CD19-targeted chimeric antigen receptor and its use in immunotherapy

Abstract

To provide polynucleotides, immune cell-based compositions comprising chimeric antigen receptors, and methods of treating cancer, for immunotherapies that employ engineered immune cells.SOLUTION: Provided for herein in several embodiments are immune cell-based (e.g., natural killer (NK) cell) compositions comprising CD19-directed chimeric antigen receptors. In some embodiments, the anti-CD19 binder portion of the CAR is humanized. In several embodiments, the humanized anti-CD19 CAR expressing cells exhibit enhanced expression of the CAR as well as enhanced cytotoxicity and / or persistence. Several embodiments include methods of use of the anti-CD19 CAR expressing immune cells in immunotherapy.SELECTED DRAWING: Figure 33-1

Description

[Technical Field]

[0001] Related applications This application claims priority under U.S. Provisional Patent Applications 62 / 814,180 (filed March 5, 2019), 62 / 895,910 (filed September 4, 2019), and 62 / 932,165 (filed November 7, 2019), the entire contents of each of these applications being incorporated herein by reference.

[0002] field Some embodiments of the methods and compositions provided herein relate to a CD19-targeted receptor. In some embodiments, the receptor is a chimeric receptor. Another embodiment relates to a method of using the chimeric receptor in immunotherapy. [Background technology]

[0003] background Further knowledge is being gained about various cancers and what characteristics of cancer cells can be used to specifically distinguish them from healthy cells, and therapeutic agents that utilize these differential characteristics of cancer cells are under development. Immunotherapy using manipulated immune cells is one attempt at treating cancer.

[0004] Incorporating ASCII text file data through citation. This application incorporates the following sequence listing, submitted concurrently with this application, by referencing the following ASCII text file: File name: NKT033WO_ST25.txt; Created February 8, 2020, 434KB in size. [Overview of the Initiative]

[0005] overview Immunotherapy, in which immune cells are engineered to express specific targeting and / or effector molecules that specifically identify and respond to diseased or damaged cells, represents a new technological advance in disease treatment. This represents a promising advance, at least in part, due to its ability to specifically target diseased or damaged cells, in contrast to many traditional approaches such as chemotherapy, where all cells are affected and the desired outcome is the maintenance of enough healthy cells for the patient's survival. One approach in immunotherapy is the recombinant expression of chimeric receptors by immune cells to achieve targeted recognition and destruction of abnormal cells of interest.

[0006] In some embodiments, immune cells and populations of immune cells expressing a CD19-directed chimeric receptor are provided, the chimeric receptor comprising an extracellular anti-CD19 binding moiety, a hinge and / or a transmembrane domain and an intracellular signaling domain. Furthermore, polynucleotides encoding a CD19-directed chimeric antigen receptor comprising an extracellular anti-CD19 binding moiety, a hinge and / or a transmembrane domain and an intracellular signaling domain are provided (and vectors for introducing these into cells).

[0007] In some embodiments, a polynucleotide encoding a CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety includes a heavy chain variable (VH) domain and a light chain variable (VL) domain of a single-strand variable fragment (scFv)), a hinge, a transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain includes an OX40 subdomain and a CD3 zeta subdomain).

[0008] In some embodiments, a polynucleotide encoding a CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain of a single-strand variable fragment (scFv), where the encoded VH domain comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs: 133, 134, and 135, and the encoded VL domain comprises at least one light chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs: 127, 128, and 129), a hinge domain, a transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain comprises an OX40 subdomain and a CD3 zeta subdomain).

[0009] In some embodiments, the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15). However, in some embodiments, a different polynucleotide is used to encode mbIL15. In some embodiments, the transmembrane domain is derived from or contains the CD8 alpha transmembrane domain. In some embodiments, the CD8 alpha transmembrane domain is encoded by SEQ ID NO: 3. In some embodiments, the hinge is derived from or contains the CD8 alpha hinge. In some embodiments, the CD8 alpha hinge is encoded by SEQ ID NO: 1. In some embodiments, the OX40 subdomain is encoded by a sequence having at least 90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 5. In some embodiments, the CD3 zeta subdomain is encoded by a sequence having at least 90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 7. In some embodiments, mbIL15 is encoded by a sequence having at least 90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 11. In some embodiments, the OX40 domain is encoded by SEQ ID NO: 5, the CD3 zeta subdomain is encoded by SEQ ID NO: 7, and / or mbIL15 (encoded separately or bicistronically) is encoded by SEQ ID NO: 11. In some embodiments, the encoded OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 6, the encoded CD3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 8, and / or the encoded mbIL15 (encoded separately or bicistronically) comprises the amino acid sequence of SEQ ID NO: 12.

[0010] In some embodiments, the VH domain includes a VH domain selected from SEQ ID NOs: 120, 121, 122, and 123, and the VL domain includes a VL domain selected from SEQ ID NOs: 117, 118, and 119. In some embodiments, the polynucleotide encodes a VL domain including at least one light chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs: 127, 128, and 129. In some embodiments, the polynucleotide encodes a VH domain including at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs: 133, 134, and 135. In some embodiments, the polynucleotide is designed (e.g., manipulated) for reducing the potential antigenicity of the encoded protein and / or for one or more features of the encoded protein (e.g., target recognition and / or binding features). Thus, according to some embodiments, the anti-CD19 binding moiety does not include a certain sequence. For example, according to some embodiments, the polynucleotide does not encode one or more of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some embodiments, the encoded VH domain contains an amino acid sequence that has at least 90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 120. In some embodiments, the encoded VH domain contains the amino acid sequence of SEQ ID NO: 120. In some embodiments, the encoded VL domain contains an amino acid sequence that has at least 90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 118. In some embodiments, the encoded VL domain contains the amino acid sequence of SEQ ID NO: 118. In some embodiments, the VH domain is derived from and modified from a parent amino acid sequence.For example, mutations, shortenings, elongations, conservative substitutions, or other modifications are introduced to increase the domain's affinity for its target, increase its avidity to the target, and / or decrease the potential antigenicity of the sequence. In some embodiments, the VH domain is derived from a humanized VH domain amino acid sequence shown in SEQ ID NO: 33. Similarly, in some embodiments, the VL domain is derived from a humanized VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the polynucleotide encodes a CD19-directed chimeric antigen receptor encoded by a sequence having at least 90% (e.g., 90-95%, 95%, 96%, 97%, 98%, or 99%) sequence identity with at least the amino acid sequence shown in SEQ ID NO: 187. In some embodiments, the polynucleotide encodes a CD19-directed chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO: 187.

[0011] In some embodiments, the polynucleotide does not encode or does not contain the DAP10 domain. In some embodiments, the polynucleotide does not encode or does not contain the DAP12 domain. In some embodiments, the intracellular signaling domain includes further subdomains that favorably enhance cytotoxic signal generation by cells expressing the construct. In some embodiments, the polynucleotide further encodes one or more of CD44 and CD27 as signaling subdomains. In some embodiments, the polynucleotide optionally further encodes a detection tag or other portion (e.g., a marker) that enables detection of the expression of the polynucleotide-encoded protein by host cells.

[0012] The use of polynucleotides disclosed herein in the manufacture of pharmaceuticals for enhancing the cytotoxicity of NK cells in mammals requiring treatment, in the manufacture of pharmaceuticals for treating cancer in mammals requiring treatment, and / or for treating cancer in mammals requiring treatment is also provided herein.

[0013] Also provided herein are engineered immune cells that express a CD19-directed chimeric antigen receptor encoded by the polynucleotides disclosed herein. In some embodiments, the engineered immune cells are natural killer (NK) cells. In certain embodiments, the engineered cells are T cells, although combinations of NK cells and T cells (and optionally other immune cell types) are used in some embodiments. In some embodiments, the immune cells are allogeneic to the subject receiving the cells. Also provided herein is the use of immune cells that express a CD19-directed chimeric antigen receptor encoded by the polynucleotides disclosed herein for the treatment of cancer in a mammal in need of treatment. Also provided herein is the use of immune cells that express a CD19-directed chimeric antigen receptor encoded by the polynucleotides disclosed herein for the manufacture of a medicament for treating cancer in a mammal in need of treatment.

[0014] In some embodiments, methods for treating cancer using the polynucleotides disclosed herein are provided. For example, in some embodiments, the method comprises administering to a subject having cancer a composition comprising a population of immune cells expressing the CD19-targeted chimeric antigen receptor disclosed herein. In some embodiments, the CAR comprises an extracellular anti-CD19 binding moiety comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain of a single-strand variable fragment (scFv), a hinge such as a CD8 alpha hinge, a transmembrane domain such as a CD8 alpha transmembrane domain, and an intracellular signaling domain comprising an OX40 subdomain and a CD3 zeta subdomain, wherein the cell also expresses membrane-bound interleukin-15 (mbIL15). In some embodiments, the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 5, the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 7, and / or mbIL15 is encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 11. In some embodiments, the encoded VH domain contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 120, and the encoded VL domain contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 118. In some embodiments, the polynucleotide encodes a CD19-targeted chimeric antigen receptor having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 187. As described above, in some embodiments, the immune cells expressing such CD19 CAR constructs are natural killer (NK) cells. In some embodiments, the immune cells are T cells. In some embodiments, a combination of NK cells and T cells (and other immune cells as desired) is used. In some embodiments, the cells are allogeneic cells from a non-subject donor. In some embodiments, the cells are autologous cells from the subject. A mixture of allogeneic and autologous cells may also be used in some embodiments. In some embodiments, the administered population is approximately 2 × 10⁶ 6The cell dose includes kilograms of body weight of the subject. In some embodiments, the administration is intravenous. In some embodiments, the method further includes one or more administrations of interleukin-2 to the subject. In some embodiments, the method also includes the administration of other treatments to the subject. For example, in some embodiments, the method includes the administration of a chemotherapeutic treatment to the subject before cell administration. In some embodiments, the chemotherapeutic treatment induces lymphocyte depletion in the subject. In some embodiments, the subject is administered a combination of cyclophosphamide and fludarabine before cell administration. In some embodiments, the cyclophosphamide dose is approximately 400 to approximately 600 mg / m². 2 It is administered in the following doses. In some embodiments, fludarabine is approximately 25-25 mg / m². 2 It is administered in doses of [specify dose]. In some embodiments, lymphocyte depletion chemotherapy is administered several days before the administration of engineered immune cells and may be administered multiple times as desired. For example, in some embodiments, lymphocyte depletion chemotherapy is administered at least 5 days, 4 days and / or 3 days before the administration of engineered immune cells disclosed herein.

[0015] In some embodiments, a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor is provided, comprising a humanized anti-CD19 binding moiety, a costimulatory domain, and a signaling domain. In some embodiments, the costimulatory domain comprises OX40. In some embodiments, the humanized anti-CD19 binding moiety comprises a humanized scFv, where one or more of the heavy and light chains are humanized. In some embodiments, one or more of the CDRs of the heavy and / or light chains are humanized. For example, in some embodiments, a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (where the anti-CD19 binding moiety comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, where the VH domain comprises a VH domain selected from SEQ ID NOs: 120, 121, 122, and 123, and the VL domain comprises a VL domain selected from SEQ ID NOs: 117, 118, and 119), a hinge and / or a transmembrane domain, and an intracellular signaling domain.

[0016] In some embodiments, a polynucleotide encoding a humanized chimeric antigen receptor (CAR) is provided, where the CAR comprises a humanized anti-CD19 binding domain, a transmembrane domain, a primary intracellular signaling domain comprising the native intracellular signaling domain of CD3 zeta or a functional fragment thereof, and a co-stimulatory domain comprising the native intracellular signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, ICOS, and 4-1BB or a functional fragment thereof, and the anti-CD19 binding domain comprises a light chain complementarity determining region 1 (LC CDR1) of SEQ ID NO: 124, 127, or 130, a light chain complementarity determining region 2 (LC CDR2) of SEQ ID NO: 125, 128, or 131, and a light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 126, 129, or 132, and a heavy chain complementarity determining region 1 (HC CDR1) of SEQ ID NO: 133, 136, 139, or 142, a heavy chain complementarity determining region 2 (HC CDR2) of SEQ ID NO: 134, 137, 140, or 143, and a heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 135, 138, 141, or 144.

[0017] In some embodiments, a polynucleotide encoding a humanized CD19-directed chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding portion (where the anti-CD19 binding portion comprises a humanized scFv sequence comprising the light chain variable (VL) domain of SEQ ID NO: 117), a hinge and / or transmembrane domain, and an intracellular signaling domain. In some embodiments, the polynucleotide encodes a humanized chimeric antigen receptor of SEQ ID NO: 161, SEQ ID NO: 167, SEQ ID NO: 173, SEQ ID NO: 179, SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 197, or SEQ ID NO: 203.

[0018] In some embodiments, a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the light chain variable (VL) domain of SEQ ID NO: 118), a hinge and / or a transmembrane domain and an intracellular signaling domain. In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 163, SEQ ID NO: 169, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 187, SEQ ID NO: 193, SEQ ID NO: 199, or SEQ ID NO: 205.

[0019] In some embodiments, a polynucleotide is provided that encodes a humanized CD19-targeted chimeric antigen receptor comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the light chain variable (VL) domain of SEQ ID NO: 119), a hinge and / or a transmembrane domain and an intracellular signaling domain. In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 165, SEQ ID NO: 171, SEQ ID NO: 177, SEQ ID NO: 183, SEQ ID NO: 189, SEQ ID NO: 195, SEQ ID NO: 201, or SEQ ID NO: 207.

[0020] In some embodiments, a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 120), a hinge and / or a transmembrane domain and an intracellular signaling domain. In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 185, SEQ ID NO: 187, or SEQ ID NO: 189.

[0021] In some embodiments, a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 121), a hinge and / or a transmembrane domain and an intracellular signaling domain. In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 191, SEQ ID NO: 193, or SEQ ID NO: 195.

[0022] In some embodiments, a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 122), a hinge and / or a transmembrane domain and an intracellular signaling domain. In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 197, SEQ ID NO: 199, or SEQ ID NO: 201.

[0023] In some embodiments, a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor is provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 123), a hinge and / or a transmembrane domain and an intracellular signaling domain. In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 203, SEQ ID NO: 205, or SEQ ID NO: 207.

[0024] In some embodiments, the provided polynucleotide also encodes membrane-bound interleukin-15 (mbIL15).

[0025] In some embodiments, the intracellular signaling domain includes the OX40 subdomain. However, in some embodiments, the intracellular signaling domain includes one or more of the OX40 subdomain, CD28 subdomain, iCOS subdomain, CD28-41BB subdomain, CD27 subdomain, CD44 subdomain, or a combination thereof.

[0026] In some embodiments, the chimeric antigen receptor comprises a hinge and a transmembrane domain, where the hinge is a CD8 alpha hinge and the transmembrane domain is a CD8 alpha or NKG2D transmembrane domain. In some embodiments, the intracellular signaling domain comprises a CD3 zeta domain.

[0027] In some embodiments, the polynucleotide does not encode sequence numbers 112, 113, or 114. In some embodiments, the polynucleotide does not encode sequence number 116.

[0028] In some embodiments, engineered NK cells, engineered T cells, and / or mixed populations of NK cells and T cells are provided, expressing one or more of the humanized CD19-targeted chimeric antigen receptors provided herein.

[0029] A method for treating a subject with cancer is also provided herein, comprising administering the subject with cancer engineered NK cells and / or T cells expressing the chimeric antigen receptor disclosed herein. The use of the polynucleotides provided herein for the treatment of cancer and the use of the polynucleotides provided herein in the manufacture of cancer treatment pharmaceuticals are also provided herein.

[0030] In some embodiments, polynucleotides encoding a CD19-targeted chimeric antigen receptor are also provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety includes scFv), a transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain includes a CD28 costimulatory domain and a CD3 zeta signaling domain).

