide of the CAR comprises an antibody fragment and the second polypeptide of the CAR comprises a signaling domain derived from a cytokine receptor, such that upon dimerization, the CAR becomes a heterodimeric-signalobody CAR, e.g., a signalobody composed of at least two independent polypeptides. A "signalobody" known in the art is a single chimeric polymer composed of an antibody fragment and a signaling domain derived from a cytokine receptor. In certain cases, the heterodimeric signalobody CAR of the present disclosure, when present in the cell membrane of a eukaryotic cell, is dimerized by a dimerizing agent and activated by an antigen, e.g., an oligomerizing antigen, to induce oligomerization of the heterodimeric signalobody CAR. Such ligand-induced oligomerization of the heterodimeric signalobody CAR can enhance, e.g., increase, or perpetuate, e.g., maintain, activation. Signal transduction, e.g., ligand-induced oligomerization of the heterodimeric signalobody CAR, can transmit signals that induce a cellular response. In some cases, multiple heterodimeric signalobody CARs can be used in combination to induce a desired cellular response.

[0060] Antigen-specific targeting region The CARs of the present disclosure include members of a specific binding pair, which is typically an ASTR. Specific binding pairs include, but are not limited to, antigen - antibody binding pairs; ligand - receptor binding pairs; and the like. Accordingly, members of specific binding pairs suitable for use in the CARs of the present disclosure include antibodies, antigens, ligands, ligand - binding domains of receptors, receptors, ligand - binding domains of receptors, and ASTRs which are affibodies.

[0061] An ASTR suitable for use in the CARs of the present disclosure can be any antigen - binding polypeptide. In certain embodiments, the ASTR is an antibody such as a full - length antibody, single - chain antibody, Fab fragment, Fab’ fragment, (Fab’)2 fragment, Fv fragment, and bivalent single - chain antibody or diabody.

[0062] In some embodiments, the ASTR is a single - chain Fv (scFV). In some embodiments, the heavy chain is positioned at the N - terminus of the light chain of the CAR. In other embodiments, the light chain is positioned at the N - terminus of the heavy chain of the CAR. In any of the disclosed embodiments, the heavy chain and the light chain can be separated by a linker as discussed in more detail herein. In any of the disclosed embodiments, the heavy chain or the light chain can be at the N - terminus of the CAR and is typically at the C - terminus of another domain such as a signal sequence or peptide.

[0063] Other antibody - based recognition domains (cAB VHH (camelid antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single - domain antibody variable domain) and “camelized” antibody variable domains) are suitable for use in the CARs and methods using the CARs of the present disclosure. In some cases, T - cell receptor (TCR) - based recognition domains, for example, single - chain TCRs (scTv, VαVβ - containing single - chain two - domain TCRs) are also suitable for use.

[0064] Conditional active - type biological CAR (CAB - CAR) The CARs of the present disclosure are typically conditionally active. This property is typically a result of the conditional activity nature of the ASTR domain of the CAR. In an exemplary embodiment, the CAB-CAR of the present disclosure has a higher binding affinity for Axl or Ror2 under conditions in the tumor microenvironment than under conditions in the non-tumor microenvironment. In some embodiments, the conditions in the tumor microenvironment and the non-tumor microenvironment are both pH. Thus, the CAB-CAR typically has a higher binding affinity for Axl or Ror2 at a pH of about 6.0 - 6.8, which is the pH encountered in the tumor microenvironment, compared to a pH of 7.2 - 7.8, which is the pH encountered by the CAB-CAR in the normal physiological environment, and can thus selectively bind to Axl or Ror2 in a conditionally active manner. For example, the CAB-CAR may have a higher binding affinity for Axl or Ror2 at pH 6.7 than at pH 7.4. Additionally or alternatively, the CAB-CAR may have a higher binding affinity for Axl or Ror2 at pH 6.0 than at pH 7.4. Such conditions can be tested in an in vitro tumor surrogate assay, for example, for testing antigen binding and / or CAR activity (e.g., cell lysis), under one or more conditions found in the in vivo tumor environment that differ from the corresponding conditions in normal physiological tissue, as described in more detail below. For example, the in vitro tumor surrogate assay conditions can be a low pH (e.g., 6.0 - 6.8) compared to physiological pH (7.2 - 7.8). In an exemplary example, the tumor surrogate assay conditions are a pH of 6.7, while the corresponding physiological pH is 7.4.

[0065] In some embodiments, the CAB-CAR can be obtained by identifying the VH and / or VL of an antibody identified under physiological conditions (i.e., the parental, “wild-type” or “wt” antibody). The antibody can then be mutated and tested (evolved). One of ordinary skill in the art can utilize the methods for identifying conditional active antibodies disclosed in U.S. Patent No. 8,709,755 to identify additional conditional active antibodies and antibody fragments for use as ASTRs for the CAB-CARs of the present disclosure. Complementary determining regions (CDRs) are three-dimensional structures formed by the interaction of VH and VL. To change the binding specificity of the starting point (“wt” antibody), it would be reasonable to predict that mutating one or both of VH / VL could lead to CAB activity of the CAB-CAR. To generate a conditional active antibody, both VH and VL are typically identified under physiological conditions and then VH or VL or more typically both VH and VL are mutated and tested under non-physiological conditions such as a pH of 6.0 to 6.7 or another condition of the tumor microenvironment to generate a conditional active antibody.

[0066] Nucleic acids encoding the VH and / or VL regions of the wild-type antibody can be cloned using known methods. Variants of such wild-type VH and VL regions can then be prepared by introducing modifications into the nucleotide sequences encoding the heavy and light chain variable regions. Such modifications include, for example, deletions from residues within the amino acid sequence of the antibody or antibody fragment, and / or insertions into and / or substitutions thereof. Any combination of deletions (single or multiple), insertions (single or multiple), and substitutions (single or multiple) can be performed to arrive at a conditional active antibody fragment.

[0067] Detailed methods for making and / or isolating conditional active ASTRs are provided in another section of this specification.

[0068] Conditional active ASTR targeting Axl Exemplary embodiments of any of the various aspects provided herein include CARs having a conditional active ASTR that specifically binds to the Axl protein at a pH of 6.7 as compared to a pH of 7.4. Examples of such ASTRs and CARs containing such ASTRs are provided in the examples herein. In certain embodiments, the ASTR binds to the same epitope of Axl as an antibody comprising the heavy chain of SEQ ID NO: 79 and the light chain of SEQ ID NO: 80. In an exemplary embodiment, the ASTR binds to the same epitope of Axl as a single-chain variable antibody fragment comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80.

[0069] The ASTR can be a single-chain antibody, Fab fragment, Fab’ fragment, (Fab’)2 fragment, Fv fragment, and bivalent single-chain antibody or diabody. In an exemplary embodiment, the conditional active ASTR that binds to Axl is a single-chain variable fragment comprising a heavy chain and a light chain.

[0070] In some embodiments where the ASTR binds to Axl and, in an exemplary embodiment, binds to the same epitope as a single-chain variable antibody fragment comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80, the heavy chain variable region can comprise three complementarity determining regions, said regions having the sequences H1, H2, and H3, The H1 sequence is X1GX2TMN (SEQ ID NO: 87); The H2 sequence is LIKPSNGGTSYNQKFKG (SEQ ID NO: 88); and The H3 sequence is GX3YX4SYX5AMDY (SEQ ID NO: 89), where X1 is T or W; X2 is H or A; X3 is H or D; X4 is E or H; and X5 is E or F.

[0071] In some embodiments, ASTR binds to Axl and binds to the same epitope as a single-chain variable antibody fragment comprising an antibody heavy chain of SEQ ID NO: 79 and an antibody light chain of SEQ ID NO: 80, including the heavy chain embodiment immediately above. In some embodiments, ASTR can include a light chain variable region comprising three complementarity determining regions, said regions having sequences L1, L2, and L3, The L1 sequence is KASQDVX6SAVA (SEQ ID NO: 90); The L2 sequence is WX7X8TRX9T (SEQ ID NO: 91); and The L3 sequence is QEHFSX 10 PLX 11 (SEQ ID NO: 92); X6 is S or V; X7 is A or Q; X8 is S or D; X9 is H or D; X 10 is T or P; and X 11 is T or R.

[0072] In some embodiments, ASTR binds to Axl and binds to the same epitope as a single-chain variable antibody fragment comprising an antibody heavy chain of SEQ ID NO: 79 and an antibody light chain of SEQ ID NO: 80. In some embodiments, the heavy chain variable region can include three complementarity determining regions, said regions having sequences H1, H2, and H3, The H1 sequence is X1GX2X3MX4 (SEQ ID NO: 134); The H2 sequence is LIKX5SNGGTX6YNQKFKG (SEQ ID NO: 135); and The H3 sequence is GX7X8X9X 10 X 11 X 12 X 13 X 14 DYX 15 X 16 (SEQ ID NO: 136), X1 is T, A, or W; X2 is H or A; X3 is T or I; X4 is N or I; X5 is P or N; X6 is S, I, or T; X7 is H, D, E, P, R, or W; X8 is Y or N; X9 is E, A, D, F, G, H, I, L, M, N, R, V, or Y; X 10 is S, D, M, N, or Q; X 11 is Y, C, E, or P; X 12 is F, E, N, S, T, or V; X 13 is A, D, G, L, or Y; X 14 is M, E, or F; X 15 is W, A, D, H, L, N, P, R, or T; and X 16 is G or H.

[0073] In some embodiments where ASTR binds to Axl and, in an exemplary embodiment, binds to the same epitope as a single-chain variable antibody fragment comprising an antibody heavy chain of SEQ ID NO: 79 and an antibody light chain of SEQ ID NO: 80, including the heavy chain embodiment immediately above, ASTR can comprise a light chain variable region comprising three complementarity determining regions, said regions having sequences L1, L2, and L3, The L1 sequence is KASQDX 17 X 18 SX 19 VX 20 (SEQ ID NO: 137); The L2 sequence is X 21 X 22 X 23 TRX 24 T(SEQ ID NO: 138); and The L3 sequence is QEX 25 X 26 SX 27 X 28 X 29 X 30 (SEQ ID NO: 139), X 17 is V, D, G, N, or W; X 18 is S or V; X 19 is A, L, or M; X 20 is A, D, N, or Q; X21 is W or F;X 22 is A, I, N, P, or Q; X 23 is S or D;X 24 is H or D;X 25 is H, C, F, I, L, Q, S, T, V, or Y; X 26 is F, C, D, E, G, N, or S; X 27 is T, C, or P; X 28 is P, A, C, D, E, H, K, S, T, V, or W; X 29 is L, G, or R; and X 30 is T, I, or R.

[0074] In certain exemplary embodiments, the ASTR comprises a light chain variable region of SEQ ID NO:80 and / or a heavy chain variable region of SEQ ID NO:79. These exemplary embodiments may include a heavy chain N-terminal to the light chain or a light chain N-terminal to the heavy chain. In exemplary embodiments, the anti-AXl ASTR may comprise any of the sequences of SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:159, SEQ ID NO:160, or SEQ ID NO:161.

[0075] The heavy and light chains of any of these anti-AX1 embodiments include two variable regions, which are typically separated by a linker. The linker can be 6-100 amino acids in length. In some embodiments, the linker is Linker 1 (SEQ ID NO:53), Linker 2 (SEQ ID NO:54), or Linker 3 (SEQ ID NO:55). In exemplary embodiments, the two variable regions are a heavy chain variable region and a light chain variable region. Either the heavy chain or the light chain can be located N-terminal to the other on the ASTR. In certain exemplary embodiments, the heavy chain is N-terminal to the light chain.

[0076] Exemplary conditional active CARs (CAB-CARs) with increased binding to Axl at pH 6.7 compared to pH 7.4 are found in Example 1 of this specification. In an exemplary embodiment, the CAR or ASTR can bind to the same epitope of Axl as the single-chain variable antibody fragment comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80. In a further embodiment of such an exemplary embodiment, the anti-AXl CAR or ASTR comprises a single-chain variable fragment (scFv) or is a single-chain variable fragment. In a further exemplary example, the anti-AXl scFv comprises a heavy chain that is N-terminal to the light chain or a light chain that is N-terminal to the heavy chain. In any of the embodiments of this specification that include a CAR and that bind to the same epitope of Axl as an antibody comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80 in an exemplary embodiment, the ASTR can comprise any of SEQ ID NO: 128, 129, 159, 160, or 161. Further, any anti-AXl CAR of any of the embodiments of this specification can comprise any of the CAR components provided herein. In a particular exemplary embodiment, the anti-AXl CAR can comprise the CAR components described in Table 1 and can be any of the CARs in Table 1. With respect to any embodiment of this specification that includes an anti-AXl CAR, more typically, the CAR is a CAB-CAR, and in a non-limiting exemplary embodiment, the CAR can comprise, for example, any of the CAB-CAR components provided in Table 1 that exhibit cytotoxic activity and any of the CAB-CARs. For example, the anti-AXl CAB-CAR can comprise a CD8 signal peptide, a CD8 or CD28 stalk domain / transmembrane domain, CD137, an ICΔ co-stimulatory domain and a CD137 co-stimulatory domain, and / or a CD3ζ activation domain. Further, any exemplary CAR of any of the embodiments of this specification that includes an anti-AXl CAR and particularly an anti-AXl CAB-CAR can comprise, in a non-limiting exemplary embodiment, any of the anti-AXl CAB-CARs that exhibit conditional cytotoxic activity in Table 1. Such exemplary CAB-CARs include F1-2-1, F1-2-2, F1-2-3, F1-2-6, F1-2-8, F1-2-10, F1-2-13, F1-2-14, F1-2-15, F1-2-22, or F1-2-23 of Table 1.In any of the embodiments of the specification that include ASTR, the ASTR can include the ASTR of F1-2-1, F1-2-2, F1-2-3, F1-2-6, F1-2-8, F1-2-10, F1-2-13, F1-2-14, F1-2-15, F1-2-22, or F1-2-23. Further, any exemplary CAR of the embodiments of the specification that include an anti-AXl CAR and in particular an anti-AXl CAB-CAR includes, in a non-limiting exemplary embodiment, any of the anti-AXl CAB-CARs that exhibit high conditional cytotoxic activity in Table 1. Such exemplary CAB-CARs include F1-2-13, F1-2-15, F1-2-22, or F1-2-23. Thus, in any of the embodiments of the specification that include ASTR, the ASTR can include the ASTR of F1-2-1, F1-2-2, F1-2-3, F1-2-6, F1-2-8, F1-2-10, F1-2-13, F1-2-14, F1-2-15, F1-2-22, or F1-2-23.

[0077] The heavy chain variable region polypeptide and the light chain variable region polypeptide disclosed herein were identified from a parental antibody heavy chain variable region (SEQ ID NO: 93) and a parental antibody light chain variable region (SEQ ID NO: 94) using the method disclosed in U.S. Patent No. 8,709,755. One of ordinary skill in the art can utilize the method for identifying conditionally active antibodies disclosed in U.S. Patent No. 8,709,755 to identify additional conditionally active antibodies and antibody fragments that can be used for the ASTR of the CAB-CARs of the present disclosure.

[0078] In some embodiments, the heavy chain variable region can be SEQ ID NOs: 112-114. In some embodiments, the light chain variable region can be SEQ ID NOs: 108-111. These heavy and light chain variable regions can specifically bind to Axl. It has been revealed that an antibody containing any one of these heavy and light chain variable regions has a high binding affinity for Axl at pH 6.7 rather than at pH 7.4. pH 6.7 is the pH found in the tumor microenvironment. pH 7.4 is the pH found in the normal physiological microenvironment of non-tumor.

[0079] The CAR can also comprise variants of the heavy and light chain variable regions of the sequences of SEQ ID NOs: 108 to 114 that can specifically bind to Axl. In order to obtain these variants, it has been revealed that the complementarity determining regions (CDRs) of the heavy chain variable region (H1-H3) and the CDRs of the light chain variable region (L1-L3) need to remain unchanged. These variants of the heavy and light chain variable regions can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the heavy and light chain variable regions, or by peptide synthesis. Such modifications include, for example, deletions from residues within the amino acid sequence of the antibody or antibody fragment, and / or insertions into and / or substitutions thereof. Any combination of deletions (single or multiple), insertions (single or multiple), and substitutions (single or multiple) can be carried out to reach the final construct, provided that the final construct has at least one desired property, such as antigen binding.

[0080] Conditional active ASTR targeting Ror2 Any exemplary embodiment of any of the various aspects provided herein includes a CAR having a conditionally active ASTR that specifically binds to the Ror2 protein at a pH of 6.7 as compared to a pH of 7.4. Examples of such ASTRs and CARs that include such ASTRs are provided in the examples herein. In certain embodiments, the ASTR binds to the same epitope of Ror2 as an antibody comprising the heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83, or the ASTR binds to the same epitope of Ror2 as an antibody comprising the heavy chain of SEQ ID NO: 151. In an exemplary embodiment, the ASTR binds to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83, or the ASTR binds to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the heavy chain of SEQ ID NO: 151. In an exemplary embodiment, the ASTR binds to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the light chain of SEQ ID NO: 84, or the ASTR binds to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the heavy chain of SEQ ID NO: 151 and the light chain of SEQ ID NO: 152.

[0081] The ASTR can be a single-chain antibody, Fab fragment, Fab’ fragment, (Fab’)2 fragment, Fv fragment, and a bivalent single-chain antibody or diabody. In an exemplary embodiment, the conditionally active ASTR that binds to Ror2 is a single-chain variable fragment comprising a heavy chain and a light chain.

[0082] In some embodiments where the ASTR binds to Ror2, the ASTR can include a heavy-chain variable region having three complementarity-determining regions, said regions having H1, H2, and H3 sequences, The H1 sequence is GYTX1TEX2TX3H (SEQ ID NO: 95) or X4GYSITTGYYWN (SEQ ID NO: 96); The H2 sequence is GX5NX6NNGGTGYNQKFKG (SEQ ID NO: 97) or YITYDGSKNYNPSLKN (SEQ ID NO: 98); The H3 sequence is GSLYSYGNSYFDY (SEQ ID NO: 99) or FEGVWX7GLDY (SEQ ID NO: 100), and X1 is F or E; X2 is Y or D; X3 is M or D; X4 is T or S; X5 is E or I; X6 is T or D; and X7 is Y or G.

[0083] In some embodiments where ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, ASTR can comprise a heavy chain variable region having three complementarity determining regions, said region having H1, H2, and H3 sequences, The H1 sequence is GYTX1TEX2TX3H (SEQ ID NO: 95); The H2 sequence is GX5NX6NNGGTGYNQKFKG (SEQ ID NO: 97); and The H3 sequence is GSLYSYGNSYFDY (SEQ ID NO: 99), X1 is F or E; X2 is Y or D; X3 is M or D; X5 is E or I; and X6 is T or D.

[0084] In some embodiments where ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, ASTR can comprise a heavy chain variable region having three complementarity determining regions, said region having H1, H2, and H3 sequences, The H1 sequence is X4GYSITTGYYWN (SEQ ID NO: 96); The H2 sequence is YITYDGSKNYNPSLKN (SEQ ID NO: 98); and The H3 sequence is FEGVWX7GLDY (SEQ ID NO: 100), X4 is T or S; and X7 is Y or G.

[0085] Some embodiments in which ASTR binds to Ror2 include, but are not limited to, those having a heavy chain with the above H1, H2, and H3 sequences. In some embodiments, ASTR includes a light chain variable region having three complementarity-determining regions, said regions having L1, L2, and L3 sequences, The L1 sequence is SATSSX8SYMH (SEQ ID NO: 101) or RASESVDRYGNSFIH (SEQ ID NO: 102); The L2 sequence is X9TSNLAS (SEQ ID NO: 103) or RTYNLES (SEQ ID NO: 104); and The L3 sequence is QQRSSYPFT (SEQ ID NO: 105) or QQTNEDPWT (SEQ ID NO: 106), X8 is E or V; and X9 is G or H.

[0086] In some embodiments in which ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, ASTR can include a light chain variable region having three complementarity-determining regions, said regions having L1, L2, and L3 sequences, The L1 sequence is SATSSX8SYMH (SEQ ID NO: 101); The L2 sequence is X9TSNLAS (SEQ ID NO: 103); and The L3 sequence is QQRSSYPFT (SEQ ID NO: 105), X8 is E or V; and X9 is G or H.

[0087] In some embodiments in which ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, ASTR can include a light chain variable region having three complementarity-determining regions, said regions having L1, L2, and L3 sequences, The L1 sequence is RASESVDRYGNSFIH (SEQ ID NO: 102); The L2 sequence is RTYNLES (SEQ ID NO: 104); and The L3 sequence is QQTNEDPWT (SEQ ID NO: 106).

[0088] In some embodiments where ASTR binds to Ror2, ASTR can comprise a heavy chain variable region comprising three complementarity-determining regions, said regions having H1, H2, and H3 sequences, The H1 sequence is GYTX1TEX2X3X4H (SEQ ID NO: 140) or GYSITTGX 29 YWN (SEQ ID NO: 141); The H2 sequence is X5X6X7X8NNGGTGYNQKFKG (SEQ ID NO: 142) or YITYDGSX 30 NYNPSLKN (SEQ ID NO: 143); and The H3 sequence is X9X 10 X 11 SX 12 YX 13 YX 14 X 15 SYFX 16 X 17 X 18 (SEQ ID NO: 144) or CSX 31 X 32 X 33 X 34 VX 35 X 36 X 37 LDX 38 (SEQ ID NO: 145), X1 is F or E; X2 is Y or D; X3 is T or C; X4 is M, D, E, or Y; X5 is G or S; X6 is I or E; X7 is N, C, L, or V; X8 is T, D or E; X9 is A, M, or T; X 10 is R or H; X 11 is G or E; X 12 is L or F; X 13 is S or G; X 14 is G or D; X 15 is N or E; X 16 is D or L; X 17 is Y, C, or T; X 18 is W or L; X29 is Y, E, R, or T; X 30 is K or N; X 31 is R, G, H, W, or Y; X 32 is F, C, N, or Q; X 33 is E or S; X 34 is G, E, F, H, M, Q, or S; X 35 is W, A, I, P, Q, T, or V; X 36 is Y, G, N, or Q; X 37 is G, S, or T; and X 38 is Y or I.

[0089] In some embodiments where ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, ASTR can comprise a heavy chain variable region comprising three complementarity determining regions, said regions having H1, H2, and H3 sequences, The H1 sequence is GYTX1TEX2X3X4H (SEQ ID NO: 140); The H2 sequence is X5X6X7X8NNGGTGYNQKFKG (SEQ ID NO: 142); and The H3 sequence is X9X 10 X 11 SX 12 YX 13 YX 14 X 15 SYFX 16 X 17 X 18 (SEQ ID NO: 144), X1 is F or E; X2 is Y or D; X3 is T or C; X4 is M, D, E, or Y; X5 is G or S; X6 is I or E; X7 is N, C, L, or V; X8 is T, D or E; X9 is A, M, or T; X 10 is R or H; X 11 is G or E; X 12 is L or F; X 13 is S or G; X 14 is G or D; X15 is N or E; X 16 is D or L; X 17 is Y, C, or T; and X 18 is W or L.

[0090] In some embodiments where ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, ASTR can comprise a heavy chain variable region having three complementarity determining regions, said regions having H1, H2, and H3 sequences, The H1 sequence is GYSITTGX 29 YWN (SEQ ID NO: 141); The H2 sequence is YITYDGSX 30 NYNPSLKN (SEQ ID NO: 143); and The H3 sequence is CSX 31 X 32 X 33 X 34 VX 35 X 36 X 37 LDX 38 (SEQ ID NO: 145), X 29 is Y, E, R, or T; X 30 is K or N; X 31 is R, G, H, W, or Y; X 32 is F, C, N, or Q; X 33 is E or S; X 34 is G, E, F, H, M, Q, or S; X 35 is W, A, I, P, Q, T, or V; X 36 is Y, G, N, or Q; X 37 is G, S, or T; and X 38 is Y or I.