[0031] In some embodiments, polynucleotides encoding a CD19-targeted chimeric antigen receptor are also provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises scFv), a hinge (wherein the hinge is a CD8 alpha hinge), a transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain comprises CD3 zeta ITAM).

[0032] In some embodiments, polynucleotides encoding a CD19-targeted chimeric antigen receptor are also provided, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a variable heavy chain or a variable light chain of scFv), a hinge (wherein the hinge is a CD8 alpha hinge), a transmembrane domain (wherein the transmembrane domain comprises a CD8 alpha transmembrane domain), and an intracellular signaling domain (wherein the intracellular signaling domain comprises a CD3 zeta ITAM).

[0033] In some embodiments, the transmembrane domain includes a CD8-alpha transmembrane domain. In some embodiments, the transmembrane domain includes an NKG2D transmembrane domain. In some embodiments, the transmembrane domain includes a CD28 transmembrane domain.

[0034] In some embodiments, the intracellular signaling domain includes or further includes a CD28 signaling domain. In some embodiments, the intracellular signaling domain includes or further includes a 4-1BB signaling domain. In some embodiments, the intracellular signaling domain includes or further includes an OX40 domain. In some embodiments, the intracellular signaling domain includes or further includes a 4-1BB signaling domain. In some embodiments, the intracellular signaling domain includes or further includes a domain selected from ICOS, CD70, CD161, CD40L, CD44 and combinations thereof.

[0035] In some embodiments, the polynucleotide also encodes a cleaved epidermal growth factor receptor (EGFRt). In some embodiments, EGFRt is expressed in cells as a soluble factor. In some embodiments, EGFRt is expressed in a membrane-bound form. In some embodiments, EGFRt is engineered to provide a “suicide switch” function in engineered NK cells. In some embodiments, the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15). Also provided herein are engineered immune cells (e.g., NK cells or T cells or a mixture thereof) expressing a CD19-targeted chimeric antigen receptor encoded by the polynucleotide disclosed herein. Further provided is a method for treating a subject with cancer, comprising administering engineered immune cells expressing the chimeric antigen receptor disclosed herein to the subject with cancer. In some embodiments, the use of the polynucleotide disclosed herein in the treatment of cancer and / or in the manufacture of a cancer treatment pharmaceutical is provided.

[0036] In some embodiments, the anti-CD19 binding moiety includes a heavy-chain variable (VH) domain and a light-chain variable (VL) domain. In some embodiments, the VH domain is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to the VH domain amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the VL domain is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the anti-CD19 binding moiety is derived from the VH and / or VL sequences of SEQ ID NO: 33 or 32. For example, in some embodiments, the VH and VL sequences of SEQ ID NO: 33 and / or 32 have been subjected to a humanization campaign and are therefore more readily expressed and / or less immunogenic when administered to human subjects. Therefore, in some embodiments, the anti-CD19 binding moiety does not include SEQ ID NO: 32 and / or SEQ ID NO: 33. In some embodiments, the anti-CD19 binding moiety includes a CD19-targeting scFv, where the scFv includes a heavy-chain variable region containing the sequence of SEQ ID NO: 35 or a sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 35. In some embodiments, the anti-CD19 binding moiety includes a CD19-targeting scFv and includes a light-chain variable region containing the sequence of SEQ ID NO: 36 or a sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 36. In some embodiments, the anti-CD19 binding moiety includes a light-chain CDR containing first, second, and third complementarity-determining regions (LC CDR1, LC CDR2, and LC CDR3, respectively) and / or a heavy-chain CDR containing first, second, and third complementarity-determining regions (HC CDR1, HC CDR2, and HC CDR3, respectively). In some embodiments, various combinations of LC CDRs and HC CDRs are used. For example, in one embodiment, the anti-CD19 binding moiety includes LC CDR1, LC CDR3, HC CD2, and HC, CDR3. Other combinations are used in certain embodiments.In some embodiments, LC CDR1 includes the sequence of sequence number 37 or a sequence that is at least about 95% homologous to the sequence of sequence number 37. In some embodiments, LC CDR2 includes the sequence of sequence number 38 or a sequence that is at least about 95% (e.g., 96%, 97%, 98%, or 99%) homologous to the sequence of sequence number 38. In some embodiments, LC CDR3 includes the sequence of sequence number 39 or a sequence that is at least about 95% homologous to sequence number 39. In some embodiments, HC CDR1 includes the sequence of sequence number 40 or a sequence that is at least about 95% homologous to sequence number 40. In some embodiments, HC CDR2 includes the sequence of sequence number 41, 42, or 43 or a sequence that is at least about 95% homologous to sequence number 41, 42, or 43. In some embodiments, HC CDR3 includes the sequence of sequence number 44 or a sequence that is at least about 95% (e.g., 96%, 97%, 98%, 99%, or 99%) homologous to the sequence of sequence number 44.

[0037] In some embodiments, an anti-CD19 binding moiety is also provided, comprising a light chain variable region (VL) and a heavy chain variable region (HL), where the VL region comprises first, second, and third complementarity-determining regions (VL CDR1, VL CDR2, and VL CDR3, respectively), and the VH region comprises first, second, and third complementarity-determining regions (VH CDR1, VH CDR2, and VH CDR3, respectively). In some embodiments, the VL region comprises the sequence of SEQ ID NOs. 45, 46, 47, or 48, or a sequence homologous to at least about 95% (e.g., 96%, 97%, 98%, 99%, or 99%) of the sequence of SEQ ID NOs. 49, 50, 51, or 52, or a sequence homologous to at least about 95% (e.g., 96%, 97%, 98%, 99%, or 99%) of the sequence of SEQ ID NOs. 49, 50, 51, or 52.

[0038] In some embodiments, an anti-CD19 binding moiety is also provided, comprising a light chain CDR containing first, second, and third complementarity-determining regions (LC CDR1, LC CDR2, and LC CDR3, respectively). In some embodiments, the anti-CD19 binding moiety further comprises a heavy chain CDR containing first, second, and third complementarity-determining regions (HC CDR1, HC CDR2, and HC CDR3, respectively). In some embodiments, LC CDR1 comprises the sequence of SEQ ID NO: 53 or a sequence at least about 95% homologous to SEQ ID NO: 53. In some embodiments, LC CDR2 comprises the sequence of SEQ ID NO: 54 or a sequence at least about 95% homologous to SEQ ID NO: 54. In some embodiments, LC CDR3 comprises the sequence of SEQ ID NO: 55 or a sequence at least about 95% homologous to SEQ ID NO: 55. In some embodiments, HC CDR1 comprises the sequence of SEQ ID NO: 56 or a sequence at least about 95% homologous to SEQ ID NO: 56. In some embodiments, HC CDR2 comprises the sequence of SEQ ID NO: 57 or a sequence at least about 95% homologous to SEQ ID NO: 57. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 58 or a sequence that is at least about 95% homologous to SEQ ID NO: 58. In some embodiments, the anti-CD19 binding moiety (and thus the resulting CAR) is engineered to not include certain sequences that may increase the risk of side effects, such as immunogenicity and / or cytokine release syndrome. Therefore, according to some embodiments, the anti-CD19 binding moiety does not include one or more of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 32, or 33.

[0039] In some embodiments, the intracellular signaling domain of the chimeric receptor includes an OX40 subdomain. In some embodiments, the intracellular signaling domain further includes a CD3 zeta subdomain. In some embodiments, the OX40 subdomain includes the amino acid sequence of SEQ ID NO: 16 (or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 16), and the CD3 zeta subdomain includes the amino acid sequence of SEQ ID NO: 8 (or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 8).

[0040] In some embodiments, the hinge domain comprises a CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises the amino acid sequence of SEQ ID NO: 2 or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 2.

[0041] In some embodiments, immune cells also express membrane-bound interleukin-15 (mbIL15). In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12 or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 12.

[0042] In some embodiments, the chimeric receptor herein further comprises the extracellular domain of the NKG2D receptor. In some embodiments, immune cells express a second chimeric receptor comprising the extracellular domain, transmembrane domain, cytotoxic signaling complex, and optionally mbIL15 of the NKG2D receptor. In some embodiments, the extracellular domain of the NKG2D receptor comprises a functional fragment of NKG2D comprising the amino acid sequence of SEQ ID NO: 26 or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 26. In various embodiments, immune cells engineered to express the chimeric antigen receptor and / or chimeric receptor disclosed herein are NK cells. In some embodiments, T cells are used. In some embodiments, a combination of NK cells and T cells (and / or other immune cells) is used.

[0043] In some embodiments, a method is provided for treating cancer in a subject, comprising administering the subject engineered to target CD19-targeting immune cells disclosed herein. Also provided herein is the use of the CD19-targeting immune cells disclosed herein for the treatment of cancer. Similarly, the CD19-targeting immune cells disclosed herein are provided for the manufacture of a cancer treatment pharmaceutical. In some embodiments, the cancer to be treated is acute lymphoblastic leukemia.

[0044] Some embodiments of the methods and compositions described herein relate to immune cells. In some embodiments, the immune cells express a CD19-directed chimeric receptor comprising an extracellular anti-CD19 moiety, a hinge and / or a transmembrane domain and / or an intracellular signaling domain. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are T cells.

[0045] In one embodiment, the hinge domain includes a CD8a hinge domain. In another embodiment, the hinge domain includes an Ig4 SH domain.

[0046] In one embodiment, the transmembrane domain includes a CD8a transmembrane domain. In another embodiment, the transmembrane domain includes a CD28 transmembrane domain. In yet another embodiment, the transmembrane domain includes a CD3 transmembrane domain.

[0047] In one embodiment, the signaling domain includes an OX40 signaling domain. In another embodiment, the signaling domain includes a 4-1BB signaling domain. In another embodiment, the signaling domain includes a CD28 signaling domain. In another embodiment, the signaling domain includes an NKp80 signaling domain. In another embodiment, the signaling domain includes a CD16 IC signaling domain. In another embodiment, the signaling domain includes a CD3 zeta or CD3ζ ITAM signaling domain. In another embodiment, the signaling domain includes an mbIL-15 signaling domain. In another embodiment, the signaling domain includes a 2A cleavage domain. In another embodiment, the mIL-15 signaling domain is separated from the rest or other parts of the CD19-targeted chimeric receptor by the 2A cleavage domain.

[0048] One embodiment relates to a method comprising administering the immune cells described herein to a subject requiring treatment. In one embodiment, the subject has cancer. In one embodiment, the administration treats, inhibits, or prevents the progression of the cancer. [Brief explanation of the drawing]

[0049] [Figure 1A] Figure 1A includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor.

[0050] [Figure 1B] Figure 1B includes a diagram of a further non-limiting example of a CD19-targeted chimeric receptor.

[0051] [Figure 2] Figure 2 similarly includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor.

[0052] [Figure 3A] Figure 3A similarly includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor.

[0053] [Figure 3B] Figure 3B similarly includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor.

[0054] [Figure 3C-1] Figure 3C similarly includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor. [Figure 3C-2] Same as above.

[0055] [Figure 3D] Figure 3D includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor that similarly contains a humanized CD19-binding domain.

[0056] [Figure 3E-1] Figure 3E includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor that similarly contains a humanized CD19-binding domain. [Figure 3E-2] Same as above.

[0057] [Figure 3F] Figure 3F includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor that similarly contains a humanized CD19-binding domain.

[0058] [Figure 3G] Figure 3G includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor that similarly contains a humanized CD19-binding domain without a tag sequence.

[0059] [Figure 3H-1] Figure 3H includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor that similarly contains a humanized CD19-binding domain without a tag sequence. [Figure 3H-2] Same as above.

[0060] [Figure 3I] Figure 3I includes a diagram of a non-limiting example of a CD19-targeted chimeric receptor that similarly contains a humanized CD19-binding domain without a tag sequence.

[0061] [Figure 4] Figure 4 outlines the non-restrictive NK cell expansion protocol.

[0062] [Figure 5] Figure 5 shows a schematic of an experimental protocol for evaluating the efficacy of CD19-targeted chimeric antigen receptors according to several embodiments disclosed herein.

[0063] [Figure 6A] Figure 6A shows in vivo data on the antitumor effects of various non-limiting CD19-targeted chimeric receptors.

[0064] [Figure 6B] Figure 6B outlines the data on the antitumor effects of various non-limiting CD19-targeted chimeric receptors.

[0065] [Figure 7] Figure 7 shows data on the expression of various CD19-targeted chimeric receptor constructs by NK cells.

[0066] [Figure 8A] Figure 8A shows biofluorescence data (mean fluorescence intensity, MFI) related to the expression of selected CD19-directional chimeric receptors.

[0067] [Figure 8B] Figure 8B shows data on the expression of selected CD19-targeted chimeric receptors, expressed as the percentage of NK cells expressing the indicated receptor.

[0068] [Figure 9A] Figures 9A-9D describe the cytotoxicity (against Nalm6 cells or Raji cells) of the CD19-targeted chimeric receptor shown. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above.

[0069] [Figure 9E] Figure 9E shows schematic data on cytotoxicity (against Nalm6 cells) for CD19-targeted chimeric receptors with various effector-to-target ratios.

[0070] [Figure 9F] Figure 9F shows schematic data on cytotoxicity (against Raji cells) for CD19-targeted chimeric receptors with various effector-to-target ratios.

[0071] [Figure 10A] Figure 10A shows data regarding the increased cytotoxicity (against Nalm6 cells) of the CD19-targeted chimeric receptor shown 7 days after transduction.

[0072] [Figure 10B] Figure 10B shows data regarding the increased cytotoxicity (against Raji cells) of the CD19-targeted chimeric receptor shown 7 days after transduction.

[0073] [Figure 10C] Figure 10C shows data regarding the increased cytotoxicity (against Nalm6 cells) of the CD19-targeted chimeric receptor shown 14 days after transduction.

[0074] [Figure 10D] Figure 10D shows data regarding the increased cytotoxicity (against Raji cells) of the CD19-targeted chimeric receptor 14 days after transduction.

[0075] [Figure 11-1]Figures 11A-11E show data on cytokine release by NK cells expressing various CD19-targeted chimeric receptors when co-cultured with Nalm6 cells. Figure 11A shows granzyme B release. Figure 11B shows perforin release. Figure 11C shows TNF-alpha release. Figure 11D shows GM-CSF release. Figure 11E shows interferon-gamma release. [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0076] [Figure 12-1] Figures 12A-12E show data on cytokine release by NK cells expressing various CD19-targeted chimeric receptors when co-cultured with Raji cells. Figure 12A shows granzyme B release. Figure 12B shows perforin release. Figure 12C shows TNF-alpha release. Figure 12D shows GM-CSF release. Figure 12E shows interferon-gamma release. [Figure 12-2] Same as above. [Figure 12-3] Same as above.

[0077] [Figure 13-1] Figure 13 shows in vivo contrast-enhanced data on tumor load over time in mice treated with PBS, untransduced NK cells, or NK cells expressing the indicated CD19-targeting chimeric receptor, with the indicated number of engineered NK cells administered (M = 1 million cells). [Figure 13-2] Same as above.

[0078] [Figure 14A] Figure 14A shows the raw data regarding the detected fluorescence signal from the in vivo data shown in Figure 13, with data followed up for 25 days after Nalm6 cell administration.

[0079] [Figure 14B] Figure 14B shows the data from Figure 14A on a logarithmic scale.

[0080] [Figure 14C] Figure 14C shows data on CD3 expression in the cells expressing the construct shown.

[0081] [Figure 14D] Figure 14D shows data on CD56 expression in the cells expressing the indicated construct.

[0082] [Figure 14E] Figure 14E shows data on GFP-expressing tumor cells when the indicated construct was brought into contact with the indicated cells expressing the indicated construct.

[0083] [Figure 14F] Figure 14F shows data on CD19-expressing tumor cells when the indicated construct was brought into contact with the indicated cells expressing the indicated construct.

[0084] [Figure 15] Figure 15 shows data regarding the selected functional characteristics of the chosen humanized anti-CD19 CAR constructs.

[0085] [Figure 16] Figure 16 shows data on the expression of various humanized anti-CD19 CAR constructs disclosed in the embodiments herein.

[0086] [Figure 17A]Figures 17A-17E show cytotoxicity data. 17A shows data on the cytotoxicity of NK cells from a first donor engineered to express the selected humanized CD19 CAR construct disclosed herein, against Nalm6 cells. 17A shows data on the cytotoxicity of NK cells from a first donor engineered to express the selected humanized CD19 CAR construct disclosed herein, against Raji cells. 17C shows data on the cytotoxicity of NK cells from a second donor engineered to express the selected humanized CD19 CAR construct disclosed herein, against Nalm6 cells. 17D shows data on the cytotoxicity of NK cells from a third donor engineered to express the selected humanized CD19 CAR construct disclosed herein, against Raji cells. 17E shows data on the cytotoxicity of NK cells from a third donor engineered to express the selected humanized CD19 CAR construct disclosed herein, against Nalm6 cells. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above.

[0087] [Figure 18] Figure 18 shows summary expression data (from 3 donors) for NK cells expressing the non-limiting anti-CD19 CAR construct 3 days after transduction.

[0088] [Figure 19A]Figures 19A–19D show cytotoxicity reload data. Figure 19A shows data on the cytotoxicity of NK cells from a first donor, engineered to express the selected humanized CD19 CAR construct disclosed herein, to Raji cells added to NK cell cultures on day 7 and further on day 14. Figure 19B shows data on the cytotoxicity of NK cells from a first donor, engineered to express the selected humanized CD19 CAR construct disclosed herein, to Nalm6 cells, to Nalm6 cell NK cell cultures on day 7 and further on day 14. Figure 19C shows data on the cytotoxicity of NK cells from a second donor, engineered to express the selected humanized CD19 CAR construct disclosed herein, to Raji cells, to NK cell cultures on day 7 and further on day 14. Figure 19D shows data related to the cytotoxicity of NK cells from a second donor engineered to express the selected humanized CD19 CAR construct disclosed herein, after Nalm6 cells were added to NK cell culture on day 7 and further on day 14. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above.

[0089] [Figure 20] Figures 20A and 20B show expression data. Figure 20A shows fluorescence data from the expression of a non-limiting, exemplary humanized anti-CD19 CAR construct from two donors 10 days after transduction. Figure 20B shows data on the percentage of cells expressing CD19 (an indicator of the expression efficiency of a particular anti-CD19 CAR).

[0090] [Figure 21A]Figures 21A and 21B show cytotoxicity data. Figure 21A shows data on the cytotoxicity of NK cells (from the first donor of the two donors in Figure 20) that were engineered to express the selected humanized CD19 CAR construct disclosed herein, after being added to NK cell cultures on day 7 and further on day 14. Figure 21B shows data on the cytotoxicity of NK cells (from the second donor of the two donors in Figure 20) that were engineered to express the selected humanized CD19 CAR construct disclosed herein, after being added to NK cell cultures on day 7 and further on day 14. [Figure 21B] Same as above.

[0091] [Figure 22-1] Figures 22A-22E show data on cytokine release by NK cells expressing various CD19-targeted chimeric receptors when co-cultured with Raji cells. Figure 22A shows interferon-gamma release. Figure 22B shows GM-CSF release. Figure 22C shows tumor necrosis factor release. Figure 22D shows perforin release. Figure 22E shows granzyme release. [Figure 22-2] Same as above. [Figure 22-3] Same as above.

[0092] [Figure 23A]Figures 23A-23D show data on NK cell survival. Figure 23A shows the survival analysis of NK cells from a first donor engineered to express the anti-CD19 CAR construct of the non-limiting embodiment shown, at 7, 13, 19, and 26 days after transduction. Figure 23B shows the survival analysis of NK cells from a second donor engineered to express the anti-CD19 CAR construct of the non-limiting embodiment shown, at 7, 13, 19, and 26 days after transduction. Figure 23C shows the survival analysis of NK cells from a third donor engineered to express the anti-CD19 CAR construct of the non-limiting embodiment shown, at 11, 19, and 26 days after transduction. Figure 23D shows the survival analysis of NK cells from a fourth donor engineered to express the anti-CD19 CAR construct of the non-limiting embodiment shown, at 11, 19, and 26 days after transduction. For each experiment, the culture medium was changed twice a week. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above.

[0093] [Figure 24] Figure 24 shows data on the percentage of NK cells expressing the anti-CD19 CAR construct of the non-limiting embodiment shown 11 days after transduction.

[0094] [Figure 25-1]Figures 25A–25I show data on CD19 expression in NK cells transduced with various non-limiting embodiments of the anti-CD19 CAR constructs disclosed herein. Figure 25A shows GFP-transduced NK cells as a control. Figure 25B shows CD19 expression in NK cells transduced with NK19-1. Figure 25C shows CD19 expression in NK cells transduced with NK19H-1. Figure 25D shows CD19 expression in NK cells transduced with NK19H-2. Figure 25E shows CD19 expression in NK cells transduced with NK19H-3. Figure 25F shows CD19 expression in NK cells transduced with NK19H-4. Figure 25G shows CD19 expression in NK cells transduced with NK19H-5. Figure 25H shows CD19 expression in NK cells transduced with NK19H-11. Figure 25I shows CD19 expression in NK cells transduced with NK19H-12. [Figure 25-2] Same as above. [Figure 25-3] Same as above. [Figure 25-4] Same as above. [Figure 25-5] Same as above.

[0095] [Figure 26A]Figures 26A–26D show cytotoxicity data. Figure 26A shows data on the cytotoxicity of NK cells (from the first donor) engineered to express the selected humanized CD19 CAR construct disclosed herein against Raji cells, with Raji cells added to the NK cell culture on day 7 and again on day 14. Figure 26B shows data on the cytotoxicity of NK cells (from the first donor) engineered to express the selected humanized CD19 CAR construct disclosed herein against Nalm6 cells, with Nalm6 cells added to the NK cell culture on day 7 and again on day 14. Figure 26C shows data on the cytotoxicity of NK cells (from the second donor) engineered to express the selected humanized CD19 CAR construct disclosed herein against Raji cells, with Raji cells added to the NK cell culture on day 7 and again on day 14. Figure 26D shows data on the cytotoxicity of NK cells (from a second donor) engineered to express the selected humanized CD19 CAR construct disclosed herein against Nalm6 cells, which were added to NK cell cultures on day 7 and again on day 14. [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 26D] Same as above.

[0096] [Figure 27A-1] Figures 27A and 27B show data on CD19 expression by engineered NK cells. Figure 27A shows flow cytometry data of NK cells from donors engineered to express various non-limiting anti-CD19 CAR constructs disclosed herein. Figure 27B shows corresponding data of NK cells isolated from further donors. [Figure 27A-2] Same as above. [Figure 27A-3] Same as above. [Figure 27B-1] Same as above. [Figure 27B-2] Same as above. [Figure 27B-3] Same as above.

[0097] [Figure 28A]Figures 28A and 28B show cytotoxicity data. Figure 28A shows the number of Raji cells over time (1:1 E:T ratio) when exposed to NK cells expressing the non-limiting, exemplary anti-CD19 CAR construct shown (from the first donor in Figure 27). Figure 28B shows the number of Raji cells over time (1:1 E:T ratio) when exposed to NK cells expressing the non-limiting, exemplary anti-CD19 CAR construct shown (from the second donor in Figure 27). [Figure 28B] Same as above.

[0098] [Figure 29A] Figures 29A-29E show data on the efficacy of various humanized CD19-targeted CAR constructs and their effects in in vivo models. Figure 29A shows a schematic diagram of the experimental protocol for evaluating the efficacy of CD19-targeted CAR constructs in vivo. Figure 29B shows in vivo bioluminescence imaging of mice treated with the indicated construct after being administered NALM6 tumor cells. Figure 29C shows a line graph of bioluminescence data obtained from Figure 29B. Figure 29D shows survival curves indicating the number of days animals survived in each treatment group. Figure 29E shows the relative expression levels of the indicated construct by NK cells, measured by MFI of the tags contained in the CD19 CAR constructs. [Figure 29B-1] Same as above. [Figure 29B-2] Same as above. [Figure 29C] Same as above. [Figure 29D] Same as above. [Figure 29E] Same as above.

[0099] [Figure 30-1]Figures 30A–30F show the expression of the indicated CD19-targeted CAR construct. Figure 30A shows data on the expression of the indicated construct as the percentage of all CD56-positive cells in blood samples from mice 15 days after NK cell administration (e.g., following the schematic in Figure 29A). Figure 30B similarly shows data on the expression of the indicated construct as the percentage of CD56-positive cells expressing the Flag tag (as part of the CD19 CAR construct) 15 days after NK cell administration. Figure 30C similarly shows data on the detection of GFP+ tumor cells in samples from animals treated with the indicated construct 15 days after NK cell administration. Figures 30D, 30E, and 30F show the corresponding data 32 days after NK cell administration. [Figure 30-2] Same as above. [Figure 30-3] Same as above.

[0100] [Figure 31] Figures 31A and 31B show data on the expression of untagged CD19 CAR constructs. As described herein, in some embodiments, the CD19 CAR constructs include a Flag or other tag for detection purposes. However, all constructs disclosed herein with tags are also included in the present invention without tags. Figure 31A shows data on the time-dependent expression of selected unflag-tagged humanized anti-CD19 CAR constructs. Figure 31B shows the corresponding data based on the average detection fluorescence intensity of the indicated constructs.

[0101] [Figure 32-1] Figures 32A-32D show data from in vitro reloading experiments in which NK cells expressing a construct representing the first population of tumor cells were co-cultured with NK cells, and the NK cells were "reloaded" with a further large dose of tumor cells. Figure 32A shows data from day 10 after the start of co-culture with Raji cells. Figure 32B shows data for Raji cells at the final time, day 14. Figure 32C shows data for Nalm6 cells at day 10. Figure 32D shows data for Nalm6 cells at day 14. [Figure 32-2] Same as above.

[0102] [Figure 33-1] Figures 33A-33J relate to the evaluation of cytokine production by NK cells expressing the indicated construct. Figure 33A shows interferon-gamma expression by NK cells expressing the indicated construct after co-culture with Raji cells. Figure 33B shows GM-CSF expression by NK cells expressing the indicated construct after co-culture with Raji cells. Figure 33C shows tumor necrosis factor alpha expression by NK cells expressing the indicated construct after co-culture with Raji cells. Figure 33D shows perforin expression by NK cells expressing the indicated construct after co-culture with Raji cells. Figure 33E shows granzyme B expression by NK cells expressing the indicated construct after co-culture with Raji cells. Figure 33F shows interferon-gamma expression by NK cells expressing the indicated construct after co-culture with Nalm6 cells. Figure 33G shows GM-CSF expression by NK cells expressing the indicated construct after co-culture with Nalm6 cells. Figure 33H shows the expression of tumor necrosis factor alpha by NK cells expressing the indicated construct after co-culture with Nalm6 cells. Figure 33I shows the expression of perforin by NK cells expressing the indicated construct after co-culture with Nalm6 cells. Figure 33J shows the expression of granzyme B by NK cells expressing the indicated construct after co-culture with Nalm6 cells. [Figure 33-2] Same as above. [Figure 33-3] Same as above. [Figure 33-4] Same as above. [Figure 33-5] Same as above. [Figure 33-6] Same as above.

[0103] [Figure 34] Figure 34 shows data on the viability of NK cells expressing a humanized, untagged CD19 CAR construct over four weeks after transduction.

[0104] [Figure 35-1]Figures 35A–35D show data on the efficacy of various humanized, untagged, and CD19-targeted CAR constructs and their effects in in vivo models. Figure 35A shows a schematic diagram of an experimental protocol for evaluating the efficacy of humanized, untagged, and CD19-targeted CAR constructs in vivo. Figure 35B shows in vivo bioluminescence imaging of mice treated with the indicated constructs after being administered NALM6 tumor cells. Figure 35C shows a line graph of bioluminescence data obtained from Figure 35B. Figure 35D shows the relative expression levels of the indicated constructs by NK cells, measured by the detection of CD19-Fc fusion proteins that bind to the CD19 CAR constructs. [Figure 35-2] Same as above. [Figure 35-3] Same as above. [Figure 35-4] Same as above.

[0105] [Figure 36-1] Figures 36A–36F relate to the expression of the indicated CD19-targeted CAR construct. Figure 36A shows data on the expression of the indicated construct as a percentage of all CD56-positive cells in blood samples from mice 13 days after NK cell administration (e.g., following the schematic in Figure 35A). Figure 36B shows data on the detection of CD19+ tumor cells in samples from animals treated with the indicated construct, similarly 13 days after NK cell administration. Figure 36C shows data on the detection of GFP+ tumor cells in samples from animals treated with the indicated construct, similarly 13 days after NK cell administration. Figures 36D, 36E, and 36F show the corresponding data 27 days after NK cell administration. [Figure 36-2] Same as above. [Figure 36-3] Same as above.

[0106] [Figure 37A]Figures 37A–37C relate to the in vivo efficacy of various CD19-directed CARs according to embodiments disclosed herein. Figure 37A shows a schematic diagram of an experimental protocol for evaluating the efficacy of humanized NK cells expressing various CD19-directed CAR constructs in vivo. The various experimental groups tested are as shown. For cells designated “IL12 / IL18”, the cells were enlarged in the presence of soluble IL12 and / or IL18 as described in U.S. Provisional Patent Application 62 / 881311 (filed July 31, 2019) and Application 62 / 932,342 (filed November 7, 2019), which are incorporated herein by reference as a whole. Figures 37B and 37C show bioluminescence data from animals administered Nalm6 tumor cells and treated with the indicated constructs. [Figure 37B-1] Same as above. [Figure 37B-2] Same as above. [Figure 37B-3] Same as above. [Figure 37B-4] Same as above. [Figure 37C-1] Same as above. [Figure 37C-2] Same as above.

[0107] [Figure 38-1]Figures 38A–38J show graphs of bioluminescence data from Figure 37B. Figure 38A shows bioluminescence (as photons / second flux) from animals that received non-transduced NK cells. Figure 38B shows the flux measured in animals that received PBS as the medium. Figure 38C shows the flux measured in animals that received pre-frozen NK cells expressing NK19 NF2 CAR (as a non-limiting example of a CAR). Figure 38D shows the flux measured in animals that received pre-frozen NK cells expressing NK19 NF2 CAR (as a non-limiting example of a CAR), amplified using IL12 and / or IL18. Figures 38E and 38F show the flux measured in animals that received fresh NK cells expressing NK19 NF2 CAR (as a non-limiting example of a CAR). Figures 38G and 38H show the flux measured in animals that received pre-fresh NK cells expressing the NK19 NF2 CAR (as a non-limiting example of a CAR), amplified using IL12 and / or IL18. Figure 38I shows a line graph illustrating bioluminescence measured in various groups over the first 30 days after tumor inoculation. Figure 38J shows a line graph illustrating bioluminescence measured in various groups over the first 56 days after tumor inoculation. [Figure 38-2] Same as above. [Figure 38-3] Same as above. [Figure 38-4] Same as above. [Figure 38-5] Same as above. [Figure 38-6] Same as above.

[0108] [Figure 39] Figure 39 shows data on the time course of body weight in mice that received the treatment described. [Modes for carrying out the invention]

[0109] Detailed description Some embodiments of the methods and compositions provided herein relate to a CD19-targeted chimeric receptor. In some embodiments, the receptor is expressed in cells as described herein. Some embodiments relate to methods of using the composition or cells in immunotherapy.