[0091] In some embodiments where ASTR binds to Ror2, ASTR can comprise a light chain variable region having three complementarity determining regions, said regions having L1, L2, and L3 sequences, The L1 array is SATSSX 19 X 20 X 21 MX 22 (SEQ ID NO: 146) or RASESVDRYGNSX 39 IH (SEQ ID NO: 147); The L2 array is X 23 TSNLAS (SEQ ID NO: 148) or X 40 TYX 41 LES (SEQ ID NO: 149); and The L3 array is QX 24 X 25 SX 26 YPFX 27 X 28 (SEQ ID NO: 150) or QQX 42 NX 43 DPX 44 TX 45 (SEQ ID NO: 85), X 19 is V or E; X 20 is S or D; X 21 is Y, C, or D; X 22 is H, G, or L; X 23 is G, C, H, or P; X 24 is Q or E; X 25 is R or H; X 26 is S, D, G, I, Q, or V; X 27 is T or D; X 28 is F, D, or E; X 39 is F, S, or T; X 40 is R, C, D, E, or W; X 41 is N or D; X 42 is T, I, or P; X 43 is E or V; X 44 is W or T; and X 45 is F or T.

[0092] In some embodiments where ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, ASTR can comprise a light chain variable region comprising three complementarity determining regions, said regions having L1, L2, and L3 sequences, The L1 sequence is SATSSX 19 X 20 X 21 MX 22 (SEQ ID NO: 146); The L2 sequence is X 23 TSNLAS (SEQ ID NO: 148); and The L3 sequence is QX 24 X 25 SX 26 YPFX 27 X 28 (SEQ ID NO: 150), X 19 is V or E; X 20 is S or D; X 21 is Y, C, or D; X 22 is H, G, or L; X 23 is G, C, H, or P; X 24 is Q or E; X 25 is R or H; X 26 is S, D, G, I, Q, or V; X 27 is T or D; and X 28 is F, D, or E.

[0093] In some embodiments where ASTR binds to Ror2 and, in an exemplary embodiment, binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, ASTR can comprise a light chain variable region comprising three complementarity determining regions, said regions having L1, L2, and L3 sequences, The L1 sequence is RASESVDRYGNSX 39 IH (SEQ ID NO: 147); The L2 sequence is X 40 TYX 41 LES (SEQ ID NO: 149); and The L3 array is QQX 42 NX 43 DPX 44 TX 45 (SEQ ID NO: 85), and X 39 is F, S, or T; X 40 is R, C, D, E, or W; X 41 is N or D; X 42 is T, I, or P; X 43 is E or V; X 44 is W or T; and X 45 is F or T.

[0094] In some embodiments, a conditionally active ASTR that binds to Ror2, and in exemplary embodiments, a conditionally active ASTR that binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, comprises a heavy chain variable region having an amino acid sequence selected from the sequences of SEQ ID NOs: 115 - 119 and SEQ ID NO: 151. In these exemplary embodiments where the conditionally active ASTR comprises the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152 and optionally binds to the same epitope of Ror2 as an antibody comprising the heavy chain described in the previous sentence, the light chain can comprise the light chain of SEQ ID NO: 81, SEQ ID NOs: 122 - 124, or SEQ ID NO: 152.

[0095] In certain exemplary embodiments, a conditionally active ASTR binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, and comprises a heavy chain variable region of any one of SEQ ID NOs: 120 - 121 and 82 - 83. In these exemplary embodiments where the conditionally active ASTR comprises the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84 and optionally binds to the same epitope of Ror2 as an antibody comprising an antibody comprising the heavy chain described in the previous sentence, the light chain can comprise the light chain of SEQ ID NO: 84 or 86.

[0096] Exemplary conditional active CARs (CAB-CARs) with increased binding to Ror2 at pH 6.7 compared to pH 7.4 are found in Example 1 of this specification. In an exemplary embodiment, the CAR or ASTR can bind to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, or the CAR or ASTR can bind to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152. In a further embodiment of such exemplary embodiments, the anti-Ror2 CAR or ASTR comprises or is a single-chain variable fragment (scFv), and in a further exemplary embodiment, comprises a light chain that is N-terminal to the heavy chain or a heavy chain that is N-terminal to the light chain. In any of the embodiments of this specification that include a CAR or ASTR and bind to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, or bind to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, the ASTR can comprise any of SEQ ID NOs: 130-132, or 153-158. Further, any anti-Ror2 CAR of any of the embodiments of this specification can comprise any of the CAR components provided herein. In certain exemplary embodiments, the anti-Ror2 CAR can comprise the CAR components described in Table 2 and can be any of the CARs in Table 2. With respect to any embodiment of this specification that includes an anti-Ror2 CAR, more typically, the CAR is a CAB-CAR, and in a non-limiting exemplary embodiment, the CAR can comprise, for example, any of the CAB-CAR components provided in Table 2 that exhibit cytotoxic activity and any of the CAB-CARs. For example, the anti-Ror2 CAB-CAR can comprise a CD8 signal peptide, a CD8 or CD28 stalk domain / transmembrane domain, a CD137 co-stimulatory domain, and / or a CD3ζ activation domain.Furthermore, any exemplary CAR of any of the embodiments herein that includes an anti-Ror2 CAR and particularly an anti-Ror2 CAB-CAR includes, in non-limiting exemplary embodiments, any of the anti-Ror2 CAB-CARs that exhibit the conditional cytotoxic activity of Table 2. Such exemplary CAB-CARs include F1-1-9, F1-1-10, F1-1-11, F1-1-12, F1-1-15, F1-1-17, F1-1-18, F1-1-19, F1-1-20, F1-1-21, F1-1-23, F1-1-25, or F1-1-26. In any of the embodiments herein that include an anti-Ror2 ASTR, the ASTR can include the ASTRs of F1-1-9, F1-1-10, F1-1-11, F1-1-12, F1-1-15, F1-1-17, F1-1-18, F1-1-19, F1-1-20, F1-1-21, F1-1-23, F1-1-25, or F1-1-26 of Table 2. Furthermore, any exemplary CAR of any of the embodiments herein that includes an anti-Ror2 CAR and particularly an anti-Ror2 CAB-CAR includes, in non-limiting exemplary embodiments, any of the anti-Ror2 CAB-CARs that exhibit the high conditional cytotoxic activity of Table 2. Such exemplary CAB-CARs include F1-1-11, F1-1-12, F1-1-15, F1-1-17, F1-1-19, F1-1-20, or F1-1-23. Thus, in any of the embodiments herein that include an anti-Ror2 ASTR, the ASTR can include the ASTRs of F1-1-11, F1-1-12, F1-1-15, F1-1-17, F1-1-19, F1-1-20, F1-1-23.

[0097] More generally, with respect to any of the embodiments provided herein, regardless of whether it is against Axl or Ror2, the ASTR can be a single-chain antibody, Fab fragment, Fab’ fragment, (Fab’)2 fragment, Fv fragment, and a bivalent single-chain antibody or diabody. In an exemplary embodiment, the conditional active ASTR that binds to Ror2 is a single-chain variable fragment comprising a heavy chain and a light chain.

[0098] The heavy chain variable region polypeptides and light chain variable region polypeptides disclosed herein were identified from parental antibodies using the method disclosed in U.S. Patent No. 8,709,755. One of ordinary skill in the art can utilize the method for identifying conditionally active antibodies disclosed in U.S. Patent No. 8,709,755 to identify additional conditionally active antibodies and antibody fragments that can be used for ASTRs of the CAB-CARs of the present disclosure.

[0099] The amino acid sequences of the heavy chain variable regions of some exemplary ASTRs are shown in SEQ ID NOs: 115-121. The amino acid sequences of the light chain variable regions of these exemplary ASTRs are shown in SEQ ID NOs: 81, 86, and 122-124. These heavy and light chain variable regions can specifically bind to human Ror2. Antibodies or antibody fragments comprising any one of these heavy chain variable regions and light chain variable regions have been shown to have a higher binding affinity for Ror2 at the pH in the tumor microenvironment than at the pH in the non-tumor microenvironment or under physiological conditions. For example, the antibodies and antibody fragments have a higher binding affinity for Ror2 at pH 6.0 than at pH 7.4. In some embodiments, the antibodies and antibody fragments have a higher binding affinity for Ror2 at pH 6.7 than at pH 7.4.

[0100] The anti-Ror2 antibodies or antibody fragments have a higher binding affinity for Ror2 in tumors compared to their binding affinity for Ror2 in normal tissues. These anti-Ror2 antibodies or antibody fragments are thought to have a longer half-life and reduced side effects, as well as equivalent efficacy, compared to monoclonal anti-Ror2 antibodies known in the art. These characteristics enable the delivery of higher doses of these anti-Ror2 antibodies or antibody fragments to patients, thereby providing a more effective treatment option.

[0101] ASTR can include a heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 81, 86, and 115-124, but the present invention specifically also provides variants thereof that can bind to human Ror2. To obtain these variants, the complementarity determining regions (CDRs) of the heavy chain variable region (H1-H3) and the complementarity determining regions of the light chain variable region (L1-L3) need to remain unchanged. However, the amino acid sequences of the heavy chain variable region and the light chain of the complementarity determining regions may be mutated according to the principles of substitution, insertion, and deletion. Variants of the heavy chain variable region and the light chain variable region can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the heavy chain variable region and the light chain variable region, or by peptide synthesis. Such modifications include, for example, deletions from residues within the amino acid sequences of the heavy chain variable region and the light chain variable region, and / or insertions into and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be carried out to arrive at an ASTR for use in a CAR, provided that they have the desired properties, such as conditional antigen binding to human Ror2.

[0102] Multispecific ASTR In some embodiments, the ASTR can be multispecific, e.g., a bispecific antibody. A bispecific antibody has binding specificities for at least two different sites or targets. In certain embodiments, one of the binding specificities is for Axl or Ror2 and the remainder is for another antigen. In certain embodiments, the bispecific antibody can bind to two different epitopes of Axl or Ror2. The bispecific antibody can also be used to localize a cytotoxic agent to cells expressing Axl or Ror2. The bispecific antibody can be prepared as a full-length antibody or an antibody fragment. In certain embodiments, one of the binding specificities binds to Axl and the other binds to Ror2, where both may not be conditionally active, or one or both may be conditionally active.

[0103] ASTRs suitable for use in the CARs of the present disclosure can have various antigen-binding specificities. In an exemplary embodiment, the ASTR binds to Axl or Ror2, which are known to be expressed on certain cancer cells (i.e., are cancer-specific antigens). In some cases, the ASTR is bispecific and, in addition to an antigen-binding domain that binds to Axl or Ror2, typically in a conditionally active manner, the ASTR can include a second antigen-binding domain specific for a second antigen that is expressed (synthesized) by cancer cells, i.e., is a cancer-associated antigen. Cancer-associated antigens can be, for example, antigens associated with breast cancer cells, B-cell lymphoma, Hodgkin lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma, glioblastoma, medulloblastoma, colorectal cancer cells, etc. Cancer-associated antigens can also be expressed by non-cancer cells in some cases.

[0104] In addition to Axl or Ror2, non-limiting examples of antigens to which the bispecific ASTR of the CAR can bind include, for example, CD19, CD20, CD38, CD30, ErbB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-α2, GD2, etc.

[0105] In some cases, in addition to the specific binding pair members that bind to Axl or Ror2, the members of the specific binding pair suitable for use of the CAR with such bispecific ASTR bind to the ligand of the receptor. Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF); etc.); integrin-binding peptides (e.g., peptides containing the sequence Arg-Gly-Asp); etc.

[0106] When the member of the specific binding pair of the bispecific CAR is a ligand, the CAR can be activated in the presence of the second member of the specific binding pair. In this case, the second member of the specific binding pair is the receptor for the ligand. For example, when the ligand is VEGF, the second member of the specific binding pair can be a VEGF receptor including a soluble VEGF receptor.

[0107] As described above, in some cases, the members of the specific binding pair included in the bispecific CAR are ASTRs that are receptors, such as receptors for ligands, ligands of co-receptors, etc. The receptor can be a ligand-binding fragment of the receptor. Suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptor); killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); cytokine receptors (e.g., IL-13 receptor; IL-2 receptor; etc.); CD27; natural cytotoxic receptor (NCR) (e.g., NKP30 (NCR3 / CD337) polypeptide (HLA-B-associated transcript 3 (BAT3) and B7-H6); etc.).

[0108] Stalk region In some cases, the CAR includes a hinge domain (also referred to herein as a "spacer" or "stalk") that is located in the extracellular portion of the CAR and is inserted between the ASTR and the transmembrane domain. In an exemplary embodiment, the hinge domain is a CD8 stalk domain or a CD28 stalk domain. In some cases, the stalk domain has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the wild-type CD8 stalk region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 125)), or at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the wild-type CD28 stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 126)), or at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the wild-type immunoglobulin heavy chain hinge / stalk region. In some cases, the stalk domain is a receptor-derived hinge region (e.g., a CDS-derived hinge region) polypeptide. In the CAR, the stalk employed enables conditional activation of the antigen-specific targeting region and, typically, the entire CAR to retain its enhanced binding properties to its Ror2 or Axl during in vitro tumor surrogate assay conditions for the corresponding physiological conditions.

[0109] The stalk region can have a length of about 4 amino acids to about 50 amino acids, such as about 4aa to about 10aa, about 10aa to about 15aa, about 15aa to about 20aa, about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 40aa, or about 40aa to about 50aa.

[0110] In some cases, the hinge domain of the CAR contains at least one cysteine. For example, in some cases, the hinge domain can contain the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 62). When present, the cysteine in the hinge domain of the first CAR can be utilized to form a disulfide bond with the hinge domain of the second CAR.

[0111] Immunoglobulin hinge / stalk region amino acid sequences are known in the art. See, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As a non-limiting example, an immunoglobulin hinge region can include a domain having at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to any contiguous at least 10, 15, 20, or all of the amino acids of the following amino acid sequences: DKTHT (SEQ ID NO: 63); CPPC (SEQ ID NO: 62); CPEPKSCDTPPPCPR (SEQ ID NO: 64) (see, for example, Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO: 65); KSCDKTHTCP (SEQ ID NO: 66); KCCVDCP (SEQ ID NO: 67); KYGPPCP (SEQ ID NO: 68); EPKSCDKTHTCPPCP (SEQ ID NO: 69) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 70) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 71) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 72) (human IgG4 hinge); etc. The hinge region can include the amino acid sequence of the human IgG1, IgG2, IgG3, or IgG4 hinge region. The hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (native) hinge region. For example, His229 of the human IgG1 hinge can be substituted with Tyr such that the hinge region includes the sequence EPKSCDKTYTCPPCP. See, for example, Yan et al. (2012) J. Biol. Chem. 287:5891. The hinge region can include an amino acid sequence derived from human CD8, for example, the hinge region can include the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 73), or a variant thereof.

[0112] Transmembrane domain The CARs of the present disclosure include transmembrane domains for insertion into the eukaryotic cell membrane. The transmembrane domain can be inserted between ASTR and the co-stimulatory domain. The transmembrane domain can be inserted between the hinge region and the co-stimulatory domain, whereby the chimeric antigen receptor comprises, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus): ASTR; hinge region; transmembrane domain; and activation domain.

[0113] Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use in the aspects and embodiments disclosed herein. In certain embodiments provided herein, the TM domain for any aspect comprising a CAR provided herein is C * an alpha TM domain, CD8 TM domain, CD4 TM domain, C3Z TM domain, C28 TM domain, C134 TM domain, CD7 TM domain, CD8 TM domain, or CD28 TM domain. Exemplary embodiments of the CARs provided herein include a CD8 TM domain or a CD28 TM domain. Further non-limiting examples of TM domains suitable for any of the aspects or embodiments provided herein include domains having at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to at least 10, 15, 20, or all of the following consecutive amino acids of a TM domain: a) CD8 alpha (IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 46)); b) CD8 beta (LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 47)); c) CD4 (ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 48)); d) CD3Z (LCYLLDGILFIYGVILTALFLRV (SEQ ID NO: 49); e) CD28 (FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 50)); f) CD134 (OX40): (VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO: 51)); g) CD7 (ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 52)); h) CD8 alpha stalk and TM (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 75)), and i) CD28 stalk and TM (IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 76)).

[0114] As a non-limiting example, the transmembrane domain of an aspect of the invention can have at least 80, 90, or 95% sequence identity to the transmembrane domain of SEQ ID NO: 46, the CD8 beta transmembrane domain, the CD4 transmembrane domain, the CD3 zeta transmembrane domain, the CD28 transmembrane domain, the CD134 transmembrane domain, or the CD7 transmembrane domain.

[0115] CAR linker In some cases, the CAR includes a linker between any two adjacent domains. For example, the linker can be present between the transmembrane domain and the first co-stimulatory domain. In another example, the ASTR is an antibody and the linker can be present between the heavy and light chains. In another example, the linker can be present between the ASTR and the transmembrane domain and co-stimulatory domain. In another example, the linker can be present between the co-stimulatory domain and the intracellular activation domain of the second polypeptide.

[0116] The linker peptide can have any of a variety of amino acid sequences. Proteins can usually be linked by a spacer peptide with flexible properties, but other chemical linkages are not excluded. The linker can be a peptide about 1 to about 100 amino acids in length, or about 1 to about 25 amino acids in length. These linkers can be produced using oligonucleotides encoding the linker for binding the protein. A peptide linker having a certain degree of flexibility can be used. The linking peptide can have substantially any amino acid sequence, but it should be noted that a suitable linker has a sequence that results in a flexible peptide as a whole. The use of small amino acids such as glycine and alanine is useful for creating a flexible peptide. The preparation of such sequences is routine work for those skilled in the art.

[0117] Suitable linkers can be easily selected and can be any suitable one of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, 7 amino acids to 8 amino acids, etc., and can also be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0118] Exemplary flexible linkers include glycine polymers (G) n , glycine - serine polymers (e.g., (GS) n , GSGGS n , GGGS n , and GGGGS nincluding (n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of note. The reason is that both of these amino acids are relatively amorphous and thus can function as neutral tethers between components. Glycine polymers are of particular note. The reason is that glycine has much more access to the Φ-Ψ space than alanine and is much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 53), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 54), GGGGSGGGSGGGGS (SEQ ID NO: 55), GGSG (SEQ ID NO: 56), GGSGG (SEQ ID NO: 57), GSGSG (SEQ ID NO: 58), GSGGG (SEQ ID NO: 59), GGGSG (SEQ ID NO: 60), GSSSG (SEQ ID NO: 61), and the like. One of ordinary skill in the art will understand that the design of a peptide conjugated to any of the above elements can include a linker that is wholly or partially flexible, whereby the linker can include a flexible linker as well as one or more moieties that impart a less flexible structure.

[0119] regulatory domain The regulatory domain can alter the effect of the activation domain in the CAR, which includes enhancing or suppressing the downstream effect of the activation domain or altering the nature of the response. Regulatory domains suitable for use in the CARs of the present disclosure and included in certain exemplary embodiments of any of the aspects herein that include a CAR include co-stimulatory domains. In some embodiments, the CAR can include two or more regulatory domains (e.g., co-stimulatory domains), or the regulatory domain of the CAR (e.g., co-stimulatory domain) can be derived from two or more polypeptides. Regulatory domains (e.g., co-stimulatory domains) suitable for inclusion in the CAR can have a length of about 30 amino acids to about 70 amino acids (aa), for example, the regulatory domain (e.g., co-stimulatory domain) can have a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, or about 65 aa to about 70 aa. In other cases, the regulatory domain can have a length of about 70 aa to about 100 aa, about 100 aa to about 200 aa, or more than 200 aa.

[0120] Co-stimulatory domains typically enhance and / or alter the nature of the response to activation of the activation domain. Co-stimulatory domains suitable for use in the CARs of the present disclosure are typically receptor-derived polypeptides. In some embodiments, the co-stimulatory domain is homodimerizing. The co-stimulatory domain can be the intracellular portion of a transmembrane protein (i.e., the co-stimulatory domain can be derived from a transmembrane protein). Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD137), B7-H3, CD2, CD7, CD27, CD28, Lck-binding deficient CD28 (ICΔ), ICOS, OX40, BTLA, CD27, CD30, CD40, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, the Fc receptor gamma chain, the Fc receptor epsilon chain, or a ligand that specifically binds to CD83. For example, the co-stimulatory domain of an aspect of the present invention can have at least 80%, 90%, or 95% sequence identity to the co-stimulatory domain of 4-1BB (CD137), CD27, CD28, Lck-binding deficient CD28 (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM. In some embodiments, the CAR can have two or more co-stimulatory domains. For example, the CAR can include a co-stimulatory domain derived from ICΔ and a co-stimulatory domain derived from 4-1BB (CD137).

[0121] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD137 (also known as TNFRSF9; CD137; 4-1BB; CDw137; ILA; etc.). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the consecutive amino acids in the following amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 1). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0122] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the consecutive amino acids in the following amino acid sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 2). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0123] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein Lck-binding deficient CD28 (ICΔ). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of a continuous stretch of amino acids in the following amino acid sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQAYAAARDFAAYRS (SEQ ID NO: 3). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0124] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of a continuous stretch of amino acids in the following amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 4). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0125] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGP1L). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 5).

[0126] In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0127] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following amino acid sequence: HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 6).

[0128] In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50aa.

[0129] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids in the following amino acid sequence: CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 7).

[0130] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, DlS166E, and Ki-1). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, or about 160 aa to about 185 aa in the following amino acid sequence: RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO: 8).

[0131] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids in the following amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 9). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0132] In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable co-stimulatory domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a continuous stretch of at least 10, 15, 20, or all of the amino acids in the following amino acid sequence: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 10). In some of these embodiments, the co-stimulatory domains of both the first and second polypeptides have a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

[0133] Intracellular activation domain Intracellular activation domains suitable for use in the CARs of the present disclosure, upon activation, result in the production of one or more cytokines; increased cell death and / or increased proliferation of CD8 + T cells, CD4 + T cells, natural killer T cells, γδ T cells, and / or neutrophils. In some embodiments, the intracellular activation domain comprises at least one (e.g., one, two, three, four, five, six, etc.) of the ITAM motifs described below. The intracellular activation domain is referred to herein as an activating domain or an activation domain. Intracellular activation domains for use in genetically modified signaling polypeptides can include intracellular signaling domains of several types of immune signaling receptors, including intracellular signaling proteins such as CD3, B7 family costimulatory molecules, and tumor necrosis factor receptor (TNFR) superfamily receptors; signaling domains used by NK and NKT cells such as NKp30 (B7-H6), DAP12, NKG2D, NKp44, NKp46, DAP10, and CD3Z; and signaling domains of human immunoglobulin receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs) such as FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, and FcRL5. Thus, in certain embodiments of the CAR for any aspect of the present disclosure, the intracellular activation domain is a signaling domain derived from NKp30 (B7-H6), DAP12, NKG2D, NKp44, NKp46, DAP10, CD3z, FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, or FcRL5. These are referred to herein as the NKp30 (B7-H6) activation domain, DAP12 activation domain, NKG2D activation domain, NKp44 activation domain, NKp46 activation domain, DAP10 activation domain, CD3Z activation domain, FcγRI activation domain, FcγRIIA activation domain, FcγRIIC activation domain, FcγRIIIA activation domain, or FcRL5 activation domain, respectively.

[0134] In some embodiments, the intracellular activation domain comprises a DAP10 / CD28 type signaling chain. In some embodiments, the intracellular activation domain is not covalently bound to the membrane-bound CAR, but instead diffuses in the cytoplasm. By way of non-limiting example, the intracellular activation domain of any aspect of the invention comprising the present CAR can be a CD3Z activation domain, a CD3D activation domain, a CD3E activation domain, a CD3G activation domain, a CD79A activation domain, a DAP12 activation domain, an FCERlG activation domain, a DAP10 / CD28 activation domain, or a ZAP70 activation domain. By way of non-limiting example, the intracellular activation domain of any aspect of the present invention comprising the CAR can have at least 80%, 90%, or 95% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, DAP12, FCERlG, DAP10 / CD28, or ZAP70 domains described below.

[0135] ITAM Intracellular activation domains suitable for use with the CARs of the present disclosure include immunoreceptor tyrosine activation motif (ITAM)-containing intracellular signaling polypeptides. The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid. In some cases, the intracellular activation domain of the CAR comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motifs are repeated twice within the intracellular activation domain, and the entities of the first and second ITAM motifs are separated from each other by 6-8 amino acids, for example, (YX1X2L / I)(X3) n (YX1X2L / I) (where n is an integer from 6 to 8 and each of the six to eight X3s can be any amino acid) are separated as such. In some cases, the intracellular activation domain of the CAR comprises three ITAM motifs.