[0110] The term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including but not limited to reductions in tumor volume, cancer cell count, metastasis, life expectancy, cancer cell proliferation, cancer cell survival, or improvement of various physiological symptoms associated with cancer. The "anti-cancer effect" can also be manifested by the SIR's ability to prevent cancer onset in the first place.

[0111] cell type Some embodiments of the methods and compositions provided herein relate to cells such as immune cells. For example, immune cells can be engineered to include a chimeric receptor, such as a CD19-targeted chimeric receptor, or to include a nucleic acid encoding the chimeric receptor, as described herein.

[0112] Traditional cancer treatments utilize surgical approaches, radiation therapy, chemotherapy, or a combination of these methods. Research has deepened our understanding of some aspects of the mechanisms of certain cancers, and this knowledge has been utilized in targeted cancer therapies. Targeted therapies are cancer treatments that use drugs that target specific genes or proteins found in cancer cells or cells that support cancer growth (such as vascular cells) to reduce or stop cancer cell proliferation. More recently, genetic engineering has enabled the development of approaches that leverage certain aspects of the immune system to fight cancer. In some cases, the patient's own immune cells are modified to specifically eradicate that patient's type of cancer. Various types of immune cells, such as T cells or natural killer (NK) cells, may be used, as will be described in more detail later.

[0113] To facilitate cancer immunotherapy, polynucleotides, polypeptides, and vectors encoding chimeric antigen receptors (CARs) comprising a target-binding moiety (e.g., a ligand expressed by cancer cells or an extracellular binding factor for a CD19-directed chimeric receptor) and a cytotoxic signaling complex are provided herein. For example, one embodiment comprises a polynucleotide, polypeptide, or vector encoding a CD19-directed chimeric receptor that facilitates the targeting of immune cells to cancer and the exertion of cytotoxic effects in cancer cells. Also provided are engineered immune cells (e.g., T cells or NK cells) expressing such CARs. In some embodiments, polynucleotides, polypeptides, and vectors encoding constructs comprising an extracellular domain including two or more subdomains, for example, a first CD19-targeting subdomain containing the CD19-binding moiety disclosed herein and a second subdomain containing a type C lectin-like receptor, and a cytotoxic signaling complex. Also provided are engineered immune cells (e.g., T cells or NK cells) expressing such bispecific constructs. Engineered immune cells (e.g., T cells or NK cells) expressing multispecific constructs and / or having the ability to bind to multiple target markers are also provided. Methods for treating cancer and other uses of such cells for cancer immunotherapy are also provided here.

[0114] Immunotherapy: Manipulated Cells In some embodiments, immune system cells are engineered to enhance their cytotoxic effects against target cells, such as tumor cells. For example, immune system cells may be engineered to contain the CD19-directed chimeric receptor described herein. In some embodiments, leukocytes or white blood cells are used because their innate function is to defend the body from the proliferation of abnormal cells and infectious diseases. There are diverse types of leukocytes that play specific roles in the human immune system and are therefore a preferred starting point for the cell manipulations disclosed herein. Leukocytes include granulocytes and agranulocytes (those with or without granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells such as those described below or elsewhere herein may be engineered to contain a chimeric receptor or nucleic acid encoding a chimeric receptor, such as the CD19-directed chimeric receptor, and / or to co-express a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0115] Monocytes for immunotherapy Monocytes are a subtype of leukocyte. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are involved in the adaptive immune system and play major roles in phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of taking up cellular material or whole cells, followed by the digestion and destruction of phagocytic cellular material. In some embodiments, monocytes are used in conjunction with one or more further engineered cells disclosed herein. Some embodiments of the methods and compositions described herein relate to monocytes containing a CD19-directed chimeric receptor or nucleic acid encoding a CD19-directed chimeric receptor. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express a CD19-directed chimeric receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0116] Lymphocytes for immunotherapy Lymphocytes, another major subtype of leukocytes, include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity). B cells are manipulated by several embodiments disclosed herein, while several embodiments also relate to manipulated T cells or manipulated NK cells (a mixture of T cells and NK cells is used in some embodiments). Some embodiments of the methods and compositions described herein relate to lymphocytes containing a CD19-directed chimeric receptor or nucleic acid encoding a CD19-directed chimeric receptor. Several embodiments of the methods and compositions disclosed herein relate to lymphocytes manipulated to express a CD19-directed chimeric receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0117] T cells for immunotherapy T cells are distinguishable from other lymphocyte subtypes (e.g., B cells or NK cells) based on the presence of T cell receptors on their cell surface. T cells can be divided into various distinct subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosa-associated invariant T cells, and gamma delta T cells. In some embodiments, a specific subtype of T cells is manipulated. In some embodiments, a mixed pool of T cell subtypes is manipulated. In some embodiments, the type of T cells manipulated to express the cytotoxic receptor complex disclosed herein is not particularly selected. In some embodiments, expansion / harvesting of T cells with a specific marker profile is performed using certain techniques, such as the use of cytokine stimulation. For example, in some embodiments, activation of certain human T cells, e.g., CD4+ T cells, CD8+ T cells, is achieved by the use of CD3 and / or CD28 as stimulating molecules. In some embodiments, a method is provided for treating or preventing cancer or infectious disease, comprising administering a therapeutically effective amount of T cells expressing the cytotoxic receptor complex and / or homing moiety described herein. In some embodiments, the engineered T cells are autologous cells, while in some embodiments, the T cells are allogeneic cells. Some embodiments of the methods and compositions described herein relate to T cells comprising a CD19-directed chimeric receptor or a nucleic acid encoding a CD19-directed chimeric receptor. Some embodiments of the methods and compositions disclosed herein relate to T cells engineered to express a CD19-directed chimeric receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0118] NK cells for immunotherapy In some embodiments, methods are provided for treating or preventing cancer or infectious diseases, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing the cytotoxic receptor complex and / or homing moiety described herein. In some embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In some embodiments, NK cells are preferred because of their relatively high potential innate cytotoxicity. In some embodiments, the engineered cells disclosed herein are unexpectedly beneficial in that they allow for further upregulation of the cytotoxic activity of NK cells and make their activity against target cells (e.g., tumors or other diseased cells) more effective. Some embodiments of the methods and compositions disclosed herein relate to NKs comprising a CD19-directed chimeric receptor or a nucleic acid encoding a CD19-directed chimeric receptor. Some embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express a CD19-directed chimeric receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0119] Hematopoietic stem cells for cancer immunotherapy In some embodiments, hematopoietic stem cells (HSCs) are used in the immunotherapy methods disclosed herein. In some embodiments, the cells are engineered to express a homing moiety and / or cytotoxic receptor complex. In some embodiments, HSCs are used to leverage their engraftment capacity for long-term hematopoietic cell production, providing a sustained source of targeted anti-cancer effector cells to fight cancer, for example, to induce remission. In some embodiments, this ongoing production helps to counteract the anergy or exhaustion of other cell types by the tumor microenvironment, for example. In some embodiments, allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In some embodiments, HSCs are used in combination with one or more further engineered cell types disclosed herein. Some embodiments of the methods and compositions described herein relate to stem cells, such as hematopoietic stem cells, comprising a CD19-directed chimeric receptor or a nucleic acid encoding a CD19-directed chimeric receptor. Some embodiments of the methods and compositions disclosed herein relate to stem cells, such as hematopoietic stem cells, engineered to express a CD19-directed chimeric receptor and a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0120] Extracellular domain (tumor binding factor) Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as CD19-targeted chimeric receptors, which include an extracellular domain. In some embodiments, the extracellular domain includes a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain) as described herein. In some embodiments, the antigen-binding domain is derived from or includes a wild-type or non-wild-type sequence of an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single-domain antibody (SDAB), vH or vL domain, camel VHH domain or non-immunoglobulin scaffold such as DARPIN, afibody, affin, adnectin, afitin, repebody, finomer, alphabody, avimer, atrimer, centinrin, pronectin, antikalin, Knitz domain, armadillo repeat protein, autoantigen, receptor or ligand. In some embodiments, the tumor-binding domain includes one or more antigen-binding domains. In one embodiment, the antigen-binding domain is operably bound to the NH2 terminus of a TCR domain (e.g., the constant chain of TCR-alpha, TCR-beta I, TCR-beta II, pre-TCR-alpha, pre-TCR-alpha-Del48, TCR-gamma, or TCR-delta) either directly or via a linker as desired.

[0121] Antigen-binding protein In some embodiments, antigen-binding proteins are provided. The term “antigen-binding protein” as used herein should be given its usual meaning and should also include a protein comprising an antigen-binding fragment that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding fragment to assume a conformation that facilitates the binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen (e.g., CD19) or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises a total of three CDRs from the heavy chain of the antibody that binds to the antigen or from the light chain of the antibody that binds to the antigen. In further embodiments, the antigen-binding fragment comprises a total of six CDRs from the antibody that binds to the antigen (three from the heavy chain and three from the light chain). In some embodiments, the antigen-binding fragment comprises one, two, three, four, five, or six CDRs from the antibody that binds to the antigen, and in some embodiments, the CDRs can be any combination of heavy chain and / or light chain CDRs. In some embodiments, the antigen-binding fragment is an antibody fragment.

[0122] Non-exclusive examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, single-strand variable fragments (scFv), nanobodies (e.g., the VH domain of camel heavy-chain antibodies; VHH fragments), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments. These molecules may originate from any mammalian, such as humans, mice, rats, rabbits or pigs, dogs or camels. Antibody fragments can compete with intact (e.g., native) antibodies for binding to target antigens, and fragments can be produced by modification of intact antibodies (e.g., enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technology or peptide synthesis. Antigen-binding proteins may include, for example, alternative protein scaffolds or artificial scaffolds onto which CDRs or CDR derivatives are implanted. Such scaffolds include, but are not limited to, antibody-derived scaffolds, which include mutations introduced to stabilize the three-dimensional structure of antigen-binding proteins, and fully synthetic scaffolds, which include, for example, biocompatible polymers. Furthermore, peptide antibody mimes ("PAMs") and antibody mimeograph-based scaffolds that utilize fibronectin as a scaffold may be used.

[0123] In one embodiment, the antigen-binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, the antigen-binding protein may include, but is not limited to, bispecific antibodies; intrabodies; domain antibodies (a single VL or VH domain or two or more VH domains linked by a peptide linker); maxibodies (two scFvs fused to an Fc region); triabodies; tetrabodies; minibodies (scFv fused to a CH3 domain); peptidebodies (one or more peptides bound to an Fc region); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form an antigen-binding domain pair); small module immunotherapies; and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0124] In one embodiment, the antigen-binding protein has the structure of an immunoglobulin. The term “immunoglobulin” as used herein should be given its usual meaning, and should also include a tetrameric molecule in which each tetramer contains two identical pairs of polypeptide chains, each pair having one “light” chain (approximately 25 kDa) and one “heavy” chain (approximately 50-70 kDa). The amino-terminus of each chain consists of a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminus of each chain defines a constant region, primarily responsible for effector function.

[0125] Within the light and heavy chains, the variable region (V) and constant region (C) are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids. The variable region of each light / heavy chain pair forms antibody binding sites, so that the intact immunoglobulin has two binding sites.

[0126] Immunoglobulin chains exhibit a relatively conserved framework region (FR) of the same general structure, linked by three hypervariable regions also called complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0127] Human light chains are classified as kappa and lambda light chains. An antibody "light chain" refers to the smaller of two polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa (K) and lambda (λ) light chains are the two main antibody light chain isotypes. Light chains may also include polypeptides containing a monoimmunoglobulin light chain variable region (VL) and a monoimmunoglobulin light chain constant domain (CL) in the direction from the amino terminus to the carboxyl terminus.

[0128] Heavy chains are classified into mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. The antibody "heavy chain" refers to the larger of the two polypeptide chains present in the antibody molecule in a naturally occurring conformation and usually determines the class to which the antibody belongs. The heavy chain may contain polypeptides comprising, from the amino terminus to the carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4).

[0129] The IgG class is further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4. The IgA class is divided into subclasses, namely IgA1 and IgA2. IgM has subclasses, including but not limited to IgM1 and IgM2. The heavy chains of IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The constant domains of immunoglobulin heavy chains include subtypes and may be derived from any immunoglobulin isotype. Antibody chains are linked by polypeptide disulfide bonds between the CL domain and the CH1 domain (e.g., between the light chain and the heavy chain) and between the hinge regions of the antibody heavy chain.

[0130] In one embodiment, the antigen-binding protein is an antibody. The term “antibody” as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be monoclonal or polyclonal, multi-chain or single-chain, or intact immunoglobulins, and may be of natural or recombinant origin. Antibodies may be tetramers of immunoglobulin molecules. Antibodies may be “humanized,” “chimeric,” or non-human. Antibodies may include any isotype of intact immunoglobulin, for example, chimeric, humanized, human, and bispecific antibodies. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains. Antibody sequences may originate from only one species, or they may be “chimeric,” i.e., different parts of the antibody may originate from two different species, as described further below. Unless otherwise specified, the term “antibody” also includes antibodies containing two substantially full-length heavy chains and two substantially full-length light chains, insofar as the antibody retains the same or similar binding and / or function as an antibody consisting of two full-length light and / or heavy chains. For example, an antibody having 1, 2, 3, 4, or 5 amino acid residue substitutions, insertions, or deletions at the N-terminus and / or C-terminus of the heavy and / or light chain is included in this definition, insofar as the antibody retains the same or similar binding and / or function as an antibody containing two full-length heavy chains and two full-length light chains. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies. In some embodiments, monoclonal antibodies and polyclonal antibodies are provided. The term “polyclonal antibody” as used herein should be given its usual meaning and should also include a population of antibodies that generally exhibit a wide range of variability in composition and binding specificity. The term “monoclonal antibody” (“mAb”) used herein should be given its usual meaning and should also refer to a group of one or more antibodies having the same sequence. Monoclonal antibodies bind to an antigen at a specific epitope of the antigen.

[0131] In one embodiment, the antigen-binding protein is an antibody fragment or antigen-binding fragment. The term “antibody fragment” refers to at least one portion of an antibody that retains the ability to specifically interact with an antigen epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of antibody fragments include, but are not limited to, multispecific antibodies formed from antibody fragments such as Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, disulfide-linked Fvs(sdFv), Fd fragment consisting of VH and CHI domains, linear antibodies, single-domain antibodies (either vL or vH) such as sdAb, camel vHH domain, and bivalent fragments containing two Fab fragments linked by disulfide crosslinking at a hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, bispecific antibodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be transplanted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent 6,703,199 describing fibronectin polypeptide minibodies). Antibody fragments may include Fab, Fab', F(ab')2, and / or Fv fragments containing at least one CDR of immunoglobulin sufficient to give specific antigen binding to a cancer antigen (e.g., CD19). Antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.

[0132] In one embodiment, Fab fragments are provided. A Fab fragment is a monovalent fragment having VL, VH, CL, and CH1 domains; an F(ab')2 fragment is a bivalent fragment having two Fab fragments linked by disulfide crosslinking at a hinge region; an Fd fragment has VH and CH1 domains; an Fv fragment has VL and VH domains of a single arm of the antibody; and a dAb fragment has an antigen-binding fragment with a VH domain, a VL domain, or a VH or VL domain. In one embodiment, these antibody fragments can be incorporated into single-domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, bispecific antibodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. In one embodiment, the antibody comprises at least one CDR described herein.

[0133] In some embodiments, single-stranded variable fragments are also provided herein. The term “single-stranded variable fragment” (“scFv”) as used herein should be given its usual meaning, and should also include a fusion protein in which VL and VH regions are linked by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, wherein the linker is long enough for the protein chain to fold back into itself to form a monovalent antigen-binding site. For clarity, unless otherwise specified, “single-stranded variable fragment” is not an antibody or antibody fragment as defined herein. A bispecific antibody is a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains linked by a linker configured to reduce or prevent pairing between two domains of the same chain, and thus cause each domain to pair with a complementary domain of the other polypeptide chain. According to some embodiments, if the two polypeptide chains of a bispecific antibody are identical, then the bispecific antibody derived from that pairing has two identical antigen-binding sites. Polypeptide chains having different sequences can be used to produce bispecific antibodies having two different antigen-binding sites. Similarly, triabodies and tetrabodies are antibodies comprising three and four polypeptide chains, which may be identical or different, forming three and four antigen-binding sites, respectively.