[0136] Suitable intracellular activation domains can be ITAM motif-containing portions derived from polypeptides containing an ITAM motif. For example, a suitable intracellular activation domain can be an ITAM motif-containing domain derived from any protein containing an ITAM motif. Thus, a suitable intracellular activation domain need not include the entire sequence of the whole protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, CD3Z (CD3 zeta); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (antigen receptor complex-associated protein alpha chain); DAP12; and FCERIG (Fc epsilon receptor I gamma chain).

[0137] In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the following consecutive amino acids, or to a consecutive stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa adjacent to any of the following amino acid sequences (2 isoforms): MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO: 11) or MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO: 12), in which the ITAM motif is shown in brackets.

[0138] Similarly, a suitable intracellular activation domain polypeptide may include the ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence. Thus, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the following consecutive amino acids, or to a consecutive stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa proximate to any of the following amino acid sequences: RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO: 13); RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO: 127); NQL[YNELNLGRREEYDVL]DKR (SEQ ID NO: 14); EGL[YNELQKDKMAEAYSEI]GMK (SEQ ID NO: 15); or DGL[YQGLSTATKDTYDAL]HMQ (SEQ ID NO: 16), wherein the ITAM motif is shown in brackets in this sequence.

[0139] In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). Thus, a suitable intracellular activation domain can contain a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the following consecutive amino acids, or to a consecutive stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa adjacent to any of the following amino acid sequences: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO: 17) or MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO: 18), in which the ITAM motif is shown in brackets.

[0140] Similarly, a suitable intracellular activation domain polypeptide can include the ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. Thus, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence: DQV[YQPLRDRDDAQYSHL]GGN (SEQ ID NO: 19), wherein the ITAM motif is shown in brackets in this sequence.

[0141] In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). Thus, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence, or to a continuous series of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa adjacent to the following amino acid sequence: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD[YEPIRKGQRDLYSGL]NQRRI (SEQ ID NO: 20), wherein the ITAM motif is shown in brackets in this sequence.

[0142] Similarly, a suitable intracellular activation domain polypeptide may comprise the ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. Thus, a suitable intracellular activation domain can comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence: NPD[YEPIRKGQRDLYSGL]NQR (SEQ ID NO: 21), wherein the ITAM motif is shown in brackets in this sequence.

[0143] In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). Thus, a suitable intracellular activation domain can comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence, or to a continuous series of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa adjacent to the following amino acid sequence: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL[YQPLKDREDDQYSHL]QGNQLRRN (SEQ ID NO: 22), wherein the ITAM motif is shown in brackets in this sequence.

[0144] Similarly, a suitable intracellular activation domain polypeptide may comprise the ITAM motif-containing portion of the full-length CD3γ amino acid sequence. Thus, a suitable intracellular activation domain can comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous stretch of amino acids in the following sequence: DQL[YQPLKDREDDQYSHL]QGN (SEQ ID NO: 23), wherein the ITAM motif is shown in brackets in this sequence.

[0145] In some cases, the intracellular activation domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig alpha; membrane-bound immunoglobulin-associated protein; surface IgM-binding protein, etc.). Thus, a suitable intracellular activation domain can comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of a continuous sequence of the following amino acids, or to a continuous sequence of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa adjacent to any of the following amino acid sequences: MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 24) or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 25), in which the ITAM motif is shown in brackets.

[0146] Similarly, a suitable intracellular activation domain polypeptide may comprise the ITAM motif-containing portion of the full-length CD79A amino acid sequence. Thus, a suitable intracellular activation domain can comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous stretch of amino acids in the following sequence: ENL[YEGLNLDDCSMYEDI]SRG (SEQ ID NO: 26), wherein the ITAM motif is shown in brackets in this sequence.

[0147] In some cases, the intracellular activation domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer cell activation receptor-associated protein; killer cell activation receptor-associated protein, etc.). For example, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of the following consecutive amino acids, or to a consecutive stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa adjacent to any of the following amino acid sequences (4 isoforms): MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO: 27), MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO: 28), MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO: 29), or MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO: 30), in which the ITAM motif is shown in brackets.

[0148] Similarly, a suitable intracellular activation domain polypeptide can include the ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Thus, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence: ESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO: 31), in which the ITAM motif is shown in brackets.

[0149] In some cases, the intracellular activation domain is derived from FCERIG (FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcRgamma; fceRIgamma; high affinity immunoglobulin epsilon receptor subunit gamma; also known as immunoglobulin E receptor, high affinity, gamma chain, etc.). For example, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence, or to a continuous series of about 50 amino acids to about 60 amino acids (aa), about 60 aa to about 70 aa, about 70 aa to about 80 aa, or about 80 aa to about 88 aa adjacent to the following amino acid sequence: MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV[YTGLSTRNQETYETL]KHEKPPQ (SEQ ID NO: 32), in which the ITAM motif is shown in brackets.

[0150] Similarly, a suitable intracellular activation domain polypeptide can include the ITAM motif-containing portion of the full-length FCER1G amino acid sequence. Thus, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence: DGV[YTGLSTRNQETYETL]KHE (SEQ ID NO: 33), in which sequence the ITAM motif is shown in brackets.

[0151] An intracellular activation domain suitable for use in the CARs of the present disclosure includes a DAP10 / CD28 type signaling chain. An example of the DAP10 signaling chain is the following amino acid sequence: RPRRSPAQDGKV[YINM]PGRG (SEQ ID NO: 34). In some embodiments, a suitable intracellular activation domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence: RPRRSPAQDGKV[YINM]PGRG (SEQ ID NO: 34), in which sequence the notable motif is shown in brackets.

[0152] An example of the CD28 signaling chain is the following amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD[YMNM]TPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 35). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids in the following sequence: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD[YMNM]TPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 35).

[0153] Intracellular activation domains suitable for use in the CARs of the present disclosure can include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of the following consecutive amino acids, or to a consecutive stretch of about 300 to about 400 amino acids, about 400 to about 500 amino acids, or about 500 to 619 amino acids proximate to the following amino acid sequence: MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA (SEQ ID NO: 36).

[0154] Additional domain The CAR can further include one or more additional polypeptide domains, such domains including, but not limited to, signal sequences; epitope tags; affinity domains; and polypeptides that generate a detectable signal. Non-limiting examples of additional domains for any aspect or embodiment provided herein include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the following sequences described below: signal sequences, epitope tags, affinity domains, or polypeptides that generate a detectable signal.

[0155] Signal sequences suitable for use in the CAR, e.g., in the first polypeptide of the CAR, include any eukaryotic signal sequence, including natural signal sequences, synthetic (e.g., artificial) signal sequences, etc. In some embodiments, for example, the signal sequence can be the CD8 signal sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 74).

[0156] Epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA SEQ ID NO: 37); FLAG (e.g., DYKDDDDK; SEQ ID NO: 38); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 39), etc.

[0157] The affinity domain includes a peptide sequence capable of interacting with a binding partner, e.g., one that is fixed on a solid support and useful for identification or purification. A DNA sequence encoding a plurality of consecutive single amino acids, e.g., histidine, when fused to an expressed protein, can be used for one-step purification of a recombinant protein by affinity binding to a resin column such as nickel sepharose. Exemplary affinity domains include His5 (HHHHH; SEQ ID NO: 40), HisX6 (HHHHHH; SEQ ID NO: 41), c-myc (EQKLISEEDL; SEQ ID NO: 39), Flag (DYKDDDDK; SEQ ID NO: 38), Strep Tag (WSHPQFEK; SEQ ID NO: 42), hemagglutinin, e.g., HA tag (YPYDVPDYA; SEQ ID NO: 37), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 43), Phe-His-His-Thr (SEQ ID NO: 44), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 45), metal binding domains, e.g., zinc binding domains or calcium binding domains derived from calcium binding proteins such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, the large subunit of calpain, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, intein, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.

[0158] Suitable detectable signal generating proteins include, for example, fluorescent proteins; enzymes that catalyze reactions that generate a detectable signal as a product; and the like.

[0159] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed monomer, J-Red, Dimer2, t-Dimer2(12), mRFPl, pocilloporin, Aequorea GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobilin proteins and B-phycoerythrin, phycoerythrin proteins conjugated with R-phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), etc. For example, any of the various fluorescent and colored proteins derived from Anthozoa described in Matz et al. (1999) Nature Biotechnol. 17:969-973 is suitable for use.

[0160] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), etc.

[0161] Recognition and / or removal domain Any CAR disclosed herein may include a recognition or elimination domain. In some embodiments, the recognition or elimination domain can be derived from the herpes simplex virus-derived enzyme thymidine kinase (HSV-tk) or inducible caspase 9, or can be a FLAG epitope (SEQ ID NO: 38). In some embodiments, the recognition or elimination domain is recognized by an antibody approved for human use by a government regulatory agency, such as, but not limited to, cetuximab, rituximab, or Herceptin. In some embodiments, the recognition or elimination domain can include a modified endogenous cell surface molecule as described in U.S. Patent No. 8,802,374. The modified endogenous cell surface molecule can be any cell surface-related receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin, or surface antigen classification (CD) that has been modified. In some embodiments, the modified endogenous cell surface molecule is a truncated tyrosine kinase receptor. In one aspect, the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor family (e.g., ErbB1, ErbB2, ErbB3, ErbB4), such as SEQ ID NO: 78. The recognition or elimination domain can be expressed as part of a single polypeptide that also includes the CAR. In some embodiments, the recognition or elimination domain can be at or near the N-terminus of the single polypeptide, such as behind the N-terminal signal peptide. In some embodiments, the recognition or elimination domain can be at or near the C-terminus of the polypeptide. In some embodiments, the recognition or elimination domain at or near the C-terminus of the polypeptide can be separated from the amino acid sequence encoding the CAR by a cleavage signal or a ribosome skipping sequence. The cleavage signal can be any cleavage signal known in the art. The ribosome skipping sequence can be any ribosome skipping sequence known in the art, such as the 2A-1 amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 77).In some embodiments, the recognition or removal domain can be between any of the domains of the CAR, for example, between the stalk and the transmembrane domain, or the recognition or removal domain can be in a linker, for example, in the linker between the heavy and light chains of a single-chain antibody.

[0162] The epidermal growth factor receptor, also known as EGFR, ErbB1, and HER1, is a cell surface receptor for extracellular ligand epidermal growth factor family members. Changes in EGFR activity are associated with certain cancers. In some embodiments, a gene encoding an EGFR polypeptide, including the human epidermal growth factor receptor (EGFR), is constructed by removing a nucleic acid sequence encoding a polypeptide comprising a membrane-distal EGF binding domain and a cytoplasmic signaling terminus, while retaining an extracellular membrane-proximal epitope recognized by an anti-EGFR antibody. In an exemplary embodiment, the antibody is a known commercially available anti-EGFR monoclonal antibody, such as cetuximab, matuzumab, necitumumab, or panitumumab.

[0163] The application of immunomagnetic selection of biotinylated cetuximab in combination with anti-biotin microbeads successfully enriches T cells transduced with an EGFRt-containing construct by lentivirus from as low as a 2% population to over 90% purity without observable toxicity to the cell preparation. The constitutive expression of this inactive EGFR molecule does not affect the T cell phenotype or effector function induced by the co-expressed chimeric antigen receptor (CAR), CD19R. Through flow cytometry analysis, EGFR was successfully utilized as an in vivo tracking marker for T cell transplantation into mice. Furthermore, EGFR was shown to have the potential of a suicide gene via the Arcitumomab (registered trademark)-mediated antibody-dependent cell cytotoxicity (ADCC) pathway. Thus, EGFR can be used as a non-immunogenic selection tool, tracking marker, and suicide gene for transduced T cells with immunotherapeutic potential. The EGFR nucleic acid can also be detected by methods well known in the art.

[0164] In some embodiments, EGFR can be expressed as part of a single polypeptide that also includes a CAR. In some embodiments, the amino acid sequence encoding the EGFR recognition domain can be separated from the amino acid sequence encoding the CAR by a cleavage signal or a ribosome skip sequence. The cleavage signal can be any cleavage signal known in the art. The ribosome skip sequence can be any ribosome skip sequence known in the art and can be, for example, the 2A-1 amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 77). In some embodiments, the polynucleotide sequence encoding the recognition domain can be present on the same transcript as the CAR but can be separated from the polynucleotide sequence encoding the CAR by an internal ribosome entry site within the sequence.

[0165] Recombination of sequences In certain cases, a CAR, for example, the polypeptide sequence of the CAR domain, can be reconfigured or deleted in a cell by use of site-specific recombination techniques. In certain embodiments, the cell activation-related response to a particular CAR can be altered by site-specific recombination, for example, the first intracellular activation domain of a CAR that induces a first activation-related response can be exchanged with a second intracellular activation domain that induces a second activation-related response. As will be apparent to those skilled in the art, site-specific recombination can be used in a cell to exchange any domain or sequence of a CAR with any other domain or sequence disclosed herein. As will be apparent to those skilled in the art, site-specific recombination can be used in a cell to delete any domain or sequence of a CAR. Such exchange and excision of sequences and domains are known in the art; see, for example, domain switching in signaling bodies described in Tone et al. (2013) Biotechnology and Bioengineering, 3219-3226. The disclosure of this reference is hereby incorporated by reference into this disclosure. The mechanisms and requirements for performing site-specific recombination in vivo are also well known in the art; see Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA). The disclosure of this reference is hereby incorporated by reference into this disclosure.

[0166] The CAR is a chimeric protein generated by fusing together all the different domains considered above to form a fusion protein. The CAR is typically generated by an expression vector containing a polynucleotide sequence encoding the different domains of the CAR considered herein. The ASTR of the present invention, which functions to recognize and bind to an antigen on a target cell, is conditionally active. Specifically, compared to the ASTR of the corresponding wild-type protein, with respect to binding to the target antigen, the ASTR has low activity or is inactive under normal physiological conditions and is active under in vitro tumor surrogate assay conditions.

[0167] A wild-type or native protein suitable for use, at least in part, as its binding domain for a target antigen can be found as the ASTR of the present invention by generating a protein library and screening that library with a protein having the desired binding affinity for the target antigen. The wild-type protein can be found by screening a cDNA library. A cDNA library is a collection of cloned cDNA (complementary DNA) fragments inserted into host cells, which together constitute part of an organism's transcriptome. cDNA is generated from fully transcribed mRNA and thus contains the coding sequences of the expressed proteins of an organism. The information in a cDNA library is a powerful and useful tool for finding proteins with desirable properties by screening the library with a protein having the desired binding affinity for the target antigen.

[0168] tumor microenvironment Cancer cells in solid tumors can form a tumor microenvironment around them, which can support the growth and metastasis of cancer cells. The tumor microenvironment is the cellular environment in which the tumor exists, and it includes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, the extracellular matrix, and mechanical cues that can promote tumorigenic transformation, support tumor growth and invasion, protect the tumor from the host immune system, promote treatment resistance, and provide a microenvironment for the seeding of dormant metastases. The tumor and the surrounding microenvironment are closely related and constantly interact. The tumor can affect its microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can affect the growth and evolution of cancer cells. See Swarts et al., “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol. 72, pages 2473-2480, 2012.

[0169] The tumor microenvironment is often hypoxic. As the tumor mass increases, the interior of the tumor grows further away from the existing blood supply, making it difficult to adequately supply oxygen to the tumor microenvironment. The oxygen partial pressure in the tumor environment is less than 5 mmHg in over 50% of locally advanced solid tumors, compared to the oxygen partial pressure of about 40 mmHg in plasma. In contrast, other parts of the body are not hypoxic. The hypoxic environment leads to genetic instability, which is associated with cancer progression through downregulation of nucleotide excision repair and mismatch repair pathways. Hypoxia also causes upregulation of hypoxia-inducible factor I alpha (HIF1-α), which is associated with induction of angiogenesis and activation of genes related to a worse prognosis and metastasis. See Weber et al., “The tumor microenvironment,” Surgical Oncology, vol. 21, pages 172 - 177, 2012 and Blagosklonny, “Antiangiogenic therapy and tumor progression,” Cancer Cell, vol. 5, pages 13 - 17, 2004.

[0170] Furthermore, tumor cells tend to rely on energy generated from lactic acid fermentation, which does not require oxygen. Therefore, tumor cells are less likely to use normal aerobic respiration that requires oxygen. As a result of the use of lactic acid fermentation, the tumor microenvironment is acidic (pH 6.5 - 6.9), in contrast to other parts of the body that are neutral or slightly alkaline. For example, the pH of human plasma is about 7.4. See Estrella et al., “Acidity Generated by the Tumor Microenvironment Drives Local Invasion,” Cancer Research, vol. 73, pages 1524 - 1535, 2013. Due to the relatively high nutrient requirements of proliferating cancer cells, the degree of nutrient availability in the tumor microenvironment is also lower compared to cells located in other parts of the body.

[0171] Furthermore, the tumor microenvironment also contains many unique cell types not normally found in other parts of the body. These cell types include endothelial cells and their precursors, pericytes, smooth muscle cells, fibroblasts, cancer-associated fibroblasts, myofibroblasts, neutrophils, eosinophils, basophils, mast cells, T and B lymphocytes, natural killer cells, and antigen-presenting cells (APCs) such as macrophages and dendritic cells (Lorusso et al., “The tumor microenvironment and its contribution to tumor evolution toward metastasis,” Histochem Cell Biol, vol. 130, pages 1091-1103, 2008).

[0172] Therefore, the tumor microenvironment has at least some physiological conditions that are different from those in other parts of the body, such as the physiological conditions in plasma. The tumor microenvironment has a lower pH (acidic) than other parts of the body, particularly plasma (pH 7.4). The tumor microenvironment has a lower oxygen concentration than other parts of the body, such as plasma. Also, the tumor microenvironment has a lower nutrient availability than other parts of the body, particularly plasma. Additionally, the tumor microenvironment also contains some unique cell types not normally found in other parts of the body, particularly plasma.

[0173] In an exemplary embodiment, the CAR of the present invention comprises a conditionally active ASTR generated from a wild-type (i.e., unmodified) biological protein such as a wild-type or natural antibody isolated from a mammalian organism that can be a candidate for tumor treatment, such as a mouse or a human. In such an exemplary embodiment, the conditionally active ASTR has lower activity than the natural or wild-type biological protein under at least one physiological condition in a part of the body other than the tumor microenvironment, such as plasma, while having higher activity than the natural or wild-type biological protein under at least one physiological condition in the tumor microenvironment. The conditionally active natural or biological protein can selectively act on cancer cells in the tumor microenvironment to treat the tumor, and thus is likely to reduce the possibility of causing side effects. In an embodiment where the natural or biological protein is an antibody against an antigen on the tumor cell and the antigen is exposed in the tumor microenvironment, the conditionally active antibody has lower affinity for the antigen than the natural or wild-type antibody in other parts of the body, while having higher affinity for the antigen than the natural or wild-type antibody in the tumor microenvironment. Such a conditionally active antibody binds weakly or not at all to other parts of the body, but has greater binding or binds strongly and firmly to the antigen in the tumor microenvironment.

[0174] In vitro tumor replacement assay In exemplary embodiments, the CAR or ASTR used in the present disclosure, and the CARs of this specification that include such ASTRs, are typically conditionally active in the tumor environment and / or in vitro tumor surrogate assay conditions. In vitro tumor surrogate assay conditions can be any conditions that are tested in vitro at values or levels of that condition found in vivo in at least some cancers, relative to values or levels of that condition found in physiological tissue under physiological conditions. The non-limiting in vitro tumor surrogate assay for cell lysis at low pH (e.g., 6.0 or 6.7) compared to physiological pH (e.g., 7.4) is provided in the examples of this specification. In vitro tumor surrogate assay conditions under which the CARs of the present disclosure can become active include, but are not limited to, high hyaluronic acid, lactic acid, and / or albumin, and normal conditions are low levels of lactic acid, hyaluronic acid, and albumin. Another in vitro tumor surrogate assay condition is pH, and in particular, specific conditions where the in vitro tumor surrogate assay condition is lower than normal physiological pH. For example, the tumor surrogate assay condition can be a pH between a lower end value of the range of 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and an upper end value of the range of 6.6, 6.7, 6.8, and 6.9. On the other hand, physiological pH can be a pH between a lower end value of the range of 7.2, 7.3, and 7.4, and an upper end value of the range of 7.5, 7.6, 7.7, and 7.8. In exemplary embodiments, the low pH of the in vitro tumor surrogate assay condition is 6.5 - 6.9, and in particular, can be 6.7. Physiological pH is 7.2 - 7.6, or in particular, can be set to pH 7.4. In another embodiment, the pH does not change, but the in vitro tumor surrogate assay conditions differ only in the concentration of lactic acid. In another embodiment, the in vitro tumor surrogate assay conditions include a high level of adenosine compared to the physiological environment. In another embodiment, the in vitro tumor surrogate assay conditions include a high level of R-2-hydroxyglutaric acid compared to the physiological environment. In other embodiments, the scFv is grafted from the heavy and light chains of monoclonal antibodies selected for microenvironment specificity through molecular evolution, such as those described in U.S. Patent No. 8,709,755B2 and International Publication No. 2016 / 033331A1.

[0175] Nucleic acid The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide of a conditionally active CAR of the present disclosure. The nucleic acid comprising a nucleotide sequence encoding a conditionally active CAR of the present disclosure is, in some embodiments, for example, DNA comprising a recombinant expression vector. The nucleic acid comprising a nucleotide sequence encoding a conditionally active CAR of the present disclosure is, in some embodiments, for example, in vitro synthesized RNA.

[0176] In some cases, the nucleic acid provides for the production of the CARs of the present disclosure, for example, production in mammalian cells. In other cases, the nucleic acid provides for the amplification of the nucleic acid encoding the CARs of the present disclosure.

[0177] The nucleotide sequence encoding the CARs of the present disclosure can be operably linked to transcriptional regulatory regions, such as promoters and enhancers, etc.

[0178] Suitable promoter and enhancer sequences are known in the art. Promoters suitable for expression in bacterial cells include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. Promoters suitable for expression in eukaryotic cells include, but are not limited to, light chain and / or heavy chain immunoglobulin gene promoter and enhancer sequences; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in long terminal repeats derived from retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

[0179] Suitable reversible promoters that include reversible inducible promoters are known in the art. Such reversible promoters can be isolated and derived from many organisms, such as eukaryotes and prokaryotes. When using a reversible promoter derived from a first organism in a second organism, for example, when the first is a prokaryote and the second is a eukaryote, or when the first is a eukaryote and the second is a prokaryote, modification of the promoter is well known in the art. Such reversible promoters, as well as systems that are based on such reversible promoters but also include additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoter responsive to alcohol trans-activator protein (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including Tet activator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoter, ethylene-regulated promoter, benzothiadiazole-regulated promoter, etc.), temperature-regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.).

[0180] In some cases, a locus, construct, or transgene containing an appropriate promoter is irreversibly switched via induction by an inducible system. Systems suitable for the induction of irreversible switches are well known in the art. As an example, the induction of an irreversible switch can utilize the Cre-lox mediated recombination method (see, e.g., Fuhrmann-Benzakein, et al., PNAS (2000) 28:e99. The disclosure of this document is incorporated herein by reference. Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc. known in the art can be used to generate an irreversibly switchable promoter. It is well known in the art to generate a promoter that is irreversibly switched using the methods, mechanisms, and requirements for site-specific recombination described elsewhere herein. See, e.g., Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA). The disclosures of these documents are incorporated herein by reference.

[0181] In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, the CD4 gene promoter can be used. See, e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved using the Neri (p46) promoter. See, e.g., Eckelhart et al. (2011) Blood 117:1565.

[0182] In some embodiments, promoters suitable for expression in yeast cells include, for example, constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHO5 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TPI promoter, and AOX1 (for example, when used in Pichia), etc. The selection of appropriate vectors and promoters is well within the scope of those of ordinary skill in the art.

[0183] Promoters suitable for use in prokaryotic host cells include, but are not limited to, bacteriophage T7 RNA polymerase promoter; trp promoter; lac operon promoter; hybrid promoters such as lac / tac hybrid promoter, tac / trc hybrid promoter, trp / lac promoter, T7 / lac promoter; trc promoter; tac promoter, etc.; araBAD promoter; in vivo regulatory promoters such as ssaG promoter, or related promoters (see, for example, US Patent Application Publication No. 2004 / 0131637), pagC promoter (Pulkkinen and Miller, J. Bacterial., 1991:173(1):86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21):10079-83), nirB promoter (Harborne et al. (1992) Mal. Micro. 6:2805-2813), etc. (see, for example, Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol. 10:888-892); sigma 70 promoter such as consensus sigma 70 promoter (see, for example, Genbank accession numbers AX798980, AX798961, and AX798183); stationary phase promoters such as dps promoter, spv promoter, etc.; promoters derived from pathogenicity island SPI-2 (see, for example, International Publication No. 96 / 17951); actA promoter (see, for example, Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); rpsM promoter (see, for example, Valdivia and Falkow (1996). Mal. Microbial. 22:367); tet promoter (see, for example, Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U.(eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162 (see); SP6 promoter (see, for example, Melton et al. (1984) Nucl. Acids Res. 12: 7035), etc. Strong promoters suitable for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators suitable for use in bacterial host cells include the lactose promoter operator (the Laci repressor protein changes its conformation upon contact with lactose, thereby avoiding binding to the operator), the tryptophan promoter operator (the TrpR repressor protein adopts a conformation that binds to the operator when complexed with tryptophan; in the absence of tryptophan, the TrpR repressor protein adopts a conformation that does not bind to the operator), and the tac promoter operator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80: 21-25), etc.

[0184] The nucleotide sequence encoding the CAR may be present within an expression vector and / or a cloning vector. If the CAR comprises two separate polypeptides, the nucleotide sequences encoding the two polypeptides may be cloned into the same or different vectors. The expression vector may contain a selectable marker, an origin of replication, and other characteristic elements that provide for the replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, etc.

[0185] A number of suitable vectors and promoters are known to those skilled in the art, and many are commercially available for the generation of such recombinant constructs. The following vectors are provided for illustrative purposes. Bacterial vectors: pBs, phagescript, PsiXl74, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic vectors: pWLneo, pSV2cat, pOG44, PXRl, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0186] Expression vectors usually have convenient restriction sites near the promoter sequence to allow insertion of a nucleic acid sequence encoding a heterologous protein. A selectable marker operable in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus-based viral vectors (e.g., see Li et al., Invest Opthalmol Vis Sci 35:2543 - 2549, 1994; Borras et al., Gene Ther 6:515 - 524, 1999; Li and Davidson, PNAS 92:7700 - 7704, 1995; Sakamoto et al., Hum Gene Ther 5:1088 - 1097, 1999; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655); adeno-associated virus (e.g., see Ali et al., Hum Gene Ther 9:81 - 86, 1998, Flannery et al., PNAS 94:6916 - 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 - 2863, 1997; Jomary et al., Gene Ther 4:683 - 690, 1997, Rolling et al., Hum Gene Ther 10:641 - 648, 1999; Ali et al., Hum Mol Genet 5:591 - 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822 - 3828; Mendelson et al., Virol. (1988) 166:154 - 165; and Flotte et al., PNAS (1993) 90:10613 - 10617); SV40; herpes simplex virus; gamma-retrovirus; human immunodeficiency virus (e.g., see Miyoshi et al., PNAS 94:10319 - 23, 1997; Takahashi et al., see J Virol 73:7812 7816, 1999; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and other retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus, etc. - derived vectors), etc. can be mentioned.

[0187] As described above, in some embodiments, the nucleic acid containing the nucleotide sequence encoding the conditional active CAR of the present disclosure is, in some embodiments, RNA, for example, RNA synthesized in vitro. Methods for in vitro synthesis of RNA are known in the art. Using any known method, RNA containing the nucleotide sequence encoding the conditional active CAR of the present disclosure can be synthesized. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. (2010) Cancer Res. 15:9053. Introduction of the RNA containing the nucleotide sequence encoding the conditional active CAR of the present disclosure into host cells can be carried out in vitro or ex vivo or in vivo. For example, in vitro or ex vivo, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated using the RNA containing the nucleotide sequence encoding the conditional active CAR of the present disclosure.

[0188] Cell Some aspects of the present disclosure include or are cells, which in an exemplary embodiment are mammalian cells and are used as packaging cells to generate viruses such as lentiviruses for the transduction of T cells and / or NK cells. Any of a variety of cells can be selected for the in vitro production of viruses such as pseudotyped retroviruses according to the present invention. Eukaryotic cells, including human, monkey, dog, cat, horse, and rodent cells, particularly mammalian cells, are typically used. In an exemplary embodiment, the cells are human cells. In a further exemplary embodiment, the cells proliferate indefinitely and are thus immortal. Examples of cells that can be advantageously used in the present invention include NIH 3T3 cells, COS cells, Madin-Darby canine kidney cells, human fetal 293T cells, and any cells derived from such cells, for example, gpnlslacZ φNX cells derived from 293T cells. Highly transfectable cells such as human fetal kidney 293T cells can be used. "Highly transfectable" means that at least about 50%, more preferably at least about 70%, and most preferably at least about 80% of the cells can express the gene of the introduced DNA.

[0189] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCLlO), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RATl cells, mouse L cells (ATCC number CCL1.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0190] Method for activating immune cells The present disclosure provides a method for activating immune cells in vitro, in vivo, or ex vivo. The method generally includes contacting the immune cells (in vitro, in vivo, or ex vivo) with Axl and / or Ror2, wherein the immune cells are genetically modified to generate (i.e., express) a conditionally active form of a chimeric antigen receptor (CAR) under the conditions of the present disclosure. In the presence of Axl and / or Ror2, the conditionally active CAR activates the immune cells, thereby generating activated immune cells. Immune cells include, for example, cytotoxic T lymphocytes, NK cells, CD4 +Included are T cells, regulatory T (Treg) cells, γδ T cells, NK-T cells, neutrophils, and the like. In an exemplary embodiment, the immune cells are T cells or NK cells, and in particular, in an exemplary embodiment, the immune cells are T cells, which include NK-T cells. In such exemplary embodiments, activation is typically activation of the cytotoxic activity of T cells or NK cells. Such methods can be carried out using a plurality of immune cells (e.g., T cells or NK cells). In a further exemplary embodiment, contacting comprises contacting a target mammalian cell expressing Axl and / or Ror2 with the immune cells. Such methods of activating T cells or NK cells can be detected by detecting the release of cytokines such as IFN-γ or IL-2 by the T cells or NK cells, an increase in the cytotoxic activity of the T cells and / or NK cells against Axl or Ror2, an increase in the intracellular expression of IFNγ and / or IL-2 in the T cells or NK cells, and an increase in the expression of CD107a and / or CD69 by the T cells or NK cells as measured by fluorescence-activated cell sorting (FACS) analysis. Examples 1, 3, and 4 herein provide details regarding some of these methods of detecting activation of these T cells and / or NK cells.

[0191] Further aspects provided herein include a method of binding immune cells (e.g., T cells or NK cells) to a target mammalian cell, the method comprising contacting the target mammalian cell with the immune cells in vitro, in vivo, or ex vivo, wherein the target mammalian cell expresses Axl and / or Ror2 and the immune cells express any CAR that binds to Axl or Ror2 provided herein. Such binding can activate the immune cells. Such methods can be carried out using a plurality of immune cells (e.g., T cells or NK cells). Such binding methods, detected by detecting the activity of T cells or NK cells by release of cytokines and increased cytotoxic activity, are provided in Examples 1, 3, and 4 herein.

[0192] In the method of binding or activating immune cells, contacting in the exemplary embodiments herein includes contacting immune cells (e.g., T cells or NK cells) in a microenvironment with a pH less than 7.4. For example, the pH may be less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or in the range of 5.8 to 7.0, in the exemplary embodiments, in the range of 6.0 to 6.8, in the range of 6.1 to 6.9, in the range of 6.2 to 6.8, or between the lower end values of the range, such as 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and the upper end values of the range, such as 6.6, 6.7, 6.8, and 6.9. In such exemplary embodiments, the CAR is any CAB-CAR that recognizes Axl or Ror2 provided herein as disclosed herein.

[0193] Contacting genetically modified immune cells (e.g., T lymphocytes, NK cells) with Axl and / or Ror2 can increase the production of cytokines by the immune cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the amount of cytokines produced by the immune cells in the absence of Axl and / or Ror2. Contacting genetically modified immune cells (e.g., T lymphocytes, NK cells) with Axl and / or Ror2 can increase the secretion of cytokines by the immune cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold compared to the amount of cytokines secreted by the immune cells in the absence of Axl and / or Ror2. Cytokines whose production can be increased include, but are not limited to, IL-2 and IFN-γ.

[0194] Contacting genetically modified immune cells (e.g., cytotoxic T lymphocytes) with AAR can increase the cytotoxic activity of the cytotoxic cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold as compared to the cytotoxic activity of the cytotoxic cells in the absence of Axl and / or Ror2.

[0195] Contacting genetically modified immune cells (e.g., cytotoxic T lymphocytes) with Axl and / or Ror2 can increase the cytotoxic activity of the cytotoxic cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold as compared to the cytotoxic activity of the cytotoxic cells in the absence of Axl and / or Ror2.

[0196] In other embodiments, for example, contacting a genetically modified host cell with an antigen can increase or decrease cell proliferation, cell survival, cell death, etc., depending on the host immune cells.

[0197] Method for producing / isolating a conditionally active antigen-specific targeting region In an exemplary embodiment, the anti-Axl and anti-Ror2 antigen receptors disclosed herein are conditionally active and exhibit increased binding to Axl or Ror2 at pH 6.7 (exemplary pH of tumor environment and / or in vitro tumor surrogate assays) compared to pH 7.4 (normal physiological conditions). In some exemplary embodiments of any aspect disclosed herein, the conditionally active anti-Axl or anti-Ror2 ASTR is identified from an initial polypeptide library that does not contain mutant / evolved members of the library prior to selection / evolution and / or does not contain mutations during or between any selection iteration rounds. In other embodiments, the conditionally active anti-Axl or anti-Ror2 ASTR is identified by methods involving mutation / evolution and, in some embodiments, starting from a wild-type antibody. Exemplary transmembrane domains and intracellular activation domains can be any of those disclosed herein for the CAR.

[0198] In one aspect, provided herein is a method of selecting a conditionally active anti-Axl or anti-Ror2 ASTR, the method comprising panning a polypeptide display library as follows: a) subjecting the polypeptides of the polypeptide display library to an Axl or Ror2 binding assay at pH 7.4 (or other normal physiological conditions) and an Axl or Ror2 binding assay at pH 6.7 (or other in vitro tumor surrogate assay conditions); and b) selecting polypeptides that exhibit increased Axl or Ror2 binding activity at pH 6.7 compared to pH 7.4 or at other in vitro tumor surrogate assay conditions compared to normal physiological conditions, thereby selecting a conditionally active antigen-specific targeting region.

[0199] In some embodiments, a single round of selection is performed to obtain a conditionally active anti-Axl or anti-Ror2 targeting region. In certain embodiments, the screening or panning method is repeated after identifying free antibodies that bind to the antigen under in vitro tumor surrogate assay conditions but do not bind under physiological conditions, or cells expressing test antibodies having these properties, or phage coated with test antibodies having such properties in an initial or previous round. In some methods, the collected phage are used to infect cells that can be further infected with helper phage to amplify the collected phage. In other methods where the antibodies on the cell surface are tested, the collected cells are grown and the antibodies expressed by the cells can be "amplified" by amplifying the antibodies in cells encoding the polypeptide. In some embodiments, amplification is performed by growing the cells expressing the identified antibodies without performing a process of mutating the antibodies encoding the identified antibodies between rounds. Thus, the antibodies collected in the previous round are subsequently enriched by amplifying the cells containing the antibodies encoding these collected antibodies.

[0200] The panning or screening method can be performed once or repeated 1 to 1000 times. In an exemplary embodiment, panning is repeated 1 to 20 times or 2 to 10 times or 2 to 5 times.

[0201] In other methods, conditionally active anti-Axl or anti-Ror2 ASTRs are generated using mutagenesis / evolution between one or multiple rounds of panning rounds. In one method, a wild-type protein (e.g., an antibody) generates, for example, a polypeptide or protein library, and the polypeptide or protein library is identified by screening with a polypeptide or protein having the desired binding affinity for the target antigen. In some embodiments where the wild-type protein is an antibody, the wild-type antibody can be found by generating and screening a polyclonal or monoclonal antibody library, such as a phage display antibody library, e.g., a phage display humanized antibody library.

[0202] The evolved anti-Axl or Ror2 ASTRs can be generated by subjecting a wild-type protein, or a nucleic acid sequence encoding the wild-type protein, to a mutagenesis process to produce a population of mutant polypeptides that can be identified by screening for mutant ASTRs having increased activity (e.g., improved binding affinity for the target antigen) under tumor environments and / or in vitro tumor surrogate assay conditions compared to normal physiological environments. Examples of such methods are provided in International Application No. 2016 / 033331 (“CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTORS FOR MODIFIED T-CELLS”) or U.S. Patent No. 8,709,755.

[0203] Conditionally active anti-Axl or anti-Ror2 ASTRs identified using the methods provided herein are typically polypeptides, more specifically, polypeptide antibodies, and in exemplary embodiments, are single-chain antibodies further discussed in more detail herein. These polypeptides can bind to Axl or Ror2 with higher or lower affinity under in vitro tumor surrogate assay conditions versus normal physiological conditions, and in exemplary embodiments, can bind with higher affinity under in vitro tumor surrogate assay conditions than under normal conditions. In some embodiments, these polypeptides can bind to their cognate antigens with 10%, 20%, 25%, 50%, 75%, 90%, 95% or 99% higher affinity under in vitro tumor surrogate assay conditions than under physiological (i.e., normal) conditions. In some embodiments, ASTRs identified using the methods provided herein do not bind to their cognate antigens under normal physiological conditions to any detectable level above the background level obtained using a negative control such as a negative control antibody.

[0204] Nucleotide sequences isolated by the methods provided herein that encode conditionally active anti-Axl or anti-Ror2 ASTRs can be determined by nucleotide sequencing of the nucleotides of harvested cells expressing conditionally active anti-Axl or anti-Ror2 antigen-specific targeting. Subsequently, using this nucleotide sequence information, a polynucleotide encoding a polypeptide comprising a conditionally active anti-Axl or anti-Ror2 antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain can be generated to create a conditionally active anti-Axl or anti-Ror2 biological chimeric antigen receptor (CAB-CAR). The conditionally active anti-Axl or anti-Ror2 antigen-specific targeting region can be cloned into a CAR construct expression system and used to generate recombinant lentiviruses containing the CAR in their genomes, and then, as shown in Example 1 herein, this recombinant lentivirus can be used to transduce T cells to test for CAR-mediated Axl or Ror2-expressing target cell killing in a tumor-selective environment compared to normal physiological conditions.

[0205] Method for generating conditionally activatable cells The present disclosure provides a method for generating conditionally activatable cells. The method generally involves genetic modification of mammalian cells using an expression vector (e.g., a plasmid or virus) or RNA (e.g., in vitro transcribed RNA) that contains a nucleotide sequence encoding a conditionally active CAR of the present disclosure. The genetically modified cells are conditionally activatable in the presence of Axl and / or Ror2. The genetic modification can be performed in vivo, in vitro, or ex vivo. The cells are generally immune cells (e.g., T lymphocytes, T-helper cells, or NK cells), stem cells, progenitor cells, etc. In an exemplary embodiment, the cells are T cells.

[0206] In some cases, the genetic modification is performed ex vivo. For example, T lymphocytes, stem cells, T-helper cells, or NK cells are obtained from an individual, and the cells obtained from the individual are genetically modified to express the CAR of the present disclosure. The genetically modified cells are conditionally activatable in the presence of Axl and / or Ror2. In some cases, the genetically modified cells are activated ex vivo. In other cases, the genetically modified cells are introduced into an individual (e.g., the individual from whom the cells were obtained), and the genetically modified cells are activated in vivo. For example, if Axl and / or Ror2 is present on the cell surface of an individual, there is no need to administer an antigen. The genetically modified cells contact an antigen present on the cell surface of the individual, and the genetically modified cells are activated. For example, if the genetically modified cells are T lymphocytes, the genetically modified cells can exhibit cytotoxicity against cells expressing Axl and / or Ror2 on their surface to which the CAR binds.

[0207] In one aspect, provided herein is a method for ex vivo generation of conditionally activatable T cells and / or NK cells comprising a chimeric antigen receptor (CAR) against conditionally binding Axl or Ror2, the method comprising: a) concentrating peripheral blood mononuclear cells (PBMCs) to isolate PBMCs containing T cells and / or NK cells from isolated blood; b) Activating the T cells and / or NK cells of the enriched PBMCs under effective conditions; c) Transducing the activated T cells and / or NK cells with replication-incompetent recombinant retroviral particles under effective conditions, thereby generating genetically modified T cells and / or NK cells, wherein the replication-incompetent recombinant retroviral particles each contain a retroviral genome comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and / or NK cells, and according to any of the embodiments provided herein, the first nucleic acid sequence among the one or more nucleic acid sequences encodes a CAB-CAR; and d) Expanding the genetically modified T cells and / or NK cells, thereby generating conditionally activatable T cells and / or NK cells.

[0208] In some embodiments of the above aspects, the method further comprises recovering the expanded genetically modified T cells and / or NK cells. In some embodiments of the above aspects, the method further comprises obtaining a blood sample from the subject prior to enriching the PBMCs. In further embodiments, the method further comprises introducing the recovered and expanded genetically modified T cells and / or NK cells into the subject. In further embodiments, the genetically modified T cells and / or NK cells are present in the subject for 1, 2, 3, 4, 5, 6, 7, or 14 days after they are introduced into the subject.

[0209] Blood sample collection The PBMC-containing blood can be collected or obtained from the subject by any suitable method known in the art. For example, the blood can be collected by venipuncture or any other blood collection method by which a sample of blood and / or PBMCs is collected. In some embodiments, the PBMCs can be obtained by apheresis, as discussed below.

[0210] Enrichment of PBMCs In an ex vivo method of generating conditionally activatable T cells and / or NK cells, peripheral blood mononuclear cells (PBMCs) containing T cells and / or NK cells are isolated from other components of a blood sample in a concentration step. Concentration of PBMCs from other blood components and blood cells can be carried out using any method known in the art, for example, apheresis and / or density gradient centrifugation. In some embodiments, Ficoll-Paque (GE Healthcare) can be used. In some embodiments, an automated apheresis separator is used, which takes blood from a subject, passes the blood through a device that selects specific cell types (e.g., PBMCs, etc.), and returns the rest to the subject. Density gradient centrifugation can be carried out after apheresis. In some embodiments, PBMCs are concentrated and isolated using a leukoreduction filter device. In some embodiments, magnetic bead activated cell sorting is then used to purify specific cell populations, e.g., T cells and / or NK cells, from PBMCs according to the cell phenotype (i.e., positive selection). In some embodiments, monocytes and / or macrophages can be removed from PBMCs using methods known in the art. Depending on the subject to be treated, the cells can be allogeneic and / or autologous. During the PBMC concentration process, one or more washes can be performed as known in the art before the concentrated PBMCs are isolated and then activated. The wash solution can be any solution suitable for washing blood and / or PBMCs. According to methods known in the art, the isolated PBMCs can be resuspended in any suitable basal medium used for culturing T cells and / or NK cells. In some embodiments, the medium can be supplemented with HSA, human AB+ serum, subject-derived serum, and / or a serum replacement.

[0211] Activation of PBMCs The ex vivo method of generating conditionally activatable T cells and / or NK cells provided herein typically involves activating or stimulating isolated PBMCs with one or more activating agents to generate activated T cells and / or NK cells. Activation can be performed on freshly isolated PBMCs or PBMCs cryopreserved previously. If cryopreserved cells are used, the cells can be thawed using a development protocol prior to use.

[0212] During activation, there is typically a medium such as those known in the art for ex vivo processes (non-limiting examples include X-VIVO15 (Lonza) or CTS medium (Thermo Fisher)). In some embodiments, the medium can be supplemented with HSA, human AB+ serum, subject-derived serum, and / or a serum replacement. In an exemplary embodiment, the medium can be supplemented with a serum replacement such as CTS Serum Replacement (Thermo Fisher). In some embodiments, the medium can be supplemented with HSA, human AB+ serum, subject-derived serum, and / or a serum replacement.

[0213] Any combination of one or more activating agents can be added to the medium to generate activated T cells and / or NK cells. Typically, a reaction mixture is formed and activation is performed. In some embodiments, the reaction mixture can be formed by adding one or more activating agents to the medium. In some embodiments disclosed herein, one or more activating agents are used in an effective amount such that activated T cells and / or NK cells are generated.

[0214] In embodiments for generating conditionally activatable T cells and / or NK cells, it is notable that such activation can include activating the cells at a pH below 7.0 using Axl or Ror2, such as isolated soluble Axl or Ror2. However, in methods for generating conditionally activatable T cells and / or NK cells, activation typically utilizes more general activators. Thus, in some embodiments, the activator can be a polypeptide or antibody (e.g., anti-CD2, anti-CD3, and / or anti-CD28) or a functional fragment thereof that targets or binds to a T cell stimulating or co-stimulating molecule, a T cell cytokine, or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), a natural ligand for a T cell stimulating or co-stimulating molecule, a phosphoantigen, or an aminobisphosphonate such as zoledronic acid. Various antibodies and functional fragments thereof are known in the art to activate or stimulate T cells and / or NK cells. In some embodiments, one or more antibodies or functional fragments thereof can be immobilized on a solid surface such as beads.

[0215] Transduction of T cells and / or NK cells The ex vivo methods provided herein for generating conditionally activatable T cells and / or NK cells typically involve the step of transforming or transducing activated T cells and / or NK cells. In some embodiments of such methods, T cells and / or NK cells are contacted with an expression vector, such as a replication-incompetent recombinant retroviral particle, ex vivo to genetically modify the T cells and / or NK cells. Without being bound by theory, during the contact time, the replication-incompetent recombinant retroviral particle binds to the T cells and / or NK cells, at which point the retrovirus and host cell membranes initiate fusion. Thereafter, through the process of transduction, genetic material enters the T cells and / or NK cells from the replication-incompetent recombinant retroviral particle and is typically integrated into the host cell DNA. Thus, such methods involve genetic modification of T cells and / or NK cells by transduction. Methods for transducing T cells and / or NK cells ex vivo with replication-incompetent recombinant retroviral particles, such as replication-incompetent recombinant lentiviral particles, are known in the art. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9):689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2):497-505. In some embodiments, T cells and / or NK cells can be contacted with replication-incompetent recombinant retroviral particles. In an exemplary embodiment, T cells and / or NK cells can be contacted with replication-incompetent recombinant lentiviral particles.

[0216] Proliferation of Transduced T Cells and / or NK Cells In an exemplary embodiment of the ex vivo method of generating conditionally activatable T cells and / or NK cells provided herein, the transduced T cells and / or NK cells are expanded prior to recovery. In any of the embodiments disclosed herein, a medium for activation and transduction is present and, after transduction, the medium can be further added or exchanged to effect expansion. In some embodiments, the medium can be added to the reaction mixture formed during activation. The medium for expansion typically includes a basal medium known in the art for ex vivo processes, particularly for T cells and / or NK cells, such as those used for activation and transduction (non-limiting examples include X-VIVO15 (Lonza) or Optimizer CTS medium (Thermo Fisher)). In some embodiments, the medium can be supplemented with a serum replacement such as HSA, human AB+ serum, subject-derived serum, and / or CTS Serum Replacement (Thermo Fisher). Cytokines such as IL-2, IL-7, or IL-15, or those found in HSA, can be added to the medium before, during, and / or after activation, transduction, and expansion. Cell expansion can be carried out over a specific number of days. In some embodiments, expansion can be carried out for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, expansion can be carried out between 4, 5, 6, 7, or 8 days as the lower end of the range and 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days as the upper end of the range. In certain exemplary embodiments, expansion is carried out for 6-12 days, or 8-10 days.