[0134] In some embodiments, antigen-binding proteins contain one or more CDRs. The term “CDR” as used herein should be given its usual meaning and should also include complementarity-determining regions (also referred to as “minimum recognition units” or “hypervariable regions”) within antibody variable sequences. CDRs enable antigen-binding proteins to specifically bind to a particular antigen of interest. There are three heavy-chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light-chain variable region CDRs (CDRL1, CDRL2, and CDRL3). Each of the two chains' CDRs is generally aligned by a framework region to form a structure that specifically binds to a particular epitope or domain of the target protein. From the N-terminus to the C-terminus, both naturally occurring light-chain and heavy-chain variable regions generally correspond to the following order of elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids occupying the positions of each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity-determining region (CDR) and framework region (FR) of a given antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). One or more CDRs can be incorporated into a molecule by covalent or non-covalent bonds to produce an antigen-binding protein.In some embodiments, the antigen-binding protein provided herein includes a heavy chain variable region selected from SEQ ID NOs: 104 and 106. In some embodiments, the antigen-binding protein provided herein includes a light chain variable region selected from SEQ ID NOs: 105 and 107.

[0135] In some embodiments, the antigen-binding protein is modified from its original sequence for purposes such as expression, functional enhancement, or reducing the host's immune response to the antigen-binding protein. In some embodiments, the antigen-binding protein includes a light chain variable region selected from SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO: 119 and / or a sequence having at least 90% identity and / or homology (e.g., 90-95%, 95%, 96%, 97%, 98%, 99%). In some embodiments, the light chain variable region differs from the sequence of SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119 by more than 5% (e.g., 5-7%, 5-10%, 10-20%, or more) in a ligand-binding function (or other functionality) similar to, substantially similar to, or identical to, SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 119. In some embodiments, the antigen-binding protein includes a heavy chain variable region selected from SEQ ID NOs: 120, 121, 122, and 123, and / or a sequence having at least 90% identity and / or homology (e.g., 90-95%, 95%, 96%, 97%, 98%, 99%). In some embodiments, the heavy chain variable region differs from the sequence of SEQ ID NOs: 120, 121, 122, or 123 by more than 5% (e.g., 5-7%, 5-10%, 10-20%, or more) in a ligand-binding function (or other functionality) similar to, substantially similar to, or identical to, SEQ ID NOs: 120, 121, 122, or 123. By some embodiment, any combination of heavy chain and light chain regions may be used (e.g., in a set of scFv). In some embodiments, the antigen-binding protein comprises one or more CDRs selected from SEQ ID NOs: 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 134, 136, 137, 138, 139, 140, 141, 142, 143, and 144.

[0136] In a further embodiment, the CDR is selected in any combination from SEQ ID NOs: 108, 109, 110, 111, 112, 113, 114, and 115. In one embodiment, the CDR is rotated to point towards CD19 and produce a CAR containing SEQ ID NO: 116.

[0137] In some embodiments, the antigen-binding protein comprises a heavy chain having the sequence of SEQ ID NO: 88. In some embodiments, the heavy chain binds (for example, as scFv) to one of the light chains of SEQ ID NO: 89, SEQ ID NO: 90 and / or SEQ ID NO: 91. In some embodiments, the antigen-binding protein comprises one or more CDRs selected from SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100.

[0138] In some embodiments, the antigen-binding protein includes the light chain region of an FMC63 antibody having the sequence of SEQ ID NO: 150. In some embodiments, the antigen-binding protein includes the light chain region of an FMC63 antibody having the sequence of SEQ ID NO: 148. In some embodiments, a linker is used between the heavy and light chains, and in some embodiments, the linker includes the sequence of SEQ ID NO: 149. In some embodiments, such heavy and light chains are used in combination with a CD28 costimulatory domain, such as that of SEQ ID NO: 153. Often, a spacer is used as another component of the CAR. For example, in some embodiments, a spacer including the sequence of SEQ ID NO: 151 is used. In some embodiments, a transmembrane domain having the sequence of SEQ ID NO: 152 is used. In some embodiments, the CAR includes a nucleic acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 147.

[0139] In one embodiment, the antigen-binding protein provided herein comprises one or more CDRs as part of a large polypeptide chain. In one embodiment, the antigen-binding protein co-possesses one or more CDRs to another polypeptide chain. In one embodiment, the antigen-binding protein incorporates one or more CDRs non-covalently. In one embodiment, the antigen-binding protein may comprise at least one of the CDRs described herein, incorporated into a biocompatible framework structure. In one embodiment, the biocompatible framework structure comprises a polypeptide or a portion thereof sufficient to form a conformationally stable structural support or framework or scaffold, which can present a sequence of one or more amino acids that bind to an antigen (e.g., a CDR, a variable region, etc.) in a localized surface region. Such a structure may be a naturally occurring polypeptide or polypeptide "folded structure" (structural motif) or may have one or more modifications to a naturally occurring polypeptide or folded structure, such as the addition, deletion, and / or substitution of amino acids. In one embodiment, the scaffold may be derived from polypeptides of a diverse range of different species (or more than one species), such as humans, non-human primates or other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0140] In some embodiments, the biocompatible framework structure is based on a protein scaffold or backbone other than immunoglobulin domains. In some such embodiments, these framework structures are based on fibronectin, ankyrin, lipocalin, neocardinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and / or tendamistat domain.

[0141] In one embodiment, an antigen-binding protein having more than one binding site is provided. In some embodiments, the binding sites are identical to each other, while in other embodiments, the binding sites are different to each other. For example, an antibody generally has two identical binding sites, but a “bispecific” or “bifunctional” antibody has two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes that may be present on the same or different protein targets. This is particularly advantageous in some embodiments because a bispecific chimeric antigen receptor may confer the ability of engineered cells to target multiple tumor markers, such as CD19 and further tumor markers, e.g., CD123, NKG2D, or any other marker disclosed herein or recognized in the art as a tumor-specific or tumor-associated antigen.

[0142] The term “chimeric antibody” as used herein should be given its usual meaning and should also include antibodies that include one or more regions from one antibody and one or more regions from one or more other antibodies. In some embodiments, one or more CDRs are derived from an anti-cancer antigen (e.g., CD19) antibody. In some embodiments, all CDRs are derived from an anti-cancer antigen antibody (e.g., anti-CD19 antibody). In some embodiments, CDRs from more than one anti-cancer antigen antibody are mixed and fitted into a chimeric antibody. For example, a chimeric antibody may include CDR1 from the light chain of a first anti-cancer antigen antibody, CDR2 and CDR3 from the light chain of a second anti-cancer antigen antibody, and a heavy chain CDR from a third anti-cancer antigen antibody. Furthermore, the framework regions of the antigen-binding proteins disclosed herein may be derived from the same anti-cancer antigen (e.g., CD19) antibody, one or more different antibodies, e.g., a human antibody or a humanized antibody. In an example of a chimeric antibody, the heavy chain and / or light chain portions are identical, homologous, or derived from an antibody from a particular species, or belong to a particular antibody class or subclass, while the rest of the chain is identical, homologous, or derived from one or more antibodies from other species, or belongs to another antibody class or subclass. Provided herein are fragments of such antibodies exhibiting desired biological activity.

[0143] In one embodiment, an antigen-binding protein is provided, comprising a heavy chain variable domain having at least 90% identity to the VH domain amino acid sequence shown in SEQ ID NO: 33. In another embodiment, the antigen-binding protein comprises a heavy chain variable domain having at least 95% identity to the VH domain amino acid sequence shown in SEQ ID NO: 33. In yet another embodiment, the antigen-binding protein comprises a heavy chain variable domain having at least 96%, 97%, 98%, or 99% identity to the VH domain amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the heavy chain variable domain may have one or more further mutations in the VH domain amino acid sequence shown in SEQ ID NO: 33 (e.g., for humanization purposes), but retain specific binding to cancer antigens (e.g., CD19). In some embodiments, the heavy chain variable domain may have one or more further mutations in the VH domain amino acid sequence shown in SEQ ID NO: 33, but improve specific binding to cancer antigens (e.g., CD19).

[0144] In some embodiments, the antigen-binding protein includes a light chain variable domain having at least 90% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the antigen-binding protein includes a light chain variable domain having at least 95% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the antigen-binding protein includes a light chain variable domain having at least 96%, 97%, 98%, or 99% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the light chain variable domain may have one or more further mutations in the VL domain amino acid sequence shown in SEQ ID NO: 32 (e.g., for humanization purposes), but retain specific binding to cancer antigens (e.g., CD19). In some embodiments, the light chain variable domain may have one or more further mutations in the VL domain amino acid sequence shown in SEQ ID NO: 32, but improve specific binding to cancer antigens (e.g., CD19).

[0145] In one embodiment, the antigen-binding protein includes a heavy chain variable domain having at least 90% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33 and a light chain variable domain having at least 90% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In another embodiment, the antigen-binding protein includes a heavy chain variable domain having at least 95% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33 and a light chain variable domain having at least 95% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In yet another embodiment, the antigen-binding protein includes a heavy chain variable domain having at least 96%, 97%, 98%, or 99% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33 and a light chain variable domain having at least 96%, 97%, 98%, or 99% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32.

[0146] In one embodiment, the antigen-binding protein includes a heavy chain variable domain having the VH domain amino acid sequence shown in SEQ ID NO: 33 and a light chain variable domain having the VL domain amino acid sequence shown in SEQ ID NO: 32. In one embodiment, the light chain variable domain includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain variable domain sequence of SEQ ID NO: 32. In one embodiment, the light chain variable domain includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain variable domain sequence matching SEQ ID NO: 33.

[0147] In one embodiment, the light chain variable domain includes an amino acid sequence encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 32. In one embodiment, the light chain variable domain includes an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions with a complement of the polynucleotide encoding a light chain variable domain matching the sequence of SEQ ID NO: 32. In one embodiment, the light chain variable domain includes an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a complement of the polynucleotide encoding a light chain variable domain matching the sequence of SEQ ID NO: 32.

[0148] In one embodiment, the heavy chain variable domain includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain variable domain sequence matching the sequence of SEQ ID NO: 33. In one embodiment, the heavy chain variable domain includes an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions with a polynucleotide complement to the polynucleotide encoding the heavy chain variable domain matching the sequence of SEQ ID NO: 33. In one embodiment, the heavy chain variable domain includes an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide complement to the polynucleotide encoding the heavy chain variable domain matching the sequence of SEQ ID NO: 33.

[0149] In some embodiments, further anti-CD19 binding constructs are provided. For example, in some embodiments, a CD19-targeting scFv is provided, comprising a heavy chain variable region containing the sequence of SEQ ID NO: 35. In some embodiments, the antigen-binding protein comprises a heavy chain variable domain having at least 95% identity with the HCV domain amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the antigen-binding protein comprises a heavy chain variable domain having at least 96%, 97%, 98%, or 99% identity with the HCV domain amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the heavy chain variable domain may have one or more further mutations in the HCV domain amino acid sequence shown in SEQ ID NO: 35 (e.g., for humanization purposes), but retain specific binding to cancer antigens (e.g., CD19). In some embodiments, the heavy chain variable domain may have one or more further mutations in the HCV domain amino acid sequence shown in SEQ ID NO: 35, but improve specific binding to cancer antigens (e.g., CD19).

[0150] Furthermore, in some embodiments, the CD19-targeting scFv includes a light chain variable region containing the sequence of SEQ ID NO: 36. In some embodiments, the antigen-binding protein includes a light chain variable domain having at least 95% identity to the LCV domain amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the antigen-binding protein includes a light chain variable domain having at least 96%, 97%, 98%, or 99% identity to the LCV domain amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the light chain variable domain may have one or more further mutations in the LCV domain amino acid sequence shown in SEQ ID NO: 36 (e.g., for humanization purposes), but retain specific binding to cancer antigens (e.g., CD19). In some embodiments, the light chain variable domain may have one or more further mutations in the LCV domain amino acid sequence shown in SEQ ID NO: 36, but improve specific binding to cancer antigens (e.g., CD19).

[0151] In some embodiments, an anti-CD19 binding moiety is also provided, comprising a light chain CDR containing first, second, and third complementarity-determining regions (LC CDR1, LC CDR2, and LC CDR3, respectively). In some embodiments, the anti-CD19 binding moiety further comprises a heavy chain CDR containing first, second, and third complementarity-determining regions (HC CDR1, HC CDR2, and HC CDR3, respectively). In some embodiments, LC CDR1 comprises the sequence of SEQ ID NO: 37. In some embodiments, LC CDR1 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% homology with the sequence of SEQ ID NO: 37. In some embodiments, LC CDR2 comprises the sequence of SEQ ID NO: 38. In some embodiments, LC CDR2 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% homology with the sequence of SEQ ID NO: 38. In some embodiments, LC CDR3 comprises the sequence of SEQ ID NO: 39. In some embodiments, LC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% homology to the sequence of SEQ ID NO: 39. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 40. In some embodiments, HC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% homology to the sequence of SEQ ID NO: 40. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 37. In some embodiments, HC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% homology to the sequence of SEQ ID NO: 41, 42, or 43. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 44. In some embodiments, HC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% homology to the sequence of SEQ ID NO: 44.

[0152] In some embodiments, an anti-CD19 binding moiety is also provided that includes a light chain variable region (VL) and a heavy chain variable region (HL), where the VL region includes first, second, and third complementarity-determining regions (VL CDR1, VL CDR2, and VL CDR3, respectively), and the VH region includes first, second, and third complementarity-determining regions (VH CDR1, VH CDR2, and VH CDR3, respectively). In some embodiments, the VL region includes the sequence of SEQ ID NOs. 45, 46, 47, or 48. In some embodiments, the VL region includes an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homology to the sequence of SEQ ID NOs. 45, 46, 47, or 48. In some embodiments, the VH region includes the sequence of SEQ ID NOs. 49, 50, 51, or 52. In some embodiments, the VH region includes an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homology to the sequence of SEQ ID NOs. 49, 50, 51, or 52.

[0153] In some embodiments, an anti-CD19 binding moiety is also provided, comprising a light chain CDR including first, second, and third complementarity-determining regions (LC CDR1, LC CDR2, and LC CDR33, respectively). In some embodiments, the anti-CD19 binding moiety further comprises a heavy chain CDR including first, second, and third complementarity-determining regions (HC CDR1, HC CDR2, and HC CDR33, respectively). In some embodiments, LC CDR1 comprises the sequence of SEQ ID NO: 53. In some embodiments, LC CDR1 comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homologous to the sequence of SEQ ID NO: 53. In some embodiments, LC CDR2 comprises the sequence of SEQ ID NO: 54. In some embodiments, LC CDR2 comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homologous to the sequence of SEQ ID NO: 54. In some embodiments, LC CDR3 comprises the sequence of SEQ ID NO: 55. In some embodiments, LC CDR3 includes an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homologous to the sequence of SEQ ID NO: 55. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 56. In some embodiments, HC CDR1 includes an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homologous to the sequence of SEQ ID NO: 56. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 57. In some embodiments, HC CDR2 includes an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homologous to the sequence of SEQ ID NO: 57. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 58. In some embodiments, HC CDR3 includes an amino acid sequence that is at least about 85%, about 90%, about 95%, or about 98% homologous to the sequence of SEQ ID NO: 58.