[0217] Cell recovery The ex vivo methods for generating conditionally activatable T cells and / or NK cells provided herein typically involve, after expansion, recovering the genetically modified T cells and / or NK cells. In some embodiments, transduced T cells and / or NK cells can be concentrated or collected during recovery using methods known in the art. In some embodiments, the T cells and / or NK cells can be washed one or more times during recovery using any suitable washing solution known in the art. At the end of the recovery, the T cells and / or NK cells can be resuspended in any suitable medium known in the art. In any of the embodiments disclosed herein, the recovery of the expanded T cells and / or NK cells can be carried out based on criteria for determining completion of expansion. In some embodiments, the criteria for determining completion of expansion can be lactate concentration, cell density, or number of days of expansion.

[0218] In some embodiments, the recovered cells can be introduced, reintroduced, reinfused, infused, or readministered to a subject. In some embodiments, the recovered cells can be cryopreserved as described below prior to reintroducing them to a subject. In an exemplary embodiment, the recovered cells can be introduced, reintroduced, reinfused, infused, or readministered to a subject without first cryopreserving the cells. The subject is typically the same subject from whom the blood was collected.

[0219] Throughout this disclosure, transduced T cells and / or NK cells include progeny of transduced cells that retain at least one nucleic acid integrated into the cells during ex vivo transduction. In the methods herein that describe "reintroducing" such cells, it will be understood that such cells are not typically in a transduced state when collected from the blood of a subject.

[0220] Cell Introduction / Reintroduction In certain embodiments of the ex vivo method of generating conditionally activatable T cells and / or NK cells disclosed herein, the harvested T cells and / or NK cells can be introduced, reintroduced, reinfused, infused, or re-infused into a subject for a therapeutic effect. The number of T cells and / or NK cells to be reintroduced can be a predetermined dosage that can be a therapeutically effective amount. In some embodiments, the predetermined dosage can depend on the CAR expressed on the cells (e.g., the affinity and density of the antigen-specific targeting region on the transduced T cells and / or NK cells), the target cell type, the nature of the disease or pathological condition being treated, or a combination thereof. In some embodiments, the predetermined dosage of the harvested cells can be based on the weight of the subject, e.g., the number of cells per kilogram of the subject (cells / kg).

[0221] Cryopreservation of cells In the ex vivo method of generating conditionally activatable T cells and / or NK cells provided herein, the harvested cells generated by the methods described herein can be cryopreserved at a predetermined dosage for later use. Cryopreservation methods and reagents are well known in the art. Cryopreservation can include the step of concentrating the T cells and / or NK cells with one or more detergents and / or. The method can also include the step of forming a cryopreservation mixture that includes the T cells and / or NK cells in a diluent solution and a suitable cryopreservation solution. In some embodiments, the method can include the step of freezing a cryopreservation mixture known in the art. Methods for thawing cryopreserved T cells and / or NK cells are known in the art.

[0222] Method for regulating CAB-CAR-expressing T cell and / or NK cell activity by changing pH In certain embodiments, provided herein is a method of regulating the activation of immune cells (e.g., T cells or NK cells) by contacting the immune cells with Axl or Ror2 in a microenvironment having a pH of less than 7.0 (e.g., less than 6.9 or 6.8), and then changing the microenvironment pH such that the pH is 7.0 or greater (e.g., greater than 7.1, 7.2, or 7.3). In this case, the immune cells express any of the CAB-CARs provided herein. In an exemplary embodiment, Axl or Ror2 is expressed on the surface of a target mammalian cell. In certain embodiments, such a method of regulating activation is the same as the method of activating the immune cells provided herein, and further includes increasing the pH of the microenvironment to a pH of 7.0 or greater (e.g., greater than 7.1, 7.2, or 7.3), thereby reducing the activation of the immune cells. In an exemplary embodiment, such an increase in pH inactivates the immune cells.

[0223] In certain embodiments, provided herein is a method of regulating the binding of a CAB-CAR-expressing T cell or NK cell to a target cell and the resulting lysis / killing of the target cell by causing a change or shift in the pH within a microenvironment that includes the target cell within a target tissue or within one or more non-target (e.g., healthy / normal) tissues, thereby regulating the binding of the CAB-CAR to its cognate antigen on the target cell. In this case, the cognate antigen is an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof. Such a method typically involves changing the pH of the microenvironment by decreasing the pH or, more typically, increasing the pH after contacting a target cell, such as a mammalian cell (e.g., a human cell), with the CAB-CAR-expressing T cell or NK cell in the microenvironment. The microenvironment can be a target microenvironment, such as a tumor, or an off-target microenvironment, and off-target binding can result in side effects. In some embodiments, such a method can result in a transient reduction in tumor microenvironment-sensitive CAR-T target binding.

[0224] Thus, in one aspect, provided herein is a method of modulating the binding of conditionally active biological chimeric antigen receptor (CAB-CAR) expressing T cells or NK cells to cells expressing the cognate antigen of the CAB-CAR in a subject, comprising: a. introducing into the subject T cells and / or NK cells comprising a nucleic acid encoding a CAB-CAR, and after (and optionally, and / or during) introduction, the T cells and / or NK cells comprising a nucleic acid encoding a CAB-CAR express the CAB-CAR and bind to cells expressing the cognate antigen of the subject, wherein the cognate antigen is an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof; and b. administering to the subject an amount of an agent sufficient to increase the blood pH and / or tissue pH and / or microenvironment pH, wherein the administration is performed before, during, or after the introduction, and the increased pH of the blood, tissue, and / or microenvironment modulates the binding of the CAB-CAR expressing T cells and / or NK cells to cells expressing the cognate antigen in the blood, tissue, or microenvironment at the increased pH.

[0225] The change / shift in pH in embodiments comprising the step of administering a pH modulating agent of the present disclosure can be achieved by exposing target or non-target cells to the pH modulating agent, such as by administering the pH modulating agent to the subject. Non-limiting examples of pH modulating agents are provided herein. In certain aspects, provided herein is an agent for use in a method of modulating the binding of a CAB-CAR to its cognate antigen, or modulating the binding of CAB-CAR expressing T cells and / or NK cells to cells expressing its cognate antigen, or reducing or alleviating off-target off-tumor toxicity in a subject. Such aspects of certain embodiments are related to the treatment of tumor growth, cancer, hyperplasia, or cell proliferative disorders.

[0226] In other aspects, provided herein is the use of a pH modulating agent for use in the manufacture of a kit for controlling in vivo binding of a drug or genetically modified T cells and / or NK cells to target mammalian cells in a subject, wherein the target cells express an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof. In other aspects, provided herein is a kit comprising a container containing replication-incompetent recombinant retroviral particles and instructions for its use for practicing a method of treating tumor growth, the instructions teaching a method of controlling binding of T cells and / or NK cells to target mammalian cells by modulation of pH, wherein the target mammalian cells express an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof. Such methods can be any of the methods provided in this section of the specification for modulating CAB-CAR expressing T cell and / or NK cell activity by changing the pH. The container containing the recombinant retroviral particles can be a tube, vial, well of a plate, or other container for storage of the recombinant retroviral particles and / or the pH modulating agent. Any of these can be of industrial strength and grade. The kit can, in certain embodiments, comprise more than one container. One container / vessel can contain the recombinant retroviral particles and another container / vessel can contain the pH modulating agent. In such methods, the agent is delivered / administered in an amount sufficient to raise the blood pH and / or tissue pH and / or microenvironment pH to modulate the binding of the CAB-CAR of modified / recombinant T cells and / or NK cells expressing CAB-CAR to its cognate antigen in blood and / or tissue having the elevated pH. Non-limiting exemplary details for administering the pH modulating agent in sufficient amount over sufficient time are provided herein.

[0227] Regardless of on-target or off-target effects on the tissue, after introduction of the CAB-CAR into the subject, the target cells can be contacted with a pH-modulating agent such as a pH-modulating drug. Thus, for example, to mitigate on-target off-tumor activity and / or to inhibit target cell proliferation such as tumor cell proliferation, exemplary embodiments provided herein for modulating the binding and / or cytotoxic activity of CAB-CAR-expressing T cells that can bind (i.e., recognize) an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof can include the following steps: a. Introducing into the subject T cells and / or NK cells comprising a nucleic acid encoding a CAB-CAR, and after introduction, the T cells and / or NK cells comprising the nucleic acid encoding the CAB-CAR express the CAB-CAR, the CAB-CAR can bind to an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof, and optionally, binding to cells expressing the subject's cognate antigen, and b. Administering to the subject a drug in an amount sufficient to increase the blood pH and / or tissue pH and / or microenvironment pH to modulate the binding of the CAB-CAR-expressing T cells and / or NK cells to cells expressing the cognate antigen of the CAB-CAR in the blood, tissue, or microenvironment having the elevated pH. Depending on the particular method used to introduce the nucleic acid encoding the CAB-CAR into the T cells and / or NK cells, it will be understood that the T cells and / or NK cells may or may not express the CAB-CAR prior to its introduction into the subject. However, at some point after introduction into the subject, for example, at 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 4 days, and / or 7 days, or at a time point beyond that, the T cells and / or NK cells comprising the nucleic acid encoding the CAB-CAR express the CAB-CAR. Thereafter, such cells typically bind to target cells expressing the cognate antigen of the CAB-CAR.

[0228] Using the methods provided herein for the genetic modification and optional expansion of the subject lymphocytes, a nucleic acid sequence encoding a CAB-CAR is introduced into the genome of the subject's T cells and / or NK cells to generate T cells and / or NK cells capable of expressing the CAB-CAR, and then the T cells and / or NK cells capable of expressing the CAB-CAR can be introduced into the subject. After introduction, the T cells and / or NK cells express the CAB-CAR to contact the target cells / tissues with the CAB-CAR. The present disclosure provides details of how to carry out such methods, along with various alternatives for different CAR components, some of which can be used in some aspects of the present disclosure including modulating the binding of CAB-CAR-expressing T cells and / or NK cells to target cells expressing the cognate antigen of the CAB-CAR by changing the pH.

[0229] Such methods for genetically modifying and expanding lymphocytes typically involve contacting T cells and / or NK cells with replication-incompetent recombinant retroviral particles to transduce the T cells and / or NK cells. Such contact is typically performed ex vivo after removing lymphocytes from the subject. The T cells and / or NK cells are then introduced / reattached into the subject, usually the subject from whom they were removed. The replication-incompetent recombinant retroviral particles contain a genome having a polynucleotide encoding the CAB-CAR. Many alternative embodiments and further details regarding such replication-incompetent recombinant retroviral particles are provided in other sessions of this specification and can be used in the methods provided herein for modulating binding and the resulting lysis / killing of T cell-expressed CAB-CAR capable of binding to the Axl polypeptide or its epitope or the Ror2 polypeptide or its epitope by modulating the pH in the microenvironment of cells expressing the cognate target polypeptide recognized by the CAB-CAR in a pH-dependent manner.

[0230] Using such methods for the regulation of the binding of target cells by CAB-CAR-expressing T cells and / or NK cells, for example, off-target off-tumor toxicity can be reduced by increasing the pH of the blood and / or non-tumor tissues in a subject. For example, if the "normal" tissue pH in a subject is primarily lower, the pH of the normal tissue is increased while the pH of the tumor remains lower, and at that time, a pH-modulating agent can be delivered such that it is the pH at which CAB-CAR-expressing T cells and / or NK cells bind to target tumor cells. In these embodiments, the pH-modulating agent can be delivered to normal tissues at a lower concentration or in a targeted manner.

[0231] In some embodiments, this can be achieved while allowing the pH in the tumor microenvironment to remain low enough for CAB-CAR T cells and / or NK cells to bind to their cognate target-expressing cells within the tumor. In an exemplary aspect of the methods provided herein, the pH of the tissue remains at the pH at which CAB-CAR-expressing T cells and / or NK cells bind to their target for a sufficient period of time (e.g., 2, 4, 8, 12, or 24 hours, or 2, 4, 7, 14, 28, or 30 days, or 1, 2, 3, 4, 5, 6, 12, 24 months, or longer) for the CAB-CAR-expressing T cells and / or NK cells to contact and bind to their cognate antigen, and then the pH is shifted / changed, for example, by increasing the pH of the tissue to an extent that affects the binding of CAB-CAR-expressing T cells and / or NK cells to their target cells.

[0232] Accordingly, in one aspect, provided herein is a method for primary reduction of tumor microenvironment-sensitive CAR-T cell target binding by pharmacological alteration of blood vessel and tissue pH, wherein the CAR-T cells express a CAB-CAR capable of binding to an Axl polypeptide or an epitope thereof, or a Ror2 polypeptide or an epitope thereof. The ASTR in these microenvironment-controlled CAR-T cells provides an additional level of protection against on-target off-tumor that requires tumor local environmental conditions to enable T cell binding. Although attractive for some monoclonal antibody therapies, adoptive cell therapies can create a temporarily permissive local environment for their CAR-T targets. For example, CAR-T cells activated in tissues with low pH can further lower the pH of the microenvironment, depending on the cytoplasmic domain present in the CAR construct. In other cases, cytokine release syndrome and other morbidity associated with adoptive cell therapies can result in a decrease in the blood bicarbonate buffering capacity, leading to lactic acidosis. Adoptive cell therapy agents administered by intravenous infusion result in temporary pulmonary entrapment. In some cell therapies, the infusion rate requires continuous monitoring of dissolved oxygen (Fischer et al. Stem Cells Dev. 2009 Jun;18(5):683-691). The degree of pulmonary entrapment depends on cell size, activation state, cell dose, and infusion rate. Cruz et al. (Cytotherapy. 2010 Oct;12(6):743-749) reported adverse findings that low-dose and low-rate infusions may reduce pulmonary entrapment from over 300 T cell infusions. However, using certain high-potency CAR-T cells, even at low levels, targets such as Her2 present on the lung endothelium (Morgan et al. Mol Ther. 2010 Apr;18(4):843-851) can result in uncontrolled acute toxicity in the lung after infusion, due to the initial high CAR-T cell concentration and the presence of T cell targets in these tissues, leading to rapid patient decline.In other cases, the presence of T cell targets in other off-target tissues, such as the bile duct, can result in uncontrolled on-target off-tumor toxicity (Lamers Mol Ther. 2013 Apr;21(4):904-12), and severe organ toxicity, before other agents, such as steroids or cell-depleting epitopes, are available. Venous and arterial plasma have strong buffering capacity against acidosis, but conditions of respiratory acidosis, shock, metabolic acidosis, and ischemic acidosis can occur in patients with cancer treated with adoptive cell therapy.

[0233] In some embodiments provided herein, the binding of CAB-CAR in a subject can be modulated by administering to the subject an agent that raises or lowers the pH of blood, tissue, and / or the microenvironment. In some embodiments, off-target off-tumor toxicity in a subject can be reduced by administering to the subject an agent that raises or lowers the pH of blood and / or the pH of tissue and / or the pH of the microenvironment. In some embodiments, the binding of T cells and / or NK cells to target mammalian cells can be controlled by introducing an agent that raises or lowers the pH of blood and / or the pH of tissue and / or the pH of the microenvironment. In some embodiments, the in vivo binding of genetically modified T cells and / or NK cells to target mammalian cells in a subject can be controlled by administering a pH-modulating agent to the subject. In an exemplary embodiment, the pH of blood and / or the pH of tissue and / or the pH of the microenvironment can be raised. In some embodiments, the microenvironment can be an in vivo microenvironment. In an exemplary embodiment, the microenvironment can be a tumor microenvironment. In some embodiments, the microenvironment can include target mammalian cells that express a target antigen on their surface. In some embodiments, administration of the agent to the subject raises the pH of blood, tissue, and / or the microenvironment from a pH less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9 to a pH of at least 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, and the pH of blood, tissue, and / or the microenvironment is lower before administration of the agent than after administration of the agent. In some embodiments, administration of the agent to the subject lowers the pH of blood, tissue, or the microenvironment from a pH greater than 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6 to a pH less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, and the pH of blood, tissue, and / or the microenvironment is higher before administration of the agent than after administration of the agent.In some embodiments, administration of the agent to the subject can cause a pH shift in the subject's blood, tissue, and / or microenvironment. In some embodiments, the pH shift after administration of the agent can be a shift in either direction of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 pH units compared to the pH before administration of the agent, i.e., an increase or decrease in pH. In an exemplary embodiment, the pH shift is an increase in pH.

[0234] The CAB-CAR of the present disclosure may have reduced binding to its cognate antigen with a difference of another pH and 1 pH. In the broadest aspect, in exemplary embodiments where exemplary pH values for specific binding of the CAB-CAR have not yet been provided, and on the one hand, with respect to other embodiments in place of these values for such an aspect, the CAB-CAR may have reduced binding at a higher pH compared to a lower pH. For example, the CAB-CAR may have reduced binding to its cognate antigen at a pH greater than 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 than at a pH less than 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In other embodiments, the CAB-CAR may have reduced binding at a higher pH than at a lower pH. For example, the CAB-CAR may have reduced binding to its cognate antigen at a pH less than 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 than at a pH greater than 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In some exemplary embodiments, the CAB-CAR exhibits increased binding at a pH of 6.5 - 6.7 compared to a pH of 7.4 - 7.6. In other exemplary embodiments, the CAB-CAR exhibits increased binding at a pH of 6.7 compared to a pH of 7.4. In other embodiments, the CAB-CAR exhibits increased binding in the pH of the tumor compared to the pH of the blood. In some embodiments, the CAB-CAR may include an antigen-specific targeting region targeting region, a stalk, and an intracellular activation domain. In some embodiments, the CAB-CAR may also include a co-stimulatory domain. In some embodiments, the CAB-CAR may bind to a tumor-associated antigen. In some embodiments, the CAB-CAR binds to an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof.

[0235] In methods involving the regulation of the pH of blood, tissue, or a microenvironment, the pH of the microenvironment can be increased from less than 7.0 to greater than 7.0. For example, the pH can be increased from a pH of 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or less than 7.0 to a pH of 7.0, 7.1, 7.2, 7.3, or greater than 7.4. In some embodiments, the CAB-CAR can bind to a cognate antigen at an increased pH, but cannot bind at the pH of the microenvironment prior to the introduction of the agent. In certain embodiments, the pH can be increased from less than 7.0 to a pH of 7.1 - 8.0, or to a pH of 7.1 - 7.8, or to a pH of 7.2 - 7.8, or to a pH of 7.2 - 7.6, or to a pH of 7.3 - 7.6, to a pH of 7.4 - 7.8, or to a pH of 7.4 - 7.6. Such an increase in pH can occur in less than 1, 2, 4, 6, 8, 12, or 24 hours, or more than 1, 2, 4, 6, 8, 12, or 24 hours, depending on the type and dosage of the agent administered. In certain embodiments, the agent is administered such that in a target tissue such as a tumor, and for example, in at least a portion of the microenvironment of the target tissue (e.g., tumor), and in at least the surface of the microenvironment of the target tissue (e.g., tumor), and in an exemplary embodiment, throughout the entire microenvironment of the target tissue (e.g., tumor), the pH is greater than 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5; or remains between a lower end value of 7.0, 7.1, 7.2, 7.3 of the range and an upper end value of 7.4, 7.5, 7.6, 7.7, or 7.8 of the range. The microenvironment can be an in vivo microenvironment such as a tumor, tissue, non-tumor tissue, normal tissue, or tissue that has undergone a transient pH shift. For example, typically, tissues that undergo a transient pH shift include muscle tissue under anaerobic conditions or exercising muscle tissue or inflammatory tissue or tissue in which inflammation is occurring. In some embodiments that include target mammalian cells, the target mammalian cells can be tumor cells or non-tumor cells or normal cells.

[0236] In some embodiments, methods are provided for temporarily increasing vascular pH to reduce the affinity of a CAB-CAR that recognizes, binds to, and in some embodiments binds to an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof in a microenvironmentally controlled manner. A 0.4 U shift in blood pH can reduce by more than 10-fold the affinity of certain scFvs that form part of the CAB-CAR for their cognate antigens. In some embodiments, pH control for therapy can be achieved via the IV or oral administration route of various agents. For example, in some embodiments, inactivation of binding affinity can be achieved using bicarbonate or sodium bicarbonate. In other embodiments, tris(hydroxymethyl)aminomethane (also known as tromethamine, trometamol, and THAM) and / or Carbicarb™ (an equimolar hypertonic solution of sodium bicarbonate and sodium carbonate) can be utilized to increase the pH of the blood in an amount sufficient to reduce on-target off-tumor toxicity. In still other embodiments, small molecule proton pump inhibitors can be utilized to increase blood pH and / or tissue pH in an amount sufficient to reduce on-target off-tumor toxicity. Proton pump inhibitors that can be used in methods involving pH modulation include, but are not limited to, esomeprazole (Nexium), esomeprazole and naproxen (Vimovo), lansoprazole (Prevacid), omeprazole (Prilosec and Zegerid), and rabeprazole (Aciphex). Administration of proton pump inhibitors can be effectively used over a long period of time to modulate the binding affinity of an antigen-binding domain for its cognate antigen over days, weeks, months, or years. In other embodiments, the affinity of an antigen-binding domain for its cognate antigen can be modulated by altering blood pH and / or tissue pH to control transcription, translation, membrane expression, and the stability of transporters and pumps.Examples of such transporters and pumps for which altered expression may serve to regulate pH include, but are not limited to, proton pumps, members of the sodium-proton exchange family (NHE), the bicarbonate transporter family (BCT), and the monocarboxylic acid transporter family.

[0237] In certain embodiments, for example, a pH modulating agent such as bicarbonate, THAM, or Caricarb™ is administered before or simultaneously with the infusion of CAR-T cells of a patient expressing a conditionally active form of a biological ASTR (e.g., scFv or scFvFc). Such treatment reduces the immediate cytotoxicity that would otherwise accompany the temporary pulmonary sequestration of the CAR-T cell infusion. Thus, in certain aspects, provided herein is a method of reducing the cytotoxicity that occurs in the normal healthy tissue of a subject by administering to the subject an agent in an amount sufficient to raise the blood pH and / or tissue pH and / or microenvironment pH; and simultaneously or subsequently (e.g., 1, 2, 4, 6, 8, 12, or 24 hours later, or 1, 2, 3, 4, or 7 days later), introducing CAB-CAR-expressing T cells or NK cells into the subject. In certain embodiments, at a target time after such introduction (e.g., 1, 2, 4, 6, 8, 12, or 24 hours later, or 1, 2, 3, 4, or 7 days later), the administration of the agent is terminated over a period of time or indefinitely to alter the pH of the subject's blood, tissue, or microenvironment to regulate the binding / activity of the CAB-CAR-expressing T cells.

[0238] As will be appreciated by those skilled in the art, various effective dosing regimens can be used for the administration of agents that can modulate pH (e.g., increase the pH of the blood pH and / or tissue pH and / or microenvironment pH of interest). As used herein, administration can mean administering an agent to a subject, including injecting the agent into the subject via IV or orally administering the agent to a patient or the subject ingesting the agent. The agent can be administered to the subject or patient over various lengths of time, e.g., for at least 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 weeks; 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 18 months; or 2, 2.5, 3, 3.5, 4, 4.5, or 5 years or an indefinite period. In some embodiments, the agent can be a bicarbonate, sodium bicarbonate (NaHCO3), or a solution of sodium bicarbonate and sodium carbonate, and the parenteral or IV dosage can be: 0.2 x subject's body weight (kg) x subject's base deficit; required HCO3 (mEq) = 0.5 x body weight (kg) x [24 - serum HCO3 (mEq / L)]; or a 2 - 5 mEq / kg IV infusion over 4 - 8 hours. In some embodiments, a standard dosing regimen of bicarbonate / sodium bicarbonate, or a solution of sodium bicarbonate, can be used depending on the severity of acidosis. 50 - 150 mEq of bicarbonate diluted in 5% dextrose in 1 L of water can be administered via IV at a rate of 1 - 1.5 L / hour. In another non - limiting example, 90 - 180 mEq of bicarbonate diluted in 5% dextrose in 1 L of water can be administered via IV at a rate of 1 - 1.5 L / hour. In some embodiments where the agent is bicarbonate or sodium bicarbonate (NaHCO3), the enteral or oral dosage can be, for example, administration of 325 - 2000 mg of sodium bicarbonate to the subject 1 - 4 times / day.

[0239] In some embodiments, the agent can be tris(hydroxymethyl)aminomethane (also known as tromethamine, trometamol, and THAM), and the parenteral or IV dosage can be as follows: Required tromethamine solution (mL of 0.3 M solution) = body weight (kg) x base deficit (mEq / liter) x 1.1. In some embodiments, the IV dosage of tris(hydroxymethyl)aminomethane can be estimated from the buffer base deficit of extracellular fluid in mEq / L determined by the Siggaard-Andersen nomogram. In some embodiments, the initial dosage can be 500 mL (150 mEq) (up to a maximum of 1000 mL) of tris(hydroxymethyl)aminomethane infused by slow IV infusion, and the maximum dosage is a dosage of 500 mg / kg (227 mg / lb) over 1 hour or more.

[0240] In some embodiments, the agent can be a small molecule proton pump inhibitor and can be administered over a long treatment length. For example, the small molecule proton pump inhibitor can be administered for at least 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 weeks; 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 18 months; or 2, 2.5, 3, 3.5, 4, 4.5, or 5 years or an indefinite period. In some embodiments, the proton pump inhibitor can be esomeprazole (Nexium), and 20 mg or 40 mg of esomeprazole can be orally administered once or twice a day. In some embodiments, the proton pump inhibitor can be a combination of esomeprazole and naproxen (Vimovo), and 20 mg of esomeprazole and 375 or 500 mg of naproxen can be orally administered together twice a day. In some embodiments, the proton pump inhibitor can be lansoprazole (Prevacid), and 15, 30, or 60 mg of lansoprazole can be orally administered once or twice a day. In some embodiments, lansoprazole can be administered IV at 30 mg once a day over 30 minutes for up to 7 days. Thereafter, the subject can be switched to oral lansoprazole and treatment can continue. In some embodiments, the proton pump inhibitor can be omeprazole (Prilosec and Zegerid), and 10, 20 mg or 40 mg of omeprazole can be orally administered once or twice a day. In some embodiments, the proton pump inhibitor can be rabeprazole (Aciphex), and 20 or 60 mg of rabeprazole can be orally administered once or twice a day, or 100 mg of rabeprazole can be orally administered once a day. In any of the embodiments disclosed herein, the agents can be used in combination with each other.

[0241] In any of the embodiments disclosed herein, the pH of the subject's blood, tissue, and / or microenvironment can be measured before, during, or after administration of the agent. In some embodiments, the determination to administer or continue to administer an agent that raises or lowers the pH to the subject can be based on measurement of the pH of the subject's blood, tissue, and / or microenvironment. The pH of the subject's blood and / or the blood bicarbonate level are well known in the art. In some embodiments, positron emission tomography (PET), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), and optical imaging can be used to measure the in vivo pH of a microenvironment, e.g., in a tumor (see Zhang X, Lin Y, Gillies RJ. Tumor pH and its measurement. J Nucl Med. 2010 Aug;51(8):1167-70 for details on tumor pH measurement).

[0242] In another aspect, provided herein is a method of reducing on-target off-tumor toxicity in a subject, the method comprising: a. introducing a polynucleotide encoding a conditionally active biological chimeric antigen receptor (CAB-CAR) into T cells or NK cells of the subject to generate T cells and / or NK cells capable of expressing the CAB-CAR, wherein the CAB-CAR is capable of binding to an Axl polypeptide or an epitope thereof, or a Ror2 polypeptide or an epitope thereof; b. introducing the T cells and / or NK cells capable of expressing the CAB-CAR into the subject such that the T cells and / or NK cells express the CAB-CAR in the subject; and c. administering to the subject an amount of an agent sufficient to raise the pH of the blood and / or the tissue and / or the microenvironment, and modulating the binding of the CAB-CAR in the blood, tissue, and / or microenvironment to its cognate antigen by the elevated pH, thereby reducing the on-target off-tumor toxicity in the subject.

[0243] In the introduction step, since T cells or NK cells are genetically modified to contain a nucleic acid encoding a CAB-CAR, they can express a CAB-CAR that can bind to an Axl polypeptide or an epitope thereof or a Ror2 polypeptide or an epitope thereof. This genetic modification can be the presence of a CAB-CAR coding sequence on a vector introduced into T cells or NK cells by gene transfer or transduction. In an exemplary embodiment, the nucleic acid encoding the CAB-CAR is integrated into the genome of T cells or NK cells.

[0244] Various methods for introducing polynucleotides into T cells and / or NK cells, which are known in the art, can be used in a manner that affects the binding of the CAB-CAR T cells or NK cells provided herein to their cognate antigen on the cell surface, including changing the pH using an agent such as a pH modulating agent (which may also be referred to herein as a "pH switching mode"). Usually, a vector, in an exemplary embodiment, an expression vector is used for the delivery of the polynucleotide. Such vectors include various vectors known in the art for delivering nucleic acids to T cells and / or NK cells. Exemplary embodiments of the present invention utilize retroviral vectors and retroviral particles, and in some specific exemplary embodiments, lentiviral vectors, and in an exemplary embodiment, recombinant lentiviral particles are utilized.

[0245] Other suitable expression vectors can be used in the pH-switching embodiments provided herein. Such expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus-based viral vectors (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; see International Publication No. 94 / 12649, International Publication No. 93 / 03769; International Publication No. 93 / 19191; International Publication No. 94 / 28938; International Publication No. 95 / 11984, and International Publication No. 95 / 00655); adeno-associated viruses (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in International Publication No. 93 / 09239, Samulski et al., J.Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., see PNAS (1993) 90:10613-10617; SV40; herpes simplex virus; or retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), e.g., gamma retrovirus; or human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23,1997; Takahashi et al., J Virol 73:7812 7816,1999); etc.

[0246] In some embodiments, DNA-containing viral particles are utilized in place of recombinant retroviral particles. Such viral particles can be adenovirus, adeno-associated virus, herpes virus, cytomegalovirus, poxvirus, orthopoxvirus, influenza virus, vesicular stomatitis virus (VSV), or Sindbis virus. One of ordinary skill in the art will know how to modify the methods disclosed herein for use with different viruses and retroviruses. When viral particles containing a DNA genome are used, one of ordinary skill in the art will understand that functional units are included in such genomes and that integration of all or part of the DNA genome of such viral particles into the genomes of T cells and / or NK cells transduced with such viruses is induced. Alternatively, functional DNA can be delivered to T cells and / or NK cells that are expressing in the cell but is not integrated into the genomes of T cells and / or NK cells.

[0247] In an exemplary embodiment, the vector used in the pH-switching aspect of the present disclosure is a recombinant retroviral particle and in certain embodiments, a recombinant lentiviral particle. Such retroviral particles typically contain a retroviral genome within a capsid located within a viral envelope. The present disclosure in various sections herein provides various embodiments of recombinant retroviral particles that disclose elements that can be included on or within the surface of the recombinant retroviral particle and / or within the genome of the recombinant retroviral particle. Any of these embodiments of the recombinant retroviral particles can be used in the pH-switching aspect provided herein.

[0248] In any of the embodiments disclosed above, the cognate antigen to which the CAB-CAR binds can be the Axl polypeptide or an epitope thereof or the Ror2 polypeptide or an epitope thereof. In some embodiments, the cognate antigen is a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20, or all of a continuous series of amino acids of the Axl polypeptide or an epitope thereof or the Ror2 polypeptide or an epitope thereof. As disclosed herein, the CAB-CAR that can bind to the Axl polypeptide or an epitope thereof or the Ror2 polypeptide or an epitope thereof typically binds to its cognate antigen with a higher binding affinity at a pH of 6.7 than at a pH of 7.4. Thus, such anti-Ror2 and anti-AXL CAB-CARs of the present disclosure typically bind to their cognate antigens with a higher binding affinity in the tumor microenvironment than in normal tissues having a physiological pH.

[0249] Therapeutic methods The present disclosure provides various methods of treatment for disorders, including anti-AXL or anti-Ror2 CAB-CARs provided herein. In some embodiments, the methods utilize the fact that the CAB-CARs of the present disclosure, when present and expressed in T lymphocytes or NK cells, can mediate cytotoxicity against target cells. The CAB-CARs of the present disclosure bind to antigens present on target cells under specific target conditions, thereby mediating the death of the target cells by T lymphocytes or NK cells genetically modified to produce the CAB-CAR. The ASTR of the CAB-CAR typically binds to an antigen present on the target cell surface.

[0250] Target cells include, but are not limited to, cancer cells. Accordingly, the present disclosure provides methods for killing or inhibiting the growth of target cancer cells, the method comprising contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell, or an NK cell) genetically modified to produce the CAR, whereby the T lymphocyte or NK cell recognizes an antigen present on the surface of the target cancer cell and mediates the death of the target cell. Exemplary embodiments of such methods provide methods for treating cancer. The CAB-CARs are not limited to use for the treatment of cancer or targeting of tumors or cancer cells, but rather will be suitable for use in one or more indications including the treatment of circulatory disorders, arthritis, multiple sclerosis, autoimmune disorders, skin diseases, viral diseases and disorders, as well as in various diagnostic formats.

[0251] In certain embodiments, the present disclosure provides a method of treating cancer in a subject having cancer. Accordingly, the present disclosure provides adoptive cell therapy for cancer, particularly methods of using the anti-Axl and anti-Ror2 CAB-CARs provided herein against cancers that express Axl or Ror2. Thus, in one embodiment, the method comprises: A. introducing into peripheral blood cells obtained from a subject an expression vector configured to express a polynucleotide sequence encoding a CAB-CAR against Axl or Ror2 provided herein to generate genetically modified cytotoxic cells (such as T cells or NK cells); and B. administering the genetically modified cytotoxic cells to the subject. Detailed methods of treating T cells are provided herein for cells that provide exemplary embodiments of step A above to activate, transduce, and usually expand.

[0252] Cancers typically express Ror2 or Axl, respectively, and in exemplary embodiments, the cancer is any cancer such as renal cell carcinoma in which the cancer cells of such cancer express Ror2 and / or Axl. The CAR is any CAB-CAR that recognizes Axl or Ror2 disclosed herein, particularly a CAB-CAR that is cytotoxic to cancer cells expressing these antigens. An expression vector encoding an anti-Axl CAB-CAR or an anti-Ror2 CAB-CAR can be introduced into peripheral blood cells by transducing peripheral blood leukocytes including T cells and / or NK cells using the vector. In certain exemplary embodiments, the vector is a recombinant virus, for example, in some embodiments, a recombinant retrovirus, a recombinant lentivirus. In some embodiments, the cancer is a soft tissue sarcoma or mesothelioma expressing Ror2, and the T cells and / or NK cells of the subject (e.g., a soft tissue sarcoma patient or a mesothelioma patient) are transduced with an anti-Ror2 CAR, for example, the anti-Ror2 CAB-CAR disclosed herein.

[0253] The methods for treating disorders provided herein typically involve administering to a subject genetically modified T cells or NK cells that express anti-Axl or anti-Ror2 CAB-CAR provided herein. Administration can be, for example, intravenous, subcutaneous, or intratumoral. In methods where genetically modified T cells and / or NK cells are administered intravenously, typically 1x10 4 cells / kg to 1x10 8 cells / kg are administered in a suitable buffer for parenteral administration. In methods where genetically modified T cells and / or NK cells are administered intratumorally, typically 1x10 6 cells to 5x10 8 cells are administered in an isotonic solution.

[0254] In some embodiments, administration is performed before, concomitantly with, and / or after administration of an interleukin or a modified form thereof. For example, some embodiments provided herein include co-administration of IL-2, or a modified form of IL-2 that has sustained release and / or is biased in a direction to activate the proliferative and / or cytotoxic activity of T cells, and that binds to a specific IL-2 receptor. For example, the modified IL-2 can be, in certain embodiments, pegylated IL-2, such as NKTR-214 (Nektar Therapeutics, San Francisco, CA). In other embodiments, the modified IL-2 is ALKS4230 (Alkermes, Inc).

[0255] Cancers treatable by the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (one form of skin cancer), squamous cell carcinoma (various tissues), transitional cell carcinoma (malignant neoplasm of the bladder) including bladder cancer, bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer including small cell and non-small cell lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, intraductal carcinoma in situ of the breast or cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0256] Sarcomas to which the methods disclosed herein are applicable for treatment include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0257] Other solid tumors to which the methods disclosed herein are applicable for treatment include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0258] Leukemias to which the methods disclosed herein are applicable for treatment include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic diseases of pluripotent hematopoietic stem cells); b) acute myeloid leukemia (neoplastic transformation of hematopoietic stem cells with the ability to differentiate into pluripotent or restricted lineage cells); c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphocytic leukemia (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated using the methods include, but are not limited to, B-cell lymphomas (e.g., Burkitt lymphoma); Hodgkin lymphoma; non-Hodgkin lymphoma, and the like.

[0259] Other cancers to which the methods disclosed herein are applicable for treatment include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymal tumor (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and mediastinal neuroblastoma.

[0260] Combination therapy In some embodiments, the CAR cells are administered as adjuvant therapy to standard cancer therapies. Standard cancer therapies include surgery (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapy treatment, antibody treatment, biological response modifier treatment, and certain combinations of the foregoing methods.

[0261] Radiation therapy includes, but is not limited to, X-rays or gamma rays delivered from a source such as an externally applied beam or by implantation of a small radioactive source.

[0262] Suitable antibodies for cancer treatment include, but are not limited to, naked antibodies, e.g., trastuzumab (bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), oregovomab (Ovarex™), ramucirumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™), etc., and conjugate antibodies, e.g., gemtuzumab ozogamicin (Mylotarg™), brentuximab vedotin, 90 Y-labeled ibritumomab tiuxetan (Zevalin™), 131 I-labeled tositumomab (Bexxar™), etc. Suitable antibodies for cancer treatment include, but are not limited to, antibodies produced against tumor-associated antigens. Such antigens include, but are not limited to, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate-binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Le y, VEGF, VEGFR, integrin alpha-V-beta-3, integrin alpha-5-beta-l, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, PAP, tenascin, and the like.

[0263] Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) tyrosine kinase (RTK) activity inhibitors; (2) serine / threonine kinase activity inhibitors; (3) tumor-associated antigen antagonists, for example, antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) interferon alpha; (7) interferon gamma; (8) colony stimulating factors; (9) angiogenesis inhibitors; and (10) antagonists of tumor necrosis factor.

[0264] Chemotherapeutic agents are non-peptide (i.e., proteinaceous) compounds that reduce the growth of cancer cells and include cytotoxic agents and cell division inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.

[0265] Agents that act to reduce cell growth are known in the art and are widely used. Such agents include alkylating agents such as nitrogen mustard, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan (trademark)), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chloromethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, dacarbazine, and temozolomide.

[0266] Metabolic antagonists include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolic acid (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0267] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g., etoposide, teniposide, etc.; antibiotics, e.g., anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin, and morpholino derivatives, etc.; phenoxazone bis-cyclopeptide, e.g., dactinomycin; basic glycopeptide, e.g., bleomycin; anthraquinone glycoside, e.g., plicamycin (mithramycin); anthracenedione, e.g., mitoxantrone; aziridinopyrroloindoledione, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (tacrolimus, Prograf), rapamycin, etc.; etc.

[0268] Other antiproliferative cytotoxic drugs are navelbine, CPT-11, anastrozole, letrozole, capecitabine, raloxifene, cyclophosphamide, ifosamide, and droloxifene.

[0269] Microtubule agents having antiproliferative effects are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), lysocine (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), tritylcysteine, vinblastine sulfate, vincristine sulfate; epothilone A, epothilone B, discodermolide, estramustine, nocodazole, and natural and synthetic epothilones (including but not limited to these).

[0270] Hormonal regulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, corticosteroids such as prednisone, dexamethasone, etc.; estrogens and progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenal cortex inhibitors such as aminoglutethimide; 17α-ethinyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, drostanolone propionate, testolactone, methylprednisolone, methyltestosterone, prednisone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex®. Estrogens stimulate growth and differentiation, and thus compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0271] Other chemotherapeutic agents include metal complexes, such as, for example, cisplatin (cis-DDP), carboplatin, etc.; ureas, such as hydroxyurea; and hydrazines, such as N-methylhydrazine; epipodophyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other notable antiproliferative agents include immunosuppressants, such as mycophenolic acid, thalidomide, deoxyspergualin, azathioprine, leflunomide, mizoribine, azaspirane (SKF105685); Iressa (registered trademark) (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); etc.

[0272] "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which is understood herein to include analogs, formulations, and derivatives, such as, for example, docetaxel, Taxol (trademark), Taxotere (trademark) (a formulation of docetaxel), the 10-desacetyl analog of paclitaxel and the 3'N-desbenzoyl-3'N-t-butoxycarbonyl analog of paclitaxel) can be readily prepared using techniques known to those of ordinary skill in the art (see also International Publication Nos. 94 / 07882, 94 / 07881, 94 / 07880, 94 / 07876, 93 / 23555, 93 / 10076; U.S. Patent Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and European Patent No. 590,267), or can be obtained from a variety of commercial sources, such as, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus chinensis).

[0273] Paclitaxel is to be understood to mean not only the generally chemically available forms of paclitaxel, but also analogs and derivatives (e.g., Taxotere (trademark) docetaxel as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0274] Also included within the term "taxane" are various known derivatives including hydrophilic derivatives and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in WO 99 / 18113; other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivatives described in U.S. Patent No. 5,415,869. Taxanes further include prodrugs of paclitaxel, including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0275] Subjects Suitable for Treatment A variety of subjects are suitable for treatment using methods of treating cancer. Suitable subjects include any individual, e.g., a human or non-human animal, that has cancer, has been diagnosed with cancer, is at risk of developing cancer, has had cancer in the past and is at risk of recurrence of cancer, has been treated with an agent for cancer and did not respond to such treatment, or has been treated with an agent for cancer but has recurred after an initial response to such treatment.

[0276] Subjects suitable for treatment by immunomodulatory methods include individuals with autoimmune disorders; individuals who are organ or tissue transplant patients; etc.; individuals in an immunocompromised state; and individuals infected with a pathogen.

[0277] Exemplary Embodiments The present disclosure provides a chimeric antigen receptor (CAR) that binds to Axl and / or Ror2, a nucleic acid comprising a nucleotide sequence encoding the CAR, and a conditionally active CAR that binds to AXl and Ror2. The present disclosure provides cells genetically modified to produce the CAR, and methods of making such cells. The CARs of the present disclosure can be used in a variety of methods, and methods are also provided that include methods of practicing adoptive immunocyte therapy, such as CAR therapy, for example, CAR therapy for cancer, such as renal cell carcinoma.

[0278] Some non-limiting, exemplary embodiments that are aspects of the present disclosure are provided in the following embodiments:

[0279] Embodiment 1. A chimeric antigen receptor (CAR) for binding to Axl or Ror2, comprising: a) A conditionally active antigen-specific targeting region (ASTR) that exhibits increased binding to Axl or Ror2 at a pH of 6.7 compared to a pH of 7.4; b) A transmembrane domain; and c) An intracellular activation domain.

[0280] Embodiment A1. A chimeric antigen receptor (CAR) for binding to Axl or Ror2, comprising: a) A conditionally active antigen-specific targeting region (ASTR) that binds to Axl or Ror2 and exhibits increased activity (i.e., greater activity) in the tumor microenvironment and / or in in vitro tumor surrogate assay conditions compared to normal physiological conditions; b) A transmembrane domain; and c) An intracellular activation domain.

[0281] Embodiment A2. The CAR according to Embodiment 1 or A1, or any other embodiment provided herein, wherein the ASTR is an antibody, antigen, ligand, ligand receptor-binding domain, receptor, receptor ligand-binding domain, and affibody, unless otherwise explicitly stated.

[0282] Embodiment A3. Unless otherwise explicitly described, the ASTR is a CAR as described in Embodiment A2 or any other embodiment provided herein that is an antibody fragment.

[0283] Embodiment A4. Unless otherwise explicitly described, a conditionally active ASTR exhibits increased antigen binding in the tumor microenvironment and / or in in vitro tumor surrogate assay conditions compared to corresponding physiological conditions, where the tumor microenvironment and / or in vitro tumor surrogate assay conditions are selected from the group consisting of hypoxia, acidic pH, higher concentrations of lactic acid, higher concentrations of hyaluronic acid, higher concentrations of albumin, higher concentrations of adenosine, higher concentrations of R-2-hydroxyglutaric acid, and lower nutrient availability, and is a CAR as described in any one of Embodiments 1 or A1 - A3 or any other embodiment provided herein.

[0284] Embodiment A5. Unless otherwise explicitly described, a conditionally active ASTR exhibits increased (or higher) antigen binding at a pH of 6.7 compared to a pH of 7.4, and is a CAR as described in any one of Embodiments A1 - A4 or any other embodiment provided herein.

[0285] Embodiment A6. Unless otherwise explicitly described, the intracellular activation domain is a human CD3Z activation domain, a human CD3D activation domain, a human CD3E activation domain, a human CD3G activation domain, a human CD28 activation domain, a human CD79A activation domain, a human DAPIO activation domain, a human DAP12 activation domain, a human FCERlG activation domain, a human CD137 activation domain, or a human ZAP70 activation domain, and is a CAR as described in any one of Embodiments 1 or A1 - A5 or any other embodiment provided herein.

[0286] Embodiment A7. Unless otherwise explicitly described, a CAR according to any one of Embodiment 1 or A1 - A6, or any other embodiment provided herein, further comprising a first, second, third, or fourth co - stimulatory domain having an amino acid sequence different from the intracellular activation domain.

[0287] Embodiment A8. Unless otherwise explicitly described, a CAR according to Embodiment A7, or any other embodiment provided herein, wherein the first, second, third, and / or fourth co - stimulatory domain comprises a ligand that specifically binds to 4 - 1BB (CD137), B7 - H3, CD2, CD7, CD27, CD28, Lck - binding - deleted CD28 (ICΔ), ICOS, OX40, BTLA, CD27, CD30, CD40, GITR, HVEM, LFA - 1, LIGHT, NKG2C, PD - 1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor epsilon chain, or CD83.

[0288] Embodiment A9. Unless otherwise explicitly described, a CAR according to Embodiment A7, or any other embodiment provided herein, wherein the first co - stimulatory domain retains co - stimulatory activity and is a human CD137 co - stimulatory domain, human CD28 co - stimulatory domain, human ICΔ co - stimulatory domain, human ICOS co - stimulatory domain, human OX40 co - stimulatory domain, human BTLA co - stimulatory domain, human CD27 co - stimulatory domain, human CD30 co - stimulatory domain, human GITR co - stimulatory domain, or human HVEM co - stimulatory domain.

[0289] Embodiment B1. Unless otherwise explicitly described, a replication - incompetent recombinant retroviral particle comprising a retroviral genome containing one or more nucleic acid sequences operably linked to a promoter active in T cells and / or NK cells, encoding a CAR according to any one of Embodiment 1 or A1 - A9, or any other embodiment provided herein.

[0290] Embodiment B2. An isolated recombinant T cell or NK cell genetically modified with the replication-incompetent recombinant retroviral particles described in Embodiment B1.

[0291] Embodiment B3. An isolated recombinant T cell genetically modified with the replication-incompetent recombinant retroviral particles described in Embodiment B1.

[0292] Embodiment B4. A replication-incompetent recombinant retroviral particle described in Embodiment B1, and a reaction mixture comprising T cells and / or NK cells.

[0293] Embodiment B5. A replication-incompetent recombinant retroviral particle described in Embodiment B1, and a reaction mixture comprising T cells.

[0294] Embodiment C1. Unless otherwise explicitly stated, a genome comprising one or more nucleic acid sequences (e.g., two or more, three or more, four or more, five or more, or six or more nucleic acid sequences) operably linked to a promoter active in T cells and / or NK cells, wherein the one or more (two or more, three or more, four or more, five or more, or six or more) nucleic acid sequences encode a CAR as described in any one of Embodiments 1 or A1 - A8 or any other embodiment provided herein, an isolated (e.g., recombinant or genetically modified) T cell or NK cell.

[0295] Embodiment C2. The isolated (e.g., recombinant or genetically modified) T cell or NK cell described in Embodiment C1, wherein the isolated cell is a T cell.