[0154] Further anti-CD19 binding portions are known in the art, for example, as described in U.S. Patent 8,399,645, U.S. Patent Publication 2018 / 0153977, U.S. Patent Publication 2014 / 0271635, U.S. Patent Publication 2018 / 0251514 and U.S. Patent Publication 2018 / 0312588 (each of which is incorporated herein by reference as a whole).

[0155] Natural killer group domains that bind tumor ligands In some embodiments, engineered immune cells, such as NK cells, are utilized for their ability to recognize and destroy tumor cells. For example, engineered NK cells may contain a CD19-targeting chimeric receptor or a nucleic acid encoding the chimeric receptor. NK cells express both inhibitory and activating receptors on their cell surface. The inhibitory receptor binds to self molecules expressed on the surface of healthy cells (thus preventing an immune response against "self" cells), while the activating receptor binds to ligands expressed on abnormal cells, such as tumor cells. When the balance of activation between the inhibitory and activating receptors shifts towards the activating receptor, NK cell activation occurs, and target (e.g., tumor) cells are lysed.

[0156] Natural killer group 2 members (NKG2D) are NK cell activating receptors that recognize a variety of ligands expressed on cells. Surface expression of various NKG2D ligands is generally low in healthy cells but is upregulated by malignant transformation, for example. Non-exclusive examples of ligands recognized by NKG2D include, but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, as well as other molecules expressed on target cells that control the cytolytic or cytotoxic functions of NK cells. In some embodiments, T cells are engineered to express an extracellular domain for binding to one or more tumor ligands and activating the T cell. For example, in some embodiments, T cells are engineered to express the NKG2D receptor as a binding factor / activating moiety. In some embodiments, the engineered cells disclosed herein are engineered to express other members of the NKG2 family, e.g., NKG2A, NKG2C, and / or NKG2E. Such receptor combinations are engineered in some embodiments. Furthermore, in some embodiments, other receptors, such as killer cell immunoglobulin-like receptors (KIRs), are expressed.

[0157] In some embodiments, cells are engineered to express a cytotoxic receptor complex containing full-length NKG2D as an extracellular component to recognize ligands on the surface of tumor cells (e.g., hepatocytes). In some embodiments, full-length NKG2D has the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, full-length NKG2D or its functional fragment is human NKG2D.

[0158] In some embodiments, cells are engineered to express a cytotoxic receptor complex containing a functional fragment of NKG2D as an extracellular component for recognizing ligands on the surface of tumor cells or other affected cells. In some embodiments, the functional fragment of NKG2D has the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the NKG2D fragment is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to full-length wild-type NKG2D. In some embodiments, the fragment may have one or more further mutations from SEQ ID NO: 25, but the ligand-binding function is retained or, in some embodiments, enhanced. In some embodiments, the functional fragment of NKG2D contains the amino acid sequence of SEQ ID NO: 26. In some embodiments, the NKG2D fragment is provided as a dimer, trimer, or other concatemer form, and such embodiments result in enhanced ligand-binding activity. In some embodiments, the sequence encoding the NKG2D fragment is, if desired, fully or partially codon-optimized. In some embodiments, the sequence encoding the codon-optimized NKG2D fragment contains the sequence of SEQ ID NO: 28. Advantageously, according to some embodiments, the functional fragment lacks its native transmembrane or intracellular domain but retains the ability to bind to the NKG2D ligand and transmit activation signals by ligand binding. A further advantage of such fragments is that DAP10 expression is not required to localize NKG2D to the cell membrane. Therefore, in some embodiments, the cytotoxic receptor complex encoded by the polypeptide disclosed herein does not contain DAP10. In some embodiments, immune cells such as NK cells or T cells are engineered to express one or more chimeric receptors targeting CD19 and NGG2D ligands. In some embodiments, such cells also co-express mbIL15.

[0159] In some embodiments, the cytotoxic receptor complex is designed for dimerization. Dimerization may, by some embodiment, involve homodimers or heterodimers. In some embodiments, dimerization improves ligand recognition by the cytotoxic receptor complex (and therefore NK cells expressing the receptor), resulting in a reduction (or absence) of harmful toxic effects. In some embodiments, the cytotoxic receptor complex utilizes an internal dimer or a repeat of one or more constituent subunits. For example, in some embodiments, the cytotoxic receptor complex may optionally include a second NKG2D extracellular domain and a first NKG2D extracellular domain bound to a transmembrane / signaling domain (or another transmembrane domain with another signaling domain).

[0160] In some embodiments, various domains / subdomains are separated by the use of a linker such as a GS3 linker (nucleotide and protein of SEQ ID NOs. 15 and 16, respectively) (or a GSn linker). Other linkers used in the various embodiments disclosed herein include, but are not limited to, those encoded by SEQ ID NOs. 17, 19, 21, or 23. This provides the possibility of separating various components of the receptor complex along polynucleotides, which may improve the expression, stability, and / or functionality of the receptor complex.

[0161] Cytotoxic signaling complex Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as CD19-directed chimeric receptors, comprising a cytotoxic signaling complex. As described herein, according to some embodiments, the cytotoxic receptor complex provided comprises one or more transmembrane and / or intracellular domains that initiate a cytotoxic signaling cascade by binding of the extracellular domain to a ligand on the surface of a target cell. Some embodiments disclosed herein relate to chimeric antigen receptor constructs in which a tumor-targeting domain (or CD19-directed domain) is bound to a cytotoxic signaling complex.

[0162] In some embodiments, the cytotoxic signaling complex comprises at least one transmembrane domain, at least one costimulatory domain, and / or at least one signaling domain. In some embodiments, a domain may have more than one component; for example, a costimulatory domain may include two subdomains. Furthermore, in some embodiments, a domain may function in multiple ways; for example, a transmembrane domain may also function to provide a signaling function.

[0163] transmembrane domain Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as CD19-directed chimeric receptors, which include a transmembrane domain. Some embodiments include a transmembrane domain from NKG2D or other transmembrane proteins. In some embodiments in which a transmembrane domain is used, some of the transmembrane proteins used retain at least some of their normal transmembrane domains.

[0164] However, in some embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein typically expressed in both T cells and NK cells. In some embodiments, the transmembrane domain comprises CD8α. In some embodiments, the transmembrane domain is referred to as the "hinge." In some embodiments, the CD8α "hinge" has the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the CD8α hinge is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to CD8α having the sequence of SEQ ID NO: 1. In some embodiments, the CD8α "hinge" comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, CD8α may be cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the sequence of SEQ ID NO: 2.

[0165] In some embodiments, the transmembrane domain includes a CD8α transmembrane region. In some embodiments, the CD8α transmembrane domain has the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the CD8α hinge is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to CD8α having the sequence of SEQ ID NO: 3. In some embodiments, the CD8α transmembrane domain includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD8α hinge is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to CD8α having the sequence of SEQ ID NO: 4.

[0166] In some embodiments, the CD8 hinge / transmembrane complex is encoded by the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the CD8 hinge / transmembrane complex is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the CD8 hinge / transmembrane complex having the sequence of SEQ ID NO: 13. In some embodiments, the CD8 hinge / transmembrane complex contains the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD8 hinge / transmembrane complex hinge is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the CD8 hinge / transmembrane complex having the sequence of SEQ ID NO: 14.

[0167] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CD28 transmembrane domain complex hinge is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to the CD28 transmembrane domain having the sequence of SEQ ID NO: 30.

[0168] Co-stimulatory domain Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as CD19-directed chimeric receptors, which include a costimulatory domain. In addition to various transmembrane domains and signaling domains (and transmembrane / signaling domain combinations), in some embodiments, further co-activating molecules may be provided. These may be, for example, molecules that further enhance the activity of immune cells. In some embodiments, cytokines may be used. For example, in non-limiting examples, certain interleukins such as IL-2 and / or IL-15 may be used. In some embodiments, immune cells for therapeutic purposes are engineered to express such molecules in a secretory form. In further embodiments, such costimulatory domains are engineered to act as membrane-bound, autocrine stimulating molecules (or as paracrine stimulating factors in delivered adjacent cells). In some embodiments, NK cells are engineered to express membrane-bound interleukin 15 (mbIL15). In such embodiments, mbIL15 expression in NK cells enhances the cytotoxic effect of the engineered NK cells by enhancing NK cell proliferation and / or lifespan extension. In some embodiments, mbIL15 has the nucleic acid sequence of Sequence ID No. 11. In some embodiments, mbIL15 may be cleaved or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology with the sequence of SEQ ID NO: 11. In some embodiments, mbIL15 contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, mbIL15 is cleaved or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology with mbIL15 having the sequence of SEQ ID NO: 12.

[0169] In some embodiments, the CD19-targeted chimeric receptor or engineered cytotoxic receptor complex is encoded by a polynucleotide containing one or more cytoplasmic protease cleavage sites, e.g., a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and / or an F2A cleavage site. Such sites can be recognized and cleaved by cytoplasmic proteases, resulting in the separation (and separate expression) of various components of the polynucleotide-encoded receptor. As a result, in some embodiments, various components of the CD19-targeted chimeric receptor or engineered cytotoxic receptor complex can be delivered to NK cells or T cells by a single vector or multiple vectors. Thus, as schematically shown in the figure, the construct can be encoded by a single polynucleotide and may also contain cleavage sites such that downstream components of the construct are expressed by the cell as other proteins (as in some embodiments with IL-15). In some embodiments, the T2A cleavage site is used. In some embodiments, the T2A cleavage site has the nucleic acid sequence SEQ ID NO: 9. In some embodiments, the T2A cleavage site can be cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the sequence of SEQ ID NO: 9. In some embodiments, the T2A cleavage site contains the amino acid sequence of SEQ ID NO: 10. In some embodiments, the T2A cleavage site is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the T2A cleavage site having the sequence of SEQ ID NO: 10.

[0170] Signal transduction domains Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as CD19-targeted chimeric receptors, which include a signaling domain. For example, immune cells manipulated by some embodiments disclosed herein include at least one subunit (or fragment thereof) of a CD3 T cell receptor complex. In some embodiments, the signaling domain includes a CD3 zeta subunit. In some embodiments, the CD3 zeta is encoded by the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the CD3 zeta can be cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to the CD3 zeta having the sequence of SEQ ID NO: 7. In some embodiments, the CD3 zeta domain includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CD3 zeta domain is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homologous to the CD3 zeta domain having the sequence of SEQ ID NO: 8.

[0171] In some embodiments, unexpectedly enhanced signaling is achieved through the use of multiple signaling domains whose activities act synergistically. For example, in some embodiments, the signaling domain further includes an OX40 domain. In some embodiments, the OX40 domain is an intracellular signaling domain. In some embodiments, the OX40 intracellular signaling domain has the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the OX40 intracellular signaling domain can be cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to OX40 having the sequence of SEQ ID NO: 5. In some embodiments, the OX40 intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, the OX40 intracellular signaling domain can be cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the OX40 intracellular signaling domain having the sequence of SEQ ID NO: 6. In some embodiments, OX40 is used as the sole transmembrane / signaling domain in the construct; however, in some embodiments, OX40 may be used in conjunction with one or more other domains. For example, a combination of OX40 and CD3 zeta is used in one embodiment. As a further example, a combination of CD28, OX40, 4-1BB and / or CD3 zeta is used in one embodiment.

[0172] In some embodiments, the signaling domain includes the 4-1BB domain. In some embodiments, the 4-1BB domain is an intracellular signaling domain. In some embodiments, the 4-1BB intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 29. In some embodiments, the 4-1BB intracellular signaling domain is cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 29. In some embodiments, 4-1BB is used as the sole transmembrane / signaling domain in the construct; however, in some embodiments, 4-1BB may be used in combination with one or more other domains. For example, a combination of 4-1BB and CD3 zeta is used in some embodiments. As a further example, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used in some embodiments.

[0173] In some embodiments, the signaling domain includes a CD28 domain. In some embodiments, the CD28 domain is an intracellular signaling domain. In some embodiments, the CD28 intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CD28 intracellular signaling domain is cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% homologous to the CD28 intracellular signaling domain having the sequence of SEQ ID NO: 31. In some embodiments, CD28 is used as the sole transmembrane / signaling domain in the construct; however, in some embodiments, CD28 may be used in combination with one or more other domains. For example, a combination of CD28 and CD3 zeta is used in some embodiments. As a further example, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used in some embodiments.

[0174] Cytotoxic receptor complex constructs Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as CD19-targeted chimeric receptors, comprising cytotoxic receptor complexes or cytotoxic receptor complex constructs. As described above, a variety of cytotoxic receptor complexes (also referred to as cytotoxic receptors) are provided herein. Expression of these complexes in immune cells such as T cells and / or NK cells enables targeting and destruction of specific target cells, such as cancer cells. Non-limiting examples of such cytotoxic receptor complexes are further detailed below.

[0175] Chimeric antigen receptor cytotoxic receptor complex construct In some embodiments, a variety of cytotoxic receptor complexes (also referred to as cytotoxic receptors) having the general structure of a chimeric antigen receptor are provided herein. Figures 1A, 1B, and 2 schematically illustrate non-limiting constructs containing an anti-CD19 moiety that bind to tumor antigens or tumor-associated antigens expressed on the surface of cancer cells and activate engineered cells expressing the chimeric antigen receptor. As shown in the figures, some embodiments of the chimeric receptor include an anti-CD19 moiety, a CD8a hinge domain, an Ig4 SH domain (or hinge), a CD8a transmembrane domain, a CD28 transmembrane domain, an OX40 domain, a 4-1BB domain, a CD28 domain, a CD3ζ ITAM domain or subdomain, a CD3 zeta domain, an NKp80 domain, a CD16 IC domain, a 2A cleavage site, and a membrane-bound IL-15 domain (however, as described above, soluble IL-15 is used in some embodiments). In some embodiments, the binding and activation functions are engineered to be carried out by separate domains. Some embodiments relate to complexes having one or more anti-CD19 moieties or other binding / activating moieties. In some embodiments, the binding / activating moieties target other markers besides CD19, such as cancer targets described herein. In some embodiments, the general structure of the chimeric antigen receptor construct includes a hinge and / or transmembrane domain. In some embodiments, these may be filled with a single domain, or in some embodiments, multiple subdomains may be used. The receptor complex further includes a signaling domain that transmits post-binding signals of the homing moiety to target cells, ultimately leading to cytotoxic effects on the target cells. In some embodiments, the complex further includes a costimulatory domain that acts synergistically to enhance the function of the signaling domain. Expression of these complexes in immune cells such as T cells and / or NK cells enables targeting and destruction of specific target cells, such as cancer cells that express CD19. Some such receptor complexes include an extracellular domain containing an anti-CD19 moiety or CD19-binding moiety that binds to CD19 on the surface of the target cell and activates the manipulated cell.The CD3 zeta ITAM subdomain may act in coordination with the signaling domain. The IL-15 domain, e.g., the mbIL-15 domain, may act as a co-stimulatory domain. The IL-15 domain, e.g., the mbIL-15 domain, makes the immune cells expressing it (e.g., NK cells or T cells) particularly effective against target tumor cells. IL-15 domains, such as the mbIL-15 domain, may be encoded by other constructs in several embodiments. Furthermore, each element may be encoded by one or more other constructs. In one embodiment, the cytotoxic receptor or CD19-targeted receptor includes an amino acid sequence identical to the sequence of SEQ ID NO: 34 in a range defined by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any two of the aforementioned percentages.