[0296] Embodiment C3. An isolated (e.g., recombinant or genetically modified) T cell and / or NK cell (in certain exemplary embodiments, a T cell) comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and / or NK cells, wherein the one or more nucleic acid sequences encode a chimeric antigen receptor (CAR) that binds to Axl or Ror2 and comprises the following: a) A conditionally active antigen-specific targeting region (ASTR) that exhibits increased binding to Axl or Ror2 at a pH of 6.7 compared to a pH of 7.4; b) A transmembrane domain; and c) An intracellular activation domain.

[0297] Embodiment D1. Unless otherwise explicitly stated, contacting a target mammalian cell with a T cell and / or an NK cell in a microenvironment having a pH of less than 7.4 (e.g., the pH is less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or in the range of 5.8 - 7.0, in an exemplary embodiment, in the range of 6.0 - 6.8, 6.1 - 6.9, 6.2 - 6.8, or between the lower end values of the range of 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5 and the upper end values of the range of 6.6, 6.7, 6.8, and 6.9), wherein the T cell and / or the NK cell expresses a CAR as described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein, and the target mammalian cell expresses Axl and / or Ror2, a method of binding a T cell and / or an NK cell to a target mammalian cell.

[0298] Embodiment D2. Contacting a target mammalian cell with a T cell and / or an NK cell in a microenvironment having a pH of less than 7.4 (e.g., the pH is less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or in the range of 5.8 - 7.0, in an exemplary embodiment, in the range of 6.0 - 6.8, 6.2 - 6.8, or between the lower end values of the range of 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5 and the upper end values of the range of 6.6, 6.7, 6.8, and 6.9), wherein the target mammalian cell expresses Axl or Ror2, and the T cell and / or the NK cell expresses a chimeric antigen receptor (CAR) for binding to Axl or Ror2 respectively, and the CAR comprises the following, a method of binding a T cell and / or an NK cell to a target mammalian cell: a) A conditionally active antigen-specific targeting region (ASTR) that exhibits increased binding to Axl or Ror2 at a pH of 6.7 compared to a pH of 7.4; b) A transmembrane domain; and c) An intracellular activation domain.

[0299] Embodiment D3. The method according to any one of Embodiments D1 - D3, wherein the binding activates T cells and / or NK cells.

[0300] Embodiment D4. A method of activating a T cell or NK cell, comprising contacting a target mammalian cell with a T cell and / or NK cell in a microenvironment having a pH of less than 7.4 (e.g., the pH is less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or in the range of 5.8 - 7.0, in an exemplary embodiment, in the range of 6.0 - 6.8, in the range of 6.2 - 6.8, or between the lower end values of the range 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and the upper end values of the range 6.6, 6.7, 6.8, and 6.9), wherein the target mammalian cell expresses Axl or Ror2, the T cell or NK cell expresses a chimeric antigen receptor (CAR) for binding to Axl or Ror2 respectively, and the CAR comprises: a) A conditionally active antigen-specific targeting region (ASTR) that exhibits increased binding to Axl or Ror2 at a pH of 6.7 compared to a pH of 7.4; b) A transmembrane domain; and c) An intracellular activation domain.

[0301] Embodiment D5. The method according to Embodiment D5, wherein the CAR is as described in another embodiment, a non-limiting exemplary embodiment, Embodiment 1, or any of A1 - A8.

[0302] Embodiment D6. The method according to Embodiment D5, wherein the CAR is as described in Embodiment 1 or any of A1 - A8.

[0303] Embodiment D7. The method according to any one of Embodiments D3 to D6, wherein activation includes an increase in the expression and / or production and / or secretion of cytokines.

[0304] Embodiment D8. The method according to any one of Embodiments D3 to D7, wherein upon activation, T cells and / or NK cells increase the expression of IL-2 or IFN-γ.

[0305] Embodiment D9. The method according to Embodiment D8, wherein the expression of IL-2 or IFN-γ is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold compared to the amount of IL-2 or IFN-γ produced by T cells or NK cells before contact.

[0306] Embodiment D10. The method according to Embodiment D8, wherein the secretion of IL-2 or IFN-γ is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold compared to the amount of IL-2 or IFN-γ secreted by T cells or NK cells before contact.

[0307] Embodiment D11. The method according to any one of Embodiments D3 to D7, wherein upon activation, the cytotoxic activity of T cells or NK cells is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold compared to the cytotoxic activity of T cells or NK cells before contact.

[0308] Embodiment D12. The method according to any one of Embodiments D1 to D11, wherein the target mammalian cell is lysed after activation of T cells or NK cells.

[0309] Embodiment D13. The method according to any one of Embodiments D1 to D12, further comprising transducing T cells or NK cells with replication-incompetent recombinant retroviral particles according to any one of Embodiments B1 or B2 before contact to genetically modify the T cells or NK cells to express a CAR.

[0310] Embodiment D14. The method according to Embodiment D13, wherein the transduction is performed ex vivo.

[0311] Embodiment D15. The method according to any one of Embodiments D1 to D14, further comprising increasing the pH of the microenvironment to a pH of 7.0 or higher (e.g., above 7.1, 7.2, or 7.3), thereby reducing the activation of T cells or NK cells.

[0312] Embodiment D16. The method according to any one of Embodiments D1 to D14, further comprising increasing the pH of the microenvironment to a pH of 7.0 or higher (e.g., above 7.1, 7.2, or 7.3), thereby inactivating T cells or NK cells.

[0313] Embodiment D17. The method according to any one of Embodiments D1 to D16, wherein the microenvironment is a tumor.

[0314] Embodiment D18. The method according to Claim D17, wherein the tumor is in a human subject.

[0315] Embodiment D19. The method according to any one of Embodiments D1 to D16, wherein the microenvironment is in vitro or ex vivo.

[0316] Embodiment E1. A method of genetically modifying lymphocytes, comprising contacting T cells and / or NK cells with replication-incompetent recombinant retroviral particles containing in their genome a polynucleotide comprising one or more nucleic acid sequences (e.g., two or more, three or more, four or more, five or more, or six or more nucleic acid sequences) operably linked to a promoter active in T cells and / or NK cells, wherein the one or more (two or more, three or more, four or more, five or more, or six or more) nucleic acid sequences encode a chimeric antigen receptor as described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein, and wherein said contacting facilitates transduction of the T cells and / or NK cells by the replication-incompetent recombinant retroviral particles, thereby generating genetically modified T cells and / or NK cells.

[0317] Embodiment F1. Unless otherwise explicitly stated, a replication-incompetent recombinant retroviral particle containing in its genome a polynucleotide comprising one or more nucleic acid sequences (e.g., two or more, three or more, four or more, five or more, or six or more nucleic acid sequences) operably linked to a promoter active in T cells and / or NK cells, wherein the one or more (two or more, three or more, four or more, five or more, or six or more) nucleic acid sequences encode a chimeric antigen receptor as described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein, and a method comprising contacting T cells and / or NK cells, wherein said contacting facilitates transduction of the T cells and / or NK cells by the replication-incompetent recombinant retroviral particles, thereby generating genetically modified T cells and / or NK cells, and which is for use in a method of genetically modifying lymphocytes.

[0318] Embodiment G1. Unless otherwise explicitly stated, a method comprises contacting T cells and / or NK cells with replication-incompetent recombinant retroviral particles comprising a polynucleotide comprising one or more nucleic acid sequences (e.g., two or more, three or more, four or more, five or more, or six or more nucleic acid sequences) operably linked to a promoter active in T cells and / or NK cells in their genome, wherein the one or more (two or more, three or more, four or more, five or more, or six or more) nucleic acid sequences encode a chimeric antigen receptor as described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein, and said contacting facilitates transduction of T cells and / or NK cells by the replication-incompetent recombinant retroviral particles, thereby generating genetically modified T cells and / or NK cells, and is for use in a method of genetically modifying T cells and / or NK cells to treat tumor growth. A replication-incompetent recombinant retroviral particle for use in a method of genetically modifying T cells and / or NK cells to treat tumor growth.

[0319] Embodiment H1. Unless otherwise explicitly stated, the use of a kit comprises contacting T cells and / or NK cells with replication-incompetent recombinant retroviral particles comprising a polynucleotide comprising one or more nucleic acid sequences (e.g., two or more, three or more, four or more, five or more, or six or more nucleic acid sequences) operably linked to a promoter active in T cells and / or NK cells in their genome, wherein the one or more (two or more, three or more, four or more, five or more, or six or more) nucleic acid sequences encode a chimeric antigen receptor as described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein, and said contacting facilitates transduction of T cells and / or NK cells by the replication-incompetent recombinant retroviral particles, thereby generating genetically modified T cells and / or NK cells, and is for use in the manufacture of a kit for genetically modifying T cells and / or NK cells.

[0320] Embodiment I1. Unless otherwise explicitly stated, a replication-incompetent recombinant retroviral particle contains in its genome a polynucleotide comprising one or more nucleic acid sequences (e.g., two or more, three or more, four or more, five or more, or six or more nucleic acid sequences) operably linked to a promoter active in T cells and / or NK cells, wherein the one or more (two or more, three or more, four or more, five or more, or six or more) nucleic acid sequences encode a chimeric antigen receptor as described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein. A commercially available container containing the replication-incompetent recombinant retroviral particle and instructions for its use.

[0321] Embodiment J1. A kit comprising a container containing replication-incompetent recombinant retroviral particles and instructions for its use, wherein the instructions teach a method for binding T cells and / or NK cells to target mammalian cells, the method comprising: a) Unless otherwise explicitly stated, transducing T cells and / or NK cells with the replication-incompetent recombinant retroviral particle, which contains in its genome a polynucleotide comprising one or more nucleic acid sequences (e.g., two or more, three or more, four or more, five or more, or six or more nucleic acid sequences) operably linked to a promoter active in T cells and / or NK cells, wherein the one or more (two or more, three or more, four or more, five or more, or six or more) nucleic acid sequences encode a CAR as described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein; and b) contacting the target mammalian cell with the transduced T cells and / or NK cells in a microenvironment having a pH of less than 7.4 (e.g., the pH is less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or in the range of 5.8 to 7.0, more generally in the range of 6.0 to 6.8, 6.2 to 6.8, or between the lower end values of the range of 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and the upper end values of the range of 6.6, 6.7, 6.8, and 6.9), wherein the T cells and / or NK cells express the CAR described in any one of Embodiment 1 or A1 - A8, and the target mammalian cell expresses Axl and / or Ror2.

[0322] Embodiment K1. Unless otherwise explicitly stated, a method of activating T cells and / or NK cells, comprising contacting a target mammalian cell with the T cells and / or NK cells in a microenvironment having a pH of less than 7.4 (e.g., the pH is less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or in the range of 5.8 to 7.0, more generally in the range of 6.0 to 6.8, 6.2 to 6.8, or between the lower end values of the range of 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and the upper end values of the range of 6.6, 6.7, 6.8, and 6.9), wherein the T cells and / or NK cells express the chimeric antigen receptor described in any one of Embodiment 1 or A1 - A8 or any other embodiment provided herein, and the target mammalian cell expresses Axl and / or Ror2.

[0323] Embodiment K2. The method according to Embodiment K1, further comprising transducing the T cell or NK cell with a replication - incompetent recombinant retroviral particle described in any one of Embodiment B1 or B2 before the contact.

[0324] Embodiment K3. The method according to Embodiment K1, wherein after activation, the T cells and / or NK cells induce cytokine expression and / or production and / or secretion.

[0325] Embodiment K4. The method according to embodiment K3, wherein the cytokine is selected from the group consisting of IL-2 or IFN-γ.

[0326] Embodiment L1. Unless otherwise explicitly stated, contacting a target mammalian cell with T cells and / or NK cells in a microenvironment having a pH of less than 7.4 (e.g., the pH is less than 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or in the range of 5.8 to 7.0, more generally in the range of 6.0 to 6.8, in the range of 6.2 to 6.8, or between 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5 as the lower end values of the range and 6.6, 6.7, 6.8, and 6.9 as the upper end values of the range), wherein the T cells and / or NK cells express a chimeric antigen receptor as described in any one of embodiment 1 or A1 - A8 or any other embodiment provided herein, and the target mammalian cell expresses Axl and / or Ror2, a method for inducing the expression and / or production of cytokines in T cells and / or NK cells and / or inducing the secretion of cytokines from T cells and / or NK cells.

[0327] Embodiment L2. The method according to embodiment L1, further comprising transducing the T cells or NK cells with replication-incompetent recombinant retroviral particles as described in any one of embodiment B1 or B2 prior to contact.

[0328] Embodiment M1. An isolated nucleic acid encoding a chimeric antigen receptor for binding to Axl or Ror2, as described in any one of embodiment 1 or A1 - A8 or any other embodiment provided herein, unless otherwise explicitly stated.

[0329] Embodiment M2. The isolated nucleic acid according to embodiment M1, wherein the nucleic acid further comprises a promoter active in T cells and / or NK cells, and the nucleic acid sequence encoding the CAR is operably linked to the promoter.

[0330] Embodiment M3. The isolated nucleic acid according to embodiment M2, wherein the isolated nucleic acid sequence further encodes a recognition domain.

[0331] Embodiment M4. The isolated nucleic acid according to embodiment M3, wherein the nucleic acid encoding the recognition domain is separated from the nucleic acid encoding the CAR by a ribosome skipping sequence.

[0332] Embodiment M5. The isolated nucleic acid according to embodiment M4, wherein the ribosome skipping sequence is 2A-1.

[0333] Embodiment M6. A replication-incompetent recombinant retroviral particle comprising any of the nucleic acids according to embodiments M1 to M5.

[0334] Embodiment M7. The replication-incompetent recombinant retroviral particle according to embodiment M6, wherein the replication-incompetent recombinant retroviral particle is a lentiviral particle.

[0335] Embodiment N1. An expression vector comprising a nucleic acid encoding a chimeric antigen receptor according to embodiment 1 or any of A1 to A8, or any other embodiment provided herein, unless otherwise explicitly stated.

[0336] Embodiment O1. A mammalian cell infected with any one of the expression vectors according to embodiment N1.

[0337] Embodiment P1. A mammalian cell expressing any of the chimeric antigen receptors according to embodiment 1 or any of A1 to A8, or any other embodiment provided herein, unless otherwise explicitly stated.

[0338] Embodiment Q1. A method for producing a conditionally activatable cell comprising a chimeric antigen receptor (CAR) against conditionally binding Axl or Ror2, the method comprising genetically modifying a mammalian cell with an expression vector comprising a promoter operably linked to a nucleotide sequence encoding a CAR, wherein the CAR comprises: a) A conditionally active antigen-specific targeting region (ASTR) that binds to Axl or Ror2 and shows increased activity in the tumor environment and / or in in vitro tumor surrogate assay conditions compared to the normal physiological environment; b) A transmembrane domain; and c) An intracellular activation domain.

[0339] Embodiment R1. A method of treating cancer in a subject, comprising the following steps: a) Introducing an expression vector containing a nucleic acid encoding any of the CARs described in Embodiment 1 or any of A1 - A8, or any other embodiment provided herein, into cytotoxic cells obtained from the subject, to generate genetically modified cytotoxic cells, provided that no other meaning is explicitly described; and b) Administering the genetically modified cytotoxic cells to the subject.

[0340] Embodiment S1. A method for ex vivo generation of conditionally activatable T cells and / or NK cells comprising a chimeric antigen receptor (CAR) against conditionally binding Axl or Ror2, comprising: a) Concentrating peripheral blood mononuclear cells (PBMCs) to isolate PBMCs containing T cells and / or NK cells from the isolated blood; b) Activating the T cells and / or NK cells of the concentrated PBMCs under effective conditions; c) Transducing the activated T cells and / or NK cells with replication-incompetent recombinant retroviral particles under effective conditions, provided that no other meaning is explicitly described, thereby generating genetically modified T cells and / or NK cells, wherein the replication-incompetent recombinant retroviral particles each contain a retroviral genome comprising one or more nucleic acid sequences operably linked to an active promoter in the T cells and / or NK cells, and the first nucleic acid sequence among the one or more nucleic acid sequences encodes a CAR described in Embodiment 1 or any of A1 - A8, or any other embodiment provided herein; and d) expanding genetically modified T cells and / or NK cells, thereby producing conditionally activatable T cells and / or NK cells.

[0341] Embodiment T1. A modified T cell produced by the method according to any one of Embodiments D3, E1, K2, Q1, R1, or S1.

[0342] Embodiment U1. A modified NK cell produced by the method according to any one of Embodiments D3, E1, K2, Q1, R1, or S1.

[0343] In any of the embodiments herein that include a chimeric antigen receptor (CAR), the antigen-specific targeting region (ASTR) can be selected from an antibody, an antigen, a ligand, a ligand receptor-binding domain, a receptor, a receptor ligand-binding domain, and an affibody. In any of the embodiments herein that include a CAR, the ASTR can be an antibody selected from a full-length antibody, a single-chain antibody, a Fab fragment, a Fab’ fragment, a (Fab’)2 fragment, an Fv fragment, and a bivalent single-chain antibody or diabody. In any of the embodiments herein that include a CAR, the ASTR can include a heavy chain and a light chain derived from an antibody. In any of the embodiments herein that include a CAR having an ASTR that includes an antibody, the antibody can be a single-chain variable fragment having a heavy chain and a light chain. In any of the embodiments herein that include a CAR having an ASTR that includes a heavy chain and a light chain derived from an antibody, the heavy chain and the light chain can be separated by a linker, and the linker can be between a lower limit of 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length and an upper limit of 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 amino acids in length. In some embodiments, the ASTR can be a single-chain variable fragment that includes a heavy chain and a light chain, the heavy chain and the light chain are separated by a linker, and the linker is 6 to 100 amino acids in length. In any of the embodiments herein that include a CAR having an ASTR that includes a single-chain variable fragment that includes a heavy chain and a light chain, the heavy chain and the light chain can be separated by a linker, the linker can be 6 to 100 amino acids in length, the ASTR can include a conditional activation heavy antibody chain or a conditional activation light antibody chain, and the other heavy antibody chain or light antibody chain can be wild-type.

[0344] In any of the embodiments herein that include a CAR having an ASTR comprising heavy and light chains derived from an antibody, the antibody can be positioned N-terminal to the light chain on the chimeric antigen receptor, or the light chain can be positioned N-terminal to the heavy chain on the chimeric antigen receptor. In any of the embodiments herein that include a CAR having an ASTR comprising heavy and light chains derived from an antibody, the heavy and light chains can be derived from a conditionally active antibody. In any of the embodiments herein that include a CAR, the CAR can include a bispecific ASTR. In any of the embodiments herein that include a CAR, the ASTR can be a conditionally active ASTR. In any of the embodiments herein that include a CAR having a conditionally active ASTR, the conditionally active ASTR can exhibit increased antigen binding in the tumor microenvironment and / or in in vitro tumor surrogate assay conditions as compared to corresponding physiological conditions, where the tumor microenvironment and / or in vitro tumor surrogate assay conditions can be selected from the group consisting of low oxygen, acidic pH, higher concentrations of lactate, higher concentrations of hyaluronic acid, higher concentrations of albumin, higher concentrations of adenosine, higher concentrations of R-2-hydroxyglutaric acid, and lower nutrient availability. In any of the embodiments herein that include a CAR having a conditionally active ASTR, the conditionally active ASTR can exhibit increased antigen binding at a pH of 6.7 as compared to a pH of 7.4. In any of the embodiments herein that include a CAR having a conditionally active ASTR, the conditionally active ASTR can exhibit increased antigen binding at a pH of 6.0 (instead of or in addition to a pH of 6.7) as compared to a pH of 7.4.

[0345] In any of the embodiments herein that include a CAR, the ASTR can bind to Axl. In any of the embodiments herein that include a CAR having an ASTR that binds to Axl, the ASTR can bind to the same epitope of Axl as an antibody comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80.

[0346] In any of the embodiments herein that include a CAR having an ASTR that binds to Axl and binds to the same epitope of Axl as an antibody comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80 in an exemplary embodiment, the ASTR can include a variable region of an antibody heavy chain having three complementarity determining regions having H1, H2, and H3 sequences, where a) the H1 sequence is X1GX2TMN (SEQ ID NO: 87); b) the H2 sequence is LIKPSNGGTSYNQKFKG (SEQ ID NO: 88); and c) the H3 sequence is GX3YX4SYX5AMDY (SEQ ID NO: 89), where X1 is T or W; X2 is H or A; X3 is H or D; X4 is E or H; X5 is E or F. In any of the embodiments herein that include a CAR having an ASTR that binds to Axl and binds to the same epitope of Axl as an antibody comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80 in an exemplary embodiment, the ASTR can include a variable region of an antibody light chain having three complementarity determining regions having L1, L2, and L3 sequences, where d) the L1 sequence is KASQDVX6SAVA (SEQ ID NO: 90); e) the L2 sequence is WX7X8TRX9T (SEQ ID NO: 91); and f) the L3 sequence is QEHFSX 10 PLX 11 (SEQ ID NO: 92), where X6 is S or V; X7 is A or Q; X8 is S or D; X9 is H or D; X 10 is T or P; and X 11 is T or R. In any of the embodiments herein that include a CAR having an ASTR that binds to Axl and binds to the same epitope of Axl as an antibody comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80 in an exemplary embodiment, the ASTR can include a variable region of an antibody heavy chain having three complementarity determining regions having H1, H2, and H3 sequences, where a) the H1 sequence is X1GX2X3MX4 (SEQ ID NO: 134); b) the H2 sequence is LIKX5SNGGTX6YNQKFKG (SEQ ID NO: 135); and c) the H3 sequence is GX7X8X9X 10 X 11 X 12 X 13 X 14 DYX15 X 16 is (SEQ ID NO: 136), where X1 is T, A, or W; X2 is H or A; X3 is T or I; X4 is N or I; X5 is P or N; X6 is S, I, or T; X7 is H, D, E, P, R, or W; X8 is Y or N; X9 is E, A, D, F, G, H, I, L, M, N, R, V, or Y; X 10 is S, D, M, N, or Q; X 11 is Y, C, E, or P; X 12 is F, E, N, S, T, or V; X 13 is A, D, G, L, or Y; X 14 is M, E, or F; X 15 is W, A, D, H, L, N, P, R, or T; and X 16 is G or H. In embodiments herein that bind to Axl and include a CAR having an ASTR that binds to the same epitope of Axl as the antibody comprising the antibody heavy chain of SEQ ID NO: 79 and the antibody light chain of SEQ ID NO: 80 in an exemplary embodiment, the ASTR can include an antibody light chain variable region having three complementarity determining regions having L1, L2, and L3 sequences, d) the L1 sequence is KASQDX 17 X 18 SX 19 VX 20 (SEQ ID NO: 137); e) the L2 sequence is X 21 X 22 X 23 TRX 24 T (SEQ ID NO: 138); and f) the L3 sequence is QEX 25 X 26 SX 27 X 28 X 29 X 30 (SEQ ID NO: 139), X 17 is V, D, G, N, or W; X 18 is S or V; X 19 is A, L, or M; X 20 is A, D, N, or Q; X 21 is W or F; X 22 is A, I, N, P, or Q; X23 is S or D; X 24 is H or D; X 25 is H, C, F, I, L, Q, S, T, V, or Y; X 26 is F, C, D, E, G, N, or S; X 27 is T, C, or P; X 28 is P, A, C, D, E, H, K, S, T, V, or W; X 29 is L, G, or R; and X 30 is T, I, or R. In any of the embodiments herein that include a CAR having an ASTR that binds to Axl and includes a light chain variable region, the light chain variable region can be selected from SEQ ID NOs: 108-111. In any of the embodiments herein that include a CAR having an ASTR that binds to Axl and includes a heavy chain variable region, the heavy chain variable region can be selected from SEQ ID NOs: 112-114.

[0347] In any of the embodiments herein that include a CAB-CAR that binds to Axl, the heavy chain can be N-terminal to the light chain. In these embodiments, the ASTR can include the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: 129, or SEQ ID NO: 159.

[0348] In any of the embodiments herein that include a CAB-CAR that binds to Axl, the light chain can be N-terminal to the heavy chain. In these embodiments, the ASTR can include the amino acid sequence of SEQ ID NO: 160, or SEQ ID NO: 161.