[0176] In one embodiment, a polynucleotide encoding an anti-CD19 moiety / CD8 hinge-CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 1A, CD19-1a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD8a transmembrane domain, OX40 domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0177] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge-CD8™ / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 1A, CD19-1b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD8a transmembrane domain, OX40 domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0178] In some embodiments, polynucleotides encoding the anti-CD19 moiety / Ig4SH-CD8™ / 4-1BB / CD3 zeta-chimeric antigen receptor complex are provided (see Figure 1A, CD19-2a). The polynucleotide comprises or consists of the anti-CD19 moiety, Ig4 SH domain, CD8a transmembrane domain, 4-1BB domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from the combination of sequences disclosed herein, or a sequence obtained from the combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0179] In some embodiments, polynucleotides encoding the anti-CD19 moiety / Ig4SH-CD8™ / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 1A, CD19-2b). The polynucleotide comprises or consists of the anti-CD19 moiety, Ig4 SH domain, CD8a transmembrane domain, 4-1BB domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from the combination of sequences disclosed herein, or a sequence obtained from the combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence matching one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0180] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge-CD28™ / CD28 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 1A, CD19-3a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD28 transmembrane domain, CD28 domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0181] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge-CD28™ / CD28 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 1A, CD19-3b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD28 transmembrane domain, CD28 domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0182] In some embodiments, polynucleotides encoding an anti-CD19 moiety / Ig4SH-CD28™ / CD28 / CD3 zeta-chimeric antigen receptor complex are provided (see Figure 1A, CD19-4a). The polynucleotide comprises or consists of the anti-CD19 moiety, Ig4 SH domain, CD28 transmembrane domain, CD28 domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0183] In some embodiments, polynucleotides encoding the anti-CD19 moiety / Ig4SH-CD28™ / CD28 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 1A, CD19-4b). The polynucleotide comprises or consists of the anti-CD19 moiety, Ig4 SH domain, CD28 transmembrane domain, CD28 domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from the combination of sequences disclosed herein, or a sequence obtained from the combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0184] In some embodiments, polynucleotides encoding an anti-CD19 moiety / Ig4SH-CD8™ / OX40 / CD3 zeta-chimeric antigen receptor complex are provided (see Figure 2A, CD19-5a). The polynucleotide comprises or consists of the anti-CD19 moiety, Ig4 SH domain, CD8a transmembrane domain, OX40 domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with a sequence obtained from one or more combinations of sequence numbers described herein.

[0185] In some embodiments, polynucleotides encoding the anti-CD19 moiety / Ig4SH-CD8™ / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 2A, CD19-5b). The polynucleotide comprises or consists of the anti-CD19 moiety, Ig4 SH domain, CD8a transmembrane domain, OX40 domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from the combination of sequences disclosed herein, or a sequence obtained from the combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0186] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge-CD3α™ / CD28 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 1B, CD19-6a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD3α transmembrane domain, CD28 domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0187] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge-CD3α™ / CD28 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 1B, CD19-6b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD3α transmembrane domain, CD28 domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence matching one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0188] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge-CD28™ / CD28 / 4-1BB / CD3 zeta chimeric antigen receptor complex are provided (see Figure 1B, CD19-7a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD28 transmembrane domain, CD28 domain, 4-1BB domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence matching one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0189] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge-CD28™ / CD28 / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 1B, CD19-7b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD28 transmembrane domain, CD28 domain, 4-1BB domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence matching one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0190] In some embodiments, a polynucleotide encoding an anti-CD19 moiety / CD8 alpha hinge / CD8 alpha TM / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2, CD19-8a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD8a transmembrane domain, 4-1BB domain, and CD3 zeta domain described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein or comprises an amino acid sequence comprising a sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that is identical to one or more of the SEQ ID NOs described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity, homology and / or functional equivalence with a sequence obtained by a combination of one or more of the SEQ ID NOs described herein.

[0191] In some embodiments, polynucleotides encoding the anti-CD19 moiety / CD8 alpha hinge / CD8 alpha™ / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 2, CD19-8b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD8a transmembrane domain, 4-1BB domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from the combination of sequences disclosed herein, or a sequence obtained from the combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence matching one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0192] In some embodiments, a polynucleotide encoding an anti-CD19 moiety / CD8 alpha hinge / CD3 TM / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2, CD19-39_5a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD3 transmembrane domain, 4-1BB domain and CD3 zeta domain described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein or comprises an amino acid sequence comprising a sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that is identical to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity, homology and / or functional equivalence with a sequence obtained by a combination of one or more of the sequence numbers described herein.

[0193] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 alpha hinge / CD3™ / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 2, CD19-39_5b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD8a transmembrane domain, 4-1BB domain, CD3 zeta domain, 2A cleavage site, and mRNA domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or an amino acid sequence containing a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0194] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 alpha hinge / CD3™ / 4-1BB / NKp80 chimeric antigen receptor complex are provided (see Figure 2, CD19-39_6a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD3 transmembrane domain, 4-1BB domain, and NKp80 domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0195] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 alpha hinge / CD3™ / 4-1BB / NKp80 / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 2, CD19-39_6b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD8a transmembrane domain, 4-1BB domain, NKp80 domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0196] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 alpha hinge / CD3™ / CD16 intracellular domain / 4-1BB chimeric antigen receptor complex are provided (see Figure 2, CD19-39_10a). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD3 transmembrane domain, CD16 intracellular domain, and 4-1BB domain described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0197] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 alpha hinge / CD3™ / CD16 / 4-1BB / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 2, CD19-39_10b). The polynucleotide comprises or consists of the anti-CD19 moiety, CD8a hinge, CD8a transmembrane domain, CD16 intracellular domain, 4-1BB domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or an amino acid sequence containing a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0198] In some embodiments, polynucleotides encoding an anti-CD19 moiety / NKG2D extracellular domain / CD8 hinge-CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 2, CD19 / NKG2D-1a). The polynucleotide comprises or consists of the anti-CD19 moiety, NKG2D extracellular domain (either full-length or fragment), CD8a hinge, CD8a transmembrane domain, OX40 domain, and CD3 zeta domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or an amino acid sequence containing a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0199] In some embodiments, polynucleotides encoding an anti-CD19 moiety / NKG2D EC domain / CD8 hinge-CD8™ / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 2, CD19 / NKG2D-1b). The polynucleotide comprises or consists of the anti-CD19 moiety, NKG2D extracellular domain (either full-length or fragment), CD8a hinge, CD8a transmembrane domain, OX40 domain, CD3 zeta domain, 2A cleavage site, and mRNA-15 domain as described herein. In some embodiments, the receptor complex comprises an amino acid sequence encoded by a nucleic acid molecule containing a sequence obtained from a combination of sequences disclosed herein, or an amino acid sequence containing a sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that matches one or more of the sequence numbers described herein, such as those provided herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with the sequence obtained by a combination of one or more of the sequence numbers described herein.

[0200] In some embodiments, a polynucleotide encoding the anti-CD19 moiety / CD8 hinge / CD8™ / 4-1BB / CD3 zeta / mbIL15 chimeric antigen receptor complex is provided (see Figure 3A, NK19). The polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, 4-1BB domain, and CD3 zeta domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 85. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor comprises a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 85. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the NK19 chimeric antigen receptor contains an amino acid sequence that shares sequence identity, homology, and / or functional equivalence of at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 86. Schematic representations and their use in some embodiments provide NK19 constructs lacking the mbIL15 domain (Figure 3A, NK19 opt.).

[0201] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3A, NK19-1a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, OX40 domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3A, NK19-1b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, OX40 domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 59. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 59. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 60. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 60. In some embodiments, the CD19 scFv does not include a Flag tag.

[0202] In some embodiments, a polynucleotide encoding an anti-CD19 moiety / CD8 hinge / CD28™ / CD28 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 3A, NK19-2a). The polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD28 transmembrane domain, a CD28 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3A, NK19-2b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD28 transmembrane domain, CD28 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 61. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 61. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 62. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 62. In some embodiments, the CD19 scFv does not include a Flag tag.

[0203] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / ICOS / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3A, NK19-3a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, inducible costimulatory molecule (ICOS) signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3A, NK19-3b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, inducible costimulatory molecule (ICOS) signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 63. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 63. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 64. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 64. In some embodiments, the CD19 scFv does not include a Flag tag.

[0204] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD28 / 4-1BB / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3A, NK19-4a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3A, NK19-4b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 65. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 65. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 66. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 66. In some embodiments, the CD19 scFv does not include a Flag tag.

[0205] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / NKG2D™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3B, NK19-5a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, NKG2D transmembrane domain, OX40 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3B, NK19-5b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, NKG2D transmembrane domain, OX40 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 67. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 67. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 68. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 68. In some embodiments, the CD19 scFv does not include a Flag tag.

[0206] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3B, NK19-6a). The polynucleotide comprises or consists of an anti-CD19 scFv variable heavy chain, a CD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3B, NK19-6b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv variable heavy chain, CD8a hinge, CD8a transmembrane domain, CD40 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 69. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 69. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 70. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 70. In some embodiments, the CD19 scFv does not include a Flag tag.

[0207] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / OX40 / CD3 zeta / 2A / EGFRt chimeric antigen receptor complex are provided (see Figure 3B, NK19-7a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain, CD3 zeta domain, 2A cleavage site, and a cleaved version of the epidermal growth factor receptor (EGFRt). In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3B, NK19-7b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain, CD3 zeta domain, 2A cleavage site, a cleaved version of the epidermal growth factor receptor (EGFRt), a further 2A cleavage site, and the mbIL-15 domain described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 71. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 71. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 72. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 72. In some embodiments, the CD19 scFv does not include a Flag tag.

[0208] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3B, NK19-8a). The polynucleotide comprises or consists of an anti-CD19 scFv variable light chain, CD8a hinge, CD8a transmembrane domain, CD40 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3B, NK19-7b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv variable light chain, CD8a hinge, CD8a transmembrane domain, CD40 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 73. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 73. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 74. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 74. In some embodiments, the CD19 scFv does not include a Flag tag.

[0209] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3B, NK19-8a). The polynucleotide comprises or consists of an anti-CD19 scFv variable light chain, CD8a hinge, CD8a transmembrane domain, CD40 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3B, NK19-7b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv variable light chain, CD8a hinge, CD8a transmembrane domain, CD40 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 73. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 73. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 74. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 74. In some embodiments, the CD19 scFv does not include a Flag tag.

[0210] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD27 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3C, NK19-9a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD27 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3C, NK19-9b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD27 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 75. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 75. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 76. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 76. In some embodiments, the CD19 scFv does not include a Flag tag.

[0211] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD70 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3C, NK19-10a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD70 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3C, NK19-10b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD70 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 77. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 77. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 78. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 78. In some embodiments, the CD19 scFv does not include a Flag tag.

[0212] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD161 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3C, NK19-11a). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD161 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3C, NK19-11b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD161 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 79. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 79. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 80. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 80. In some embodiments, the CD19 scFv does not include a Flag tag.

[0213] In some embodiments, a polynucleotide encoding an anti-CD19 moiety / CD8 hinge / CD8aTM / CD40L / CD3 zeta chimeric antigen receptor complex is provided (see Figure 3C, NK19-12a). The polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, a CD40L signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3C, NK19-12b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD40L signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 81. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor comprises a sequence sharing at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 81. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the NK19 chimeric antigen receptor comprises an amino acid sequence sharing at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 82. In some embodiments, the CD19 scFv does not contain a Flag tag.

[0214] In some embodiments, polynucleotides encoding an anti-CD19 moiety / CD8 hinge / CD8a™ / CD44 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3C, NK19-13). The polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD44 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3C, NK19-13b). In such embodiments, the polynucleotide comprises or consists of the anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, CD44 signaling domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of Sequence ID No. 83. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 83. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 84. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 84. In some embodiments, the CD19 scFv does not include a Flag tag.

[0215] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3D, NK19H-1a). The polynucleotide is humanized and comprises a first humanized light chain and a first humanized heavy chain (L1 / H1), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3D, NK19H-1b). In such embodiments, the polynucleotide is humanized and comprises a first humanized light chain and a first humanized heavy chain (L1 / H1), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain and OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 160. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 160. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 161. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 161.

[0216] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3D, NK19H-2a). The polynucleotide is humanized and comprises a second humanized light chain and a first humanized heavy chain (L2 / H1), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3D, NK19H-2b). In such embodiments, the polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a second humanized light chain and a first humanized heavy chain (L2 / H1), and comprises a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain, CD3 zeta domain, 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 162. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 162. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 163. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 162.

[0217] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3D, NK19H-3a). The polynucleotide is humanized and comprises a third humanized light chain and a first humanized heavy chain (L3 / H1), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3D, NK19H-3b). In such embodiments, the polynucleotide is humanized and comprises a third humanized light chain and a first humanized heavy chain (L3 / H1), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain and OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 164. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 164. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 165. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 165.

[0218] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3D, NK19H-4a). The polynucleotide is humanized and comprises a first humanized light chain and a second humanized heavy chain (L1 / H2), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3D, NK19H-4b). In such embodiments, the polynucleotide is humanized and comprises a first humanized light chain and a second humanized heavy chain (L1 / H2), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain and OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 166. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 166. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 167. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 167.

[0219] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3E, NK19H-5a). The polynucleotide is humanized and comprises a second humanized light chain and a second humanized heavy chain (L2 / H2), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3E, NK19H-5b). In such embodiments, the polynucleotide is humanized and comprises a second humanized light chain and a second humanized heavy chain (L2 / H2), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain and OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 168. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 168. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 169. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 169.

[0220] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3E, NK19H-6a). The polynucleotide is humanized and comprises a third humanized light chain and a second humanized heavy chain (L3 / H2), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3E, NK19H-6b). In such embodiments, the polynucleotide is humanized and comprises a third humanized light chain and a second humanized heavy chain (L3 / H2), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain and OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 170. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 170. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 171. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 171.

[0221] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3E, NK19H-7a). The polynucleotide is humanized and comprises a first humanized light chain and a third humanized heavy chain (L1 / H3), and includes or comprises an anti-CD19 scFv comprising a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3E, NK19H-7b). In such embodiments, the polynucleotide is humanized as described herein and comprises a first humanized light chain and a third humanized heavy chain (L1 / H3), and includes or comprises an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain, a 2A cleavage site, a cleaved version of the epidermal growth factor receptor (EGFRt), a further 2A cleavage site and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 172. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence with SEQ ID NO: 172. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 173. In some embodiments, the NK19 chimeric antigen receptor comprises an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 174.

[0222] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3E, NK19H-8a). The polynucleotide is humanized and comprises a second humanized light chain and a third humanized heavy chain (L2 / H3), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3E, NKH19-8b). In such embodiments, the polynucleotide is humanized and comprises a second humanized light chain and a third humanized heavy chain (L2 / H3), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain and OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 174. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 174. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 175. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 175.

[0223] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3E, NK19H-9a). The polynucleotide is humanized and comprises a third humanized light chain and a third humanized heavy chain (L3 / H3), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3E, NKH19-9b). In such embodiments, the polynucleotide is humanized and comprises a third humanized light chain and a third humanized heavy chain (L3 / H3), and comprises or consists of an anti-CD19 scFv including a Flag tag, CD8a hinge, CD8a transmembrane domain and OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 176. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 176. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 177. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 177.

[0224] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3E, NKH19-10a). The polynucleotide is humanized and comprises a first humanized light chain and a fourth humanized heavy chain (L1 / H4), and includes or comprises an anti-CD19 scFv comprising a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3E, NK19H-10b). In such embodiments, the polynucleotide is humanized as described herein and comprises a first humanized light chain and a fourth humanized heavy chain (L1 / H4), and includes or comprises an anti-CD19 scFv containing a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 178. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence with SEQ ID NO: 178. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 179. In some embodiments, the NK19 chimeric antigen receptor comprises an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 179.

[0225] In some embodiments, polynucleotides encoding a Flag-tagged humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3F, NK19H-11a). The polynucleotide is humanized and comprises a second humanized light chain and a fourth humanized heavy chain (L2 / H4), and includes or comprises an anti-CD19 scFv comprising a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3F, NK19H-11b). In such embodiments, the polynucleotide is humanized as described herein and comprises a second humanized light chain and a fourth humanized heavy chain (L2 / H4), and includes or comprises an anti-CD19 scFv containing a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 180. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence with SEQ ID NO: 180. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 181. In some embodiments, the NK19 chimeric antigen receptor comprises an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 181.