[0349] In any of the embodiments of the present specification that include a CAR, ASTR can bind to Ror2. In any of the embodiments of the present specification that include a CAR having an ASTR that binds to Ror2, ASTR can bind to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and an antibody light chain of SEQ ID NO: 84, and / or ASTR can bind to the same epitope of Ror2 as a single-chain variable antibody fragment comprising an antibody heavy chain of SEQ ID NO: 151 and an antibody light chain of SEQ ID NO: 152. In any of the embodiments of the present specification that include a CAR having an ASTR that binds to Ror2, ASTR can comprise a heavy-chain variable region having three complementarity-determining regions, said region having H1, H2, and H3 sequences, where a) the H1 sequence is GYTX1TEX2TX3H (SEQ ID NO: 95) or X4GYSITTGYYWN (SEQ ID NO: 96); b) the H2 sequence is GX5NX6NNGGTGYNQKFKG (SEQ ID NO: 97) or YITYDGSKNYNPSLKN (SEQ ID NO: 98); c) the H3 sequence is GSLYSYGNSYFDY (SEQ ID NO: 99) or FEGVWX7GLDY (SEQ ID NO: 100), X1 is F or E; X2 is Y or D; X3 is M or D; X4 is T or S; X5 is E or I; X6 is T or D; and X7 is Y or G. In any of the embodiments of the present specification that include a CAR having an ASTR that binds to Ror2 and binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 151 and an antibody light chain of SEQ ID NO: 152 in an exemplary embodiment, ASTR can comprise a heavy-chain variable region having three complementarity-determining regions, said region having H1, H2, and H3 sequences, where a) the H1 sequence is GYTX1TEX2TX3H (SEQ ID NO: 95); b) the H2 sequence is GX5NX6NNGGTGYNQKFKG (SEQ ID NO: 97); and c) the H3 sequence is GSLYSYGNSYFDY (SEQ ID NO: 99), X1 is F or E; X2 is Y or D; X3 is M or D; X5 is E or I; and X6 is T or D.In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2 and binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and an antibody light chain of SEQ ID NO: 84 in an exemplary embodiment, the ASTR can include a heavy chain variable region having three complementarity determining regions, the regions having H1, H2, and H3 sequences, a) the H1 sequence is X4GYSITTGYYWN (SEQ ID NO: 96); b) the H2 sequence is YITYDGSKNYNPSLKN (SEQ ID NO: 98); and c) the H3 sequence is FEGVWX7GLDY (SEQ ID NO: 100), where X4 is T or S; and X7 is Y or G. In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2, the ASTR can include a light chain variable region having three complementarity determining regions, the regions having L1, L2, and L3 sequences, a) the L1 sequence is SATSSX8SYMH (SEQ ID NO: 101) or RASESVDRYGNSFIH (SEQ ID NO: 102); b) the L2 sequence is X9TSNLAS (SEQ ID NO: 103) or RTYNLES (SEQ ID NO: 104); and c) the L3 sequence is QQRSSYPFT (SEQ ID NO: 105) or QQTNEDPWT (SEQ ID NO: 106), where X8 is E or V; and X9 is G or H. In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2 and binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 151 and an antibody light chain of SEQ ID NO: 152 in an exemplary embodiment, the ASTR can include a light chain variable region having three complementarity determining regions, the regions having L1, L2, and L3 sequences, a) the L1 sequence is SATSSX8SYMH (SEQ ID NO: 101); b) the L2 sequence is X9TSNLAS (SEQ ID NO: 103); and c) the L3 sequence is QQRSSYPFT (SEQ ID NO: 105), where X8 is E or V; and X9 is G or H.In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2 and that binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and an antibody light chain of SEQ ID NO: 84 in an exemplary embodiment, the ASTR can include a light chain variable region having three complementarity determining regions, said regions having L1, L2, and L3 sequences, wherein: a) the L1 sequence is RASESVDRYGNSFIH (SEQ ID NO: 102); b) the L2 sequence is RTYNLES (SEQ ID NO: 104); and c) the L3 sequence is QQTNEDPWT (SEQ ID NO: 106).

[0350] In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2, the ASTR can include a heavy chain variable region comprising three complementarity determining regions, said regions having H1, H2, and H3 sequences, wherein: a) the H1 sequence is GYTX1TEX2X3X4H (SEQ ID NO: 140) or GYSITTGX 29 YWN (SEQ ID NO: 141); b) the H2 sequence is X5X6X7X8NNGGTGYNQKFKG (SEQ ID NO: 142) or YITYDGSX 30 NYNPSLKN (SEQ ID NO: 143); and c) the H3 sequence is X9X 10 X 11 SX 12 YX 13 YX 14 X 15 SYFX 16 X 17 X 18 (SEQ ID NO: 144) or CSX 31 X 32 X 33 X 34 VX 35 X 36 X 37 LDX 38 (SEQ ID NO: 145), where X1 is F or E; X2 is Y or D; X3 is T or C; X4 is M, D, E, or Y; X5 is G or S; X6 is I or E; X7 is N, C, L, or V; X8 is T, D or E; X9 is A, M, or T; X 10 is R or H; X11 is G or E; X 12 is L or F; X 13 is S or G; X 14 is G or D; X 15 is N or E; X 16 is D or L; X 17 is Y, C, or T; X 18 is W or L; X 29 is Y, E, R, or T; X 30 is K or N; X 31 is R, G, H, W, or Y; X 32 is F, C, N, or Q; X 33 is E or S; X 34 is G, E, F, H, M, Q, or S; X 35 is W, A, I, P, Q, T, or V; X 36 is Y, G, N, or Q; X 37 is G, S, or T; and X 38 is Y or I. In embodiments of the present specification that bind to Ror2 and have an ASTR that binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 151 and an antibody light chain of SEQ ID NO: 152 in exemplary embodiments, the ASTR can comprise a heavy chain variable region comprising three complementarity determining regions, said regions having H1, H2, and H3 sequences, a) the H1 sequence is GYTX1TEX2X3X4H (SEQ ID NO: 140); b) the H2 sequence is X5X6X7X8NNGGTGYNQKFKG (SEQ ID NO: 142); and c) the H3 sequence is X9X 10 X 11 SX 12 YX 13 YX 14 X 15 SYFX 16 X 17 X 18 (SEQ ID NO: 144), where X1 is F or E; X2 is Y or D; X3 is T or C; X4 is M, D, E, or Y; X5 is G or S; X6 is I or E; X7 is N, C, L, or V; X8 is T, D or E; X9 is A, M, or T; X10 is R or H; X 11 is G or E; X 12 is L or F; X 13 is S or G; X 14 is G or D; X 15 is N or E; X 16 is D or L; X 17 is Y, C, or T; and X 18 is W or L. Binds to Ror2 and, in an exemplary embodiment, has an ASTR that binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84. In any of the embodiments herein that include a CAR having an ASTR, the ASTR can include a heavy chain variable region that includes three complementarity determining regions, said regions having H1, H2, and H3 sequences, a) the H1 sequence is GYSITTGX 29 YWN (SEQ ID NO: 141); b) the H2 sequence is YITYDGSX 30 NYNPSLKN (SEQ ID NO: 143); and c) the H3 sequence is CSX 31 X 32 X 33 X 34 VX 35 X 36 X 37 LDX 38 (SEQ ID NO: 145), where X 29 is Y, E, R, or T; X 30 is K or N; X 31 is R, G, H, W, or Y; X 32 is F, C, N, or Q; X 33 is E or S; X 34 is G, E, F, H, M, Q, or S; X 35 is W, A, I, P, Q, T, or V; X 36 is Y, G, N, or Q; X 37 is G, S, or T; and X 38 is Y or I.

[0351] In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2, the ASTR can include a light chain variable region that includes three complementarity determining regions, the regions having L1, L2, and L3 sequences, where a) the L1 sequence is SATSSX 19 X 20 X 21 MX 22 (SEQ ID NO: 146) or RASESVDRYGNSX 39 IH (SEQ ID NO: 147); b) the L2 sequence is X 23 TSNLAS (SEQ ID NO: 148) or X 40 TYX 41 LES (SEQ ID NO: 149); and c) the L3 sequence is QX 24 X 25 SX 26 YPFX 27 X 28 (SEQ ID NO: 150) or QQX 42 NX 43 DPX 44 TX 45 (SEQ ID NO: 85), where X 19 is V or E; X 20 is S or D; X 21 is Y, C, or D; X 22 is H, G, or L; X 23 is G, C, H, or P; X 24 is Q or E; X 25 is R or H; X 26 is S, D, G, I, Q, or V; X 27 is T or D; X 28 is F, D, or E; X 39 is F, S, or T; X 40 is R, C, D, E, or W; X 41 is N or D; X 42 is T, I, or P; X 43 is E or V; X 44 is W or T; and X 45is F or T. In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2 and binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152 in an exemplary embodiment, the ASTR can include a light chain variable region that includes three complementarity determining regions, the regions having L1, L2, and L3 sequences, a) the L1 sequence is SATSSX 19 X 20 X 21 MX 22 (SEQ ID NO: 146); b) the L2 sequence is X 23 TSNLAS (SEQ ID NO: 148); and c) the L3 sequence is QX 24 X 25 SX 26 YPFX 27 X 28 (SEQ ID NO: 150), where X 19 is V or E; X 20 is S or D; X 21 is Y, C, or D; X 22 is H, G, or L; X 23 is G, C, H, or P; X 24 is Q or E; X 25 is R or H; X 26 is S, D, G, I, Q, or V; X 27 is T or D; and X 28 is F, D, or E. In any of the embodiments herein that include a CAR having an ASTR that binds to Ror2 and binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84 in an exemplary embodiment, the ASTR can include a light chain variable region that includes three complementarity determining regions, the regions having L1, L2, and L3 sequences, a) the L1 sequence is RASESVDRYGNSX 39 IH (SEQ ID NO: 147); b) the L2 sequence is X 40 TYX 41 LES (SEQ ID NO: 149); and c) the L3 sequence is QQX 42 NX 43 DPX 44 TX 45is (SEQ ID NO: 85), and X 39 is F, S, or T; X 40 is R, C, D, E, or W; X 41 is N or D; X 42 is T, I, or P; X 43 is E or V; X 44 is W or T; and X 45 is F or T.

[0352] A chimeric antigen receptor (CAR) comprising an ASTR that binds to Ror2, and in an exemplary embodiment, the ASTR binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and an antibody light chain of SEQ ID NO: 84. In any of the embodiments herein, the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 120, or SEQ ID NO: 121.

[0353] A chimeric antigen receptor (CAR) comprising an ASTR that binds to Ror2, and in an exemplary embodiment, the ASTR binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and an antibody light chain of SEQ ID NO: 84. In any of the embodiments herein, the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83. The chimeric antigen receptor according to claim 2931, wherein the ASTR binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and an antibody light chain of SEQ ID NO: 84, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 86.

[0354] A chimeric antigen receptor (CAR) comprising an ASTR that binds to Ror2, and in an exemplary embodiment, the ASTR binds to the same epitope of Ror2 as an antibody comprising an antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and an antibody light chain of SEQ ID NO: 84. In any of the embodiments herein, the light chain variable region can comprise the amino acid sequence of SEQ ID NO: 84.

[0355] Comprising a CAR having an ASTR that binds to Ror2, and in an exemplary embodiment, any of the embodiments herein where the ASTR binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 120, or SEQ ID NO: 121.

[0356] Comprising a CAR having an ASTR that binds to Ror2, and in an exemplary embodiment, any of the embodiments herein where the ASTR binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, the heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83.

[0357] Comprising a CAR having an ASTR that binds to Ror2, and in an exemplary embodiment, any of the embodiments herein where the ASTR binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 82 or SEQ ID NO: 83 and the antibody light chain of SEQ ID NO: 84, the ASTR can comprise the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 131, or SEQ ID NO: 132, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 157, or SEQ ID NO: 158.

[0358] Comprising a CAR having an ASTR that binds to Ror2, and in an exemplary embodiment, any of the embodiments herein where the ASTR binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, the light chain variable region can comprise the amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 152.

[0359] Comprising a CAR having an ASTR that binds to Ror2, and in an exemplary embodiment, any of the embodiments herein where the ASTR binds to the same epitope of Ror2 as an antibody comprising the antibody heavy chain of SEQ ID NO: 151 and the antibody light chain of SEQ ID NO: 152, the light chain variable region can comprise the amino acid sequence of SEQ ID NO: 152.

[0360] A CAR comprising an ASTR that binds to Ror2, and in exemplary emb...

Claims

1. An isolated nucleic acid encoding a chimeric antigen receptor for binding to Axl, wherein the chimeric antigen receptor comprises: a) an antigen-specific targeting region (ASTR), wherein the ASTR that binds to Axl comprises a heavy chain variable region comprising three complementarity-determining regions having H1, H2, and H3 sequences, and a light chain variable region comprising three complementarity-determining regions having L1, L2, and L3 sequences, wherein i) the H1 sequence is X １ GX ２ TMN (SEQ ID NO: 87); ii) the H2 sequence is LIKPSNGGTSYNQKFKGG (SEQ ID NO: 88); iii) the H3 sequence is GX ３ YX ４ SYX ５ AMDY (SEQ ID NO: 89), and Here, X １ is T, and X ２ is H, and X ３ is H, and X ４ is E, and X ５ is E; or X １ is T, and X ２ is A, and X ３ is H, and X ４ is E, and X ５ is E; or X １ is W, and X ２ is A, and X ３ is H, and X ４ is E, and X ５ is E; iv) the L1 sequence is KASQDVX ６ SAVA (SEQ ID NO: 90); v) the L2 sequence is WX ７ X ８ TRX ９ T (SEQ ID NO: 91); and vi) the L3 sequence is QEHFSX １０ PLX １１ (SEQ ID NO: 92); Here, X ６ is V, and X ７ is Q, and X ８ is D, and X ９ is H, and X １０ is T, and X １１ is T, or X ６ is V, and X ７ is Q, and X ８ is S, and X ９ is H, and X １０ is T, and X １１ is T, or X ６ is S, and X ７ is Q, and X ８ is D, and X ９ is H, and X １０ is P, and X １１ is T, or; or X ６ is V, and X ７ is Q, and X ８ is D, and X ９ is H, and X １０ is P, and X １１ is T; b) a transmembrane domain; and c) an intracellular activation domain and wherein the transmembrane domain is located between the ASTR and the intracellular activation domain, the isolated nucleic acid.

2. An isolated nucleic acid encoding a chimeric antigen receptor for binding to Axl, wherein the chimeric antigen receptor comprises: a) an antigen-specific targeting region (ASTR), wherein the ASTR that binds to Axl comprises a heavy chain variable region comprising three complementarity-determining regions having H1, H2, and H3 sequences, and a light chain variable region comprising three complementarity-determining regions having L1, L2, and L3 sequences, wherein i) the H1 sequence is X １ GX ２ TMN (SEQ ID NO: 87); ii) the H2 sequence is LIKPSNGGTSYNQKFKGG (SEQ ID NO: 88); iii) the H3 sequence is GX ３ YX ４ SYX ５ AMDY (SEQ ID NO: 89), Here, X １ is W, and X ２ is A, and X ３ is H, and X ４ is E, and X ５ is E; iv) the L1 sequence is KASQDVX ６ SAVA (SEQ ID NO: 90); v) the L2 sequence is WX ７ X ８ TRX ９ T (SEQ ID NO: 91); and vi) the L3 sequence is QEHFSX １０ PLX １１ (SEQ ID NO: 92); Here, X ６ is V; X ７ is Q; X ８ is D; X ９ is H; X １０ is P; and X １１ is T; b) a transmembrane domain; and c) an intracellular activation domain and wherein the transmembrane domain is located between the ASTR and the intracellular activation domain, the isolated nucleic acid.

3. The isolated nucleic acid of the chimeric antigen receptor according to claim 1 or 2, wherein the ASTR binds to the same epitope of Axl as a single-chain variable antibody fragment comprising the antibody heavy chain variable region of SEQ ID NO: 79 and the antibody light chain variable region of SEQ ID NO:

80.

4. The isolated nucleic acid of the chimeric antigen receptor according to claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

79.

5. The isolated nucleic acid of the chimeric antigen receptor according to claim 1 or 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

80.

6. The isolated nucleic acid of the chimeric antigen receptor according to claim 5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

79.

7. An isolated nucleic acid encoding a chimeric antigen receptor for binding to Ror2, wherein the chimeric antigen receptor comprises: a) An antigen-specific targeting region (AST), wherein the AST that binds to Ror2 comprises a heavy-chain variable region comprising three complementarity-determining regions having H1, H2, and H3 sequences, and a light-chain variable region comprising three complementarity-determining regions having L1, L2, and L3 sequences, wherein i) The H1 sequence is GYSITTGX １ YWN (SEQ ID NO: 141); ii) the H2 sequence is YITYDGSKNYNPSLKN (SEQ ID NO: 98); iii) the H3 sequence is CSX ２ X ３ X ４ X ５ VX ６ X ７ X ８ LDX ９ (SEQ ID NO: 145); Here, X １ is Y, and X ２ is H, and X ３ is F, and X ４ is E, and X ５ is G and X ６ is W; X ７ is Y; X ８ is G; X ９ is Y, or X １ is E, and X ２ is R, and X ３ is F, and X ４ is E, and X ５ is G and X ６ is W; X ７ is Y; X ８ is G; X ９ is Y; iv) the L1 sequence is RASESVDRYGNSFIH (SEQ ID NO: 102); v) the L2 sequence is RTYNLES (SEQ ID NO: 104); and vi) the L3 sequence is QQTNEDPWT (SEQ ID NO: 106), or i) The H1 sequence is GYT X １０ TEX １１ TX １２ H (SEQ ID NO: 95); ii) The H2 sequence is GX １３ NX １４ is NNGGTGYNQKFKKG (SEQ ID NO: 97); iii) the H3 sequence is GSLYSYGN SYFDY (SEQ ID NO: 99); Here, X １０ is F; X １１ is Y; X １２ is M; X １３ is I; X １４ is D; iv) the L1 sequence is SATSSX １５ SYMH (SEQ ID NO: 101); v) the L2 sequence is X １６ TSNLAS (SEQ ID NO: 103); and vi) the L3 sequence is QQRSSYPFT (SEQ ID NO: 105), Here, X １５ is V; and X １６ is H; b) A transmembrane domain; and c) An intracellular activation domain An isolated nucleic acid encoding a chimeric antigen receptor, wherein the transmembrane domain is located between the AST and the intracellular activation domain. **Claim 8** An isolated nucleic acid encoding a chimeric antigen receptor for binding to Ror2, wherein the chimeric antigen receptor is: a) An antigen-specific targeting region (AST), wherein the AST that binds to Ror2 comprises a heavy-chain variable region comprising three complementarity-determining regions having H1, H2, and H3 sequences, and a light-chain variable region comprising three complementarity-determining regions having L1, L2, and L3 sequences, wherein i) the H1 sequence is GYSITTGX １ YWN (SEQ ID NO: 141); ii) the H2 sequence is YITYDGSKNYNPSLKN (SEQ ID NO: 98); iii) The H3 sequence is CSX ２ X ３ X ４ X ５ VX ６ X ７ X ８ LDX ９ (SEQ ID NO: 145); Here, X １ is Y, and X ２ is H, and X ３ is F, and X ４ is E, and X ５ is G and X ６ is W; X ７ is Y; X ８ is G; X ９ is Y or; X １ is E, and X ２ is R, and X ３ is F, and X ４ is E, and X ５ is G and X ６ is W; X ７ is Y; X ８ is G; X ９ is Y; iv) the L1 sequence is RASESVDRYGNSFIH (SEQ ID NO: 102); v) the L2 sequence is RTYNLES (SEQ ID NO: 104); and vi) the L3 sequence is QQTNEDPWT (SEQ ID NO: 106), Here, X １ is E or Y; X ２ is R or H; X ３ is F; X ４ is E; X ５ is G; X ６ is W; X ７ is Y; X ８ is G; and X ９ is Y; where X ２ when is R, X １ is not Y, b) A transmembrane domain; and c) An intracellular activation domain An isolated nucleic acid encoding a chimeric antigen receptor, wherein the transmembrane domain is located between the AST and the intracellular activation domain. **Claim 9** The isolated nucleic acid according to claim 7 or 8, wherein the AST binds to the same epitope of Ror2 as a single-chain variable antibody fragment comprising the antibody heavy-chain variable region of SEQ ID NO: 82 and the antibody light-chain variable region of SEQ ID NO:

84. **Claim 10** The isolated nucleic acid according to claim 7 or 8, wherein the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO:

82.

11. The isolated nucleic acid according to claim 7 or 8, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

84.

12. The isolated nucleic acid according to claim 11, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

82.

13. The isolated nucleic acid encoding a chimeric antigen receptor according to any one of claims 1 to 12, wherein the heavy chain variable region and the light chain variable region are separated by a linker, and the linker is 6 to 100 amino acids in length.

14. The chimeric antigen receptor further comprises a stalk domain and a co-stimulatory domain, and the chimeric antigen receptor comprises, from the amino terminus to the carboxy terminus, the ASTR, the stalk domain, the transmembrane domain, the co-stimulatory domain, and the intracellular activation domain, wherein the stalk domain is a CD8 stalk domain or a CD28 stalk domain, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain, wherein the co-stimulatory domain is a CD137 co-stimulatory domain, an ICΔ co-stimulatory domain, a C28 co-stimulatory domain, or comprises both an ICΔ co-stimulatory domain and a CD137 co-stimulatory domain, and wherein the intracellular activation domain is a CD3Z activation domain, the isolated nucleic acid according to any one of claims 1 to 13.

15. The isolated nucleic acid according to claim 14, wherein the stalk domain is a CD28 stalk domain, the transmembrane domain is a CD28 transmembrane domain, and the co-stimulatory domain is an ICΔ co-stimulatory domain.

16. The isolated nucleic acid according to any one of claims 1 to 15, wherein the ASTR is a conditionally active ASTR that exhibits increased binding at pH 6.7 compared to pH 7.

4.

17. The ASTRs are antibodies selected from scFv, Fab fragments, Fab' fragments, (Fab') ２ fragments, Fv fragments, and bivalent single-chain antibodies or diabodies, the isolated nucleic acid according to any one of claims 1 to 16.

18. An isolated recombinant T cell and / or NK cell comprising a genome comprising a nucleic acid sequence operably linked to a promoter active in T cells and / or NK cells, wherein the nucleic acid sequence is the isolated nucleic acid according to any one of claims 1 to 17.

19. The isolated recombinant T cell and / or NK cell according to claim 18, wherein the isolated nucleic acid further encodes a removal domain, and the nucleic acid encoding the removal domain is separated from the nucleic acid encoding the chimeric antigen receptor by a ribosome skip sequence.

20. A vector comprising a nucleic acid sequence operably linked to a promoter active in T cells and / or NK cells, wherein the nucleic acid sequence is the isolated nucleic acid according to any one of claims 1 to 17.

21. A chimeric antigen receptor for binding to Axl encoded by the nucleic acid defined in any one of claims 1 to 6 and 13 to 17, or a chimeric antigen receptor for binding to Ror2 encoded by the nucleic acid according to any one of claims 7 to 17, which is used in a method for activating T cells or NK cells, the method comprising contacting a target mammalian cell with T cells and / or NK cells in a microenvironment having a pH of less than 7.0, wherein the target mammalian cell expresses Axl or Ror2, and the T cell or NK cell expresses a chimeric antigen receptor for binding to Axl encoded by the nucleic acid according to any one of claims 1 to 6 and 13 to 17, or a chimeric antigen receptor for binding to Ror2 encoded by the nucleic acid according to any one of claims 7 to 17. A chimeric antigen receptor for binding to Axl encoded by the nucleic acid defined in any one of claims 1 to 6 and 13 to 17, or a chimeric antigen receptor for binding to Ror2 encoded by the nucleic acid according to any one of claims 7 to 17.

22. The chimeric antigen receptor according to claim 21, wherein the microenvironment contains a tyrosine kinase inhibitor.

23. The chimeric antigen receptor according to claim 21, further comprising transducing the T cell or NK cell with replication-incompetent recombinant retroviral particles encoding the chimeric antigen receptor in the genome before the contacting to genetically modify the T cell or NK cell.

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