[0226] In some embodiments, polynucleotides are provided that encode a Flag tag, a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex (see Figure 3F, NK19H-12a). The polynucleotide is humanized and comprises a third humanized light chain and a fourth humanized heavy chain (L3 / H4), and includes or comprises an anti-CD19 scFv comprising a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3F, NK19H-12b). In such embodiments, the polynucleotide is humanized as described herein and comprises a third humanized light chain and a fourth humanized heavy chain (L3 / H4), and includes or comprises an anti-CD19 scFv containing a Flag tag, CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain, a 2A cleavage site and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 182. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence with SEQ ID NO: 182. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 183. In some embodiments, the NK19 chimeric antigen receptor comprises an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 183.

[0227] In some embodiments, polynucleotides encoding a chimeric antigen receptor comprising a Flag tag, a humanized anti-CD19 moiety, and multiple costimulatory domains are provided. For example, a schematic structure is anti-CD19 moiety / transmembrane domain / costimulatory domain 1 / costimulatory domain 2 / costimulatory domain 3 / signaling domain. The order of the costimulatory domains varies depending on the embodiment. For example, in some embodiments, the costimulatory domains ("CSDs") may be arranged as follows: CSD1 / CSD2, CSD2 / CSD1, CSD1 / CSD2 / CSD3, CSD1 / CSD2 / CSD3, CSD3 / CSD2 / CSD1, etc. In some embodiments, polynucleotides encoding a Flag tag, a humanized anti-CD19 moiety / CD8 hinge / CD8a™ / CD44 / OX40 / CD27 / CD3 zeta-chimeric antigen receptor complex are provided (see Figure 3F, NK19H-13a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv containing a Flag tag, CD8a hinge, CD8a transmembrane domain, CD44 costimulatory domain, OX40 costimulatory domain, CD27 costimulatory domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3F, NK19H-13b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, containing a Flag tag, CD8a hinge, CD8a transmembrane domain, CD44 costimulatory domain, OX40 costimulatory domain, CD27 costimulatory domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain.

[0228] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3G, NK19H-NF-1a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv comprising a first humanized light chain and first humanized heavy chain (L1 / H1), CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3G, NK19H-NF-1b). In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv comprising the humanized first humanized light chain and first humanized heavy chain (L1 / H1), CD8a hinge, CD8a transmembrane domain, OX40 signaling domain and CD3 zeta domain, 2A cleavage site and mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 184. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 184. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 185. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 185.

[0229] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3G, NK19H-NF-2a). The polynucleotide is humanized and comprises or comprises an anti-CD19 scFv including a second humanized light chain and a first humanized heavy chain (L2 / H1), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3G, NK19H-NF-2b). In such embodiments, the polynucleotide comprises or comprises an anti-CD19 scFv including the humanized second humanized light chain and first humanized heavy chain (L2 / H1), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 186. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 186. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 187. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 187.

[0230] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3G, NK19H-NF-3a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a third humanized light chain and a first humanized heavy chain (L3 / H1), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3G, NK19H-NF-3b). In such embodiments, the polynucleotide comprises or consists of the humanized anti-CD19 scFv described herein, including a third humanized light chain and a first humanized heavy chain (L3 / H1), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 188. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 188. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 189. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 189.

[0231] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3G, NK19H-NF-4a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a first humanized light chain and a second humanized heavy chain (L1 / H2), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3G, NK19H-NF-4b). In such embodiments, the polynucleotide comprises or consists of the humanized anti-CD19 scFv described herein, comprising a first humanized light chain and a second humanized heavy chain (L1 / H2), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 190. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 190. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 191. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 191.

[0232] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3H, NK19H-NF-5a). The polynucleotide is humanized and comprises or comprises an anti-CD19 scFv including a second humanized light chain and a second humanized heavy chain (L2 / H2), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3H, NK19H-NF-5b). In such embodiments, the polynucleotide comprises or comprises an anti-CD19 scFv including the humanized second humanized light chain and second humanized heavy chain (L2 / H2), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 192. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 192. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 193. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 193.

[0233] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3H, NK19H-NF-6a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a third humanized light chain and a second humanized heavy chain (L3 / H2), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3H, NK19H-NF-6b). In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv including the humanized third humanized light chain and second humanized heavy chain (L3 / H2), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 194. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 194. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 195. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 195.

[0234] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3G, NK19H-NF-7a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv comprising a first-humanized light chain and a third-humanized heavy chain (L1 / H3), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3H, NK19H-NF-7b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, comprising a first-humanized light chain and a third-humanized heavy chain (L1 / H3), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 196. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 196. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 197. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 197.

[0235] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3H, NK19H-NF-8a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv comprising a second humanized light chain and a third humanized heavy chain (L2 / H3), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3H, NKH19-NF-8b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv variable light chain described herein, comprising a second humanized light chain and a third humanized heavy chain (L2 / H3), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 198. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 198. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 199. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 199.

[0236] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3H, NK19H-NF-9a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a third humanized light chain and a third humanized heavy chain (L3 / H3), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3H, NKH19-NF-9b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, including a third humanized light chain and a third humanized heavy chain (L3 / H3), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 200. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 200. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 201. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 201.

[0237] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3H, NKH19-NF-10a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a first humanized light chain and a fourth humanized heavy chain (L1 / H4), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3H, NK19H-NF-10b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, including a first humanized light chain and a fourth humanized heavy chain (L1 / H4), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 202. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 202. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 203. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 203.

[0238] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3I, NK19H-NF-11a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a second humanized light chain and a fourth humanized heavy chain (L2 / H4), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3I, NK19H-NF-11b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, including a second humanized light chain and a fourth humanized heavy chain (L2 / H4), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 204. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 204. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 205. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 205.

[0239] In some embodiments, polynucleotides encoding a humanized anti-CD19 moiety / CD8 hinge / CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 3I, NK19H-12a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv including a third humanized light chain and a fourth humanized heavy chain (L3 / H4), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL-15 (see Figure 3I, NK19H-NF-12b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, including a third humanized light chain and a fourth humanized heavy chain (L3 / H4), a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 206. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor includes a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 206. In some embodiments, the chimeric receptor includes the amino acid sequence of SEQ ID NO: 207. In some embodiments, the NK19 chimeric antigen receptor includes an amino acid sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with SEQ ID NO: 207.

[0240] In some embodiments, polynucleotides encoding a chimeric antigen receptor comprising a flag tag, a humanized anti-CD19 moiety, and multiple costimulatory domains are provided. For example, a schematic structure is anti-CD19 moiety / transmembrane domain / costimulatory domain 1 / costimulatory domain 2 / costimulatory domain 3 / signaling domain. The order of the costimulatory domains varies depending on the embodiment. For example, in some embodiments, the costimulatory domains ("CSDs") may be arranged as follows: CSD1 / CSD2, CSD2 / CSD1, CSD1 / CSD2 / CSD3, CSD1 / CSD2 / CSD3, CSD3 / CSD2 / CSD1, etc. In some embodiments, polynucleotides encoding a flag tag, a humanized anti-CD19 moiety / CD8 hinge / CD8a™ / CD44 / OX40 / CD27 / CD3 zeta-chimeric antigen receptor complex are provided (see Figure 3I, NK19H-NF-13a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv containing a Flag tag, CD8a hinge, CD8a transmembrane domain, CD44 costimulatory domain, OX40 costimulatory domain, CD27 costimulatory domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3I, NK19H-NF-13b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, containing a Flag tag, CD8a hinge, CD8a transmembrane domain, CD44 costimulatory domain, OX40 costimulatory domain, CD27 costimulatory domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain.

[0241] In some embodiments, polynucleotides encoding a chimeric antigen receptor comprising a Flag tag, a humanized anti-CD19 moiety, and multiple costimulatory domains are provided. For example, a schematic structure is anti-CD19 moiety / transmembrane domain / costimulatory domain 1 / costimulatory domain 2 / costimulatory domain 3 / signaling domain. The order of the costimulatory domains varies depending on the embodiment. For example, in some embodiments, the costimulatory domains ("CSDs") may be arranged as follows: CSD1 / CSD2, CSD2 / CSD1, CSD1 / CSD2 / CSD3, CSD1 / CSD2 / CSD3, CSD3 / CSD2 / CSD1, etc. In some embodiments, polynucleotides encoding a Flag tag, a humanized anti-CD19 moiety / CD8 hinge / CD8a™ / CD44 / OX40 / CD27 / CD3 zeta-chimeric antigen receptor complex are provided (see Figure 3F, NK19H-13a). The polynucleotide is humanized and comprises or consists of an anti-CD19 scFv containing a Flag tag, CD8a hinge, CD8a transmembrane domain, CD44 costimulatory domain, OX40 costimulatory domain, CD27 costimulatory domain, and CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 3F, NK19H-13b). In such embodiments, the polynucleotide is humanized and comprises or consists of the anti-CD19 scFv described herein, containing a Flag tag, CD8a hinge, CD8a transmembrane domain, CD44 costimulatory domain, OX40 costimulatory domain, CD27 costimulatory domain, CD3 zeta domain, 2A cleavage site, and mbIL-15 domain.

[0242] It is acknowledged that for any receptor construct described herein, certain sequence variability, elongation, and / or shortening of the disclosed sequences may occur when the sequences are combined, for example, as a result of ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0243] Treatment method One embodiment relates to a method for treating, mitigating, inhibiting, or preventing cancer with cells or immune cells containing a chimeric receptor, such as a CD19-directed chimeric receptor. In one embodiment, the method includes treating or preventing cancer. In one embodiment, the method includes administering a therapeutically effective amount of immune cells expressing the CD19-directed chimeric receptor described herein. Examples of cancer types that can be treated in this manner are described herein.

[0244] In one embodiment, treatment of a subject with the genetically modified cells described herein achieves one, two, three, four, or more effects, including, for example, (i) reduction or improvement of the severity of the disease or associated symptoms; (ii) reduction of the duration of the symptoms associated with the disease; (iii) protection from the progression of the disease or associated symptoms; (iv) regression of the disease or associated symptoms; (v) protection from the progression or onset of the symptoms associated with the disease; (vi) protection from the recurrence of the symptoms associated with the disease; (vii) reduction of hospitalization of the subject; (viii) reduction of the length of hospitalization; (ix) increased survival of the subject with the disease; (x) reduction of the number of symptoms associated with the disease; and (xi) enhancement, improvement, supplementation, complementation, or augmentation of the preventive or therapeutic effects of other treatments. Administration is obtained by a variety of routes, including, but not limited to, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal, and / or local delivery to affected tissue.

[0245] Usage / Dosage Furthermore, provided herein are methods for treating a subject having cancer, comprising administering to the subject a composition comprising immune cells (e.g., NK cells and / or T cells) engineered to express a cytotoxic receptor complex as described herein. For example, certain embodiments of the compositions and methods described herein relate to the use of CD19-directed chimeric receptors or cells expressing CD19-directed chimeric receptors for the treatment of cancer patients. The use of such engineered immune cells for the treatment of cancer is also provided.

[0246] In one embodiment, the treatment of a subject with the genetically engineered cells described herein achieves one, two, three, four or more of the following effects, for example: (i) reduction or amelioration of the severity of a disease or an associated symptom; (ii) reduction of the duration of an associated symptom of a disease; (iii) protection from progression of a disease or an associated symptom; (iv) regression of a disease or an associated symptom; (v) protection from development or onset of an associated symptom of a disease; (vi) protection from recurrence of an associated symptom of a disease; (vii) reduction of hospitalization of a subject; (viii) shortening of the length of hospitalization; (ix) increased survival of a subject having a disease; (x) decrease in the number of symptoms associated with a disease; (xi) enhancement, improvement, supplementation, complementation or augmentation of the prophylactic or therapeutic effect of another treatment. Each of these comparisons is, for example, against different treatments of a disease, including cell-based immunotherapies of the disease using cells that do not express the constructs disclosed herein.

[0247] Administration Administration can be obtained by a variety of routes including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to the affected tissue. The amount of immune cells such as NK cells and / or T cells can be readily determined for a particular subject based on body size, disease type and condition, and the desired aggressiveness of the treatment, but in embodiments is in the range of about 10 5 cells / kg to about 10 12 cells / kg (e.g., 10 5 ~10 7 cells / kg, 10 7 ~10 10 cells / kg, 10 10 ~10 12 cells / kg and overlapping ranges thereof). In one embodiment, a dose escalation regimen is used. In some embodiments, for example, immune cells such as NK cells and / or T cells in the range of about 1×10 6 cells / kg to about 1×10 8 cells / kg are administered. In some embodiments, the dosage includes about 2×10 6 cells / kg and 2×10 7 cells / kg, and is in the range of about 2×10 5 cells / kg to about 2×10 8The range is cells / kg. In some embodiments, the dose is determined by the maximum number of viable manipulated cells at the time of administration. For example, in one embodiment, the single dose is approximately 2 × 10⁶ 6 , about 2×10 7 Or approximately 2 × 10 8 It contains viable, manipulated cells up to approximately 2 × 10⁶ 5 ~about 2×10 9 The present invention provides viable, manipulated cells. Various types of cancer can be treated by certain embodiments. In some embodiments, hepatocellular carcinoma is treated. Further embodiments provided herein include the following: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendiceal cancer, central nervous system cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumors (including, but not limited to, astrocytoma, spinal cord tumor, brainstem glioma, glioblastoma, craniopharyngioma, ependymoblastoma, ependymoldoma, medulloblastoma, medullary epithelioma), breast cancer, bronchial tumor, Burkitt lymphoma, cervical cancer, colon cancer, chronic lymphocytic leukemia (CLL). This includes treatment or prevention of non-exclusive exemplary cancers, including, but not limited to, chronic myeloid leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including, but not limited to, non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, prostate cancer, pituitary cancer, uterine cancer and vaginal cancer.

[0248] In one embodiment, also provided herein, is an NK cell carrying receptors encoded by nucleic acids and amino acid sequences that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and within that range) sequence identity or homology to each of the nucleic acids or amino acid sequences of SEQ ID NOs. 1 to 207 (or two or more combinations of SEQ ID NOs. 1 to 207), and that, compared to each of the SEQ ID NOs. 1 to 207 (or two or more combinations of SEQ ID NOs. 1 to 207), (i) enhances proliferation, (ii) enhances activation, and (iii) enhances the receptor encoded by the nucleic acid and amino acid sequence. Nucleic acid and amino acid sequences exhibiting one or more functions, including but not limited to: (iv) enhanced cytotoxic activity against cells presenting ligands to which cells bind; (v) enhanced homing to tumors or infection sites; (v) reduced off-target cytotoxic effects; (vi) enhanced secretion of immunostimulatory cytokines and chemokines (including, but not limited to, IFNg, TNFa, IL-22, CCL3, CCL4, and CCL5); (vii) increased ability to further stimulate innate and adaptive immune responses; and (viii) combinations thereof.

[0249] Furthermore, in some embodiments, considering the degeneracy of nucleic acid coding, amino acid sequences corresponding to the nucleic acids disclosed herein are provided. Moreover, sequences (whether nucleic acids or amino acids) that differ from those explicitly shown herein but possess functional similarity or equivalence are also considered within the scope of this disclosure. These include variants, shortenings, substitutions, or other types of modifications.

[0250] In some embodiments, the polynucleotide encoding the cytotoxic receptor complex or CD19-targeted chimeric receptor disclosed is mRNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is controllably bound to at least one regulatory element for cytotoxic receptor complex expression.

[0251] According to some embodiments, further provided are vectors comprising a polynucleotide encoding any of the polynucleotides provided herein, wherein the polynucleotide is optionally controllably bound to at least one regulatory element for the expression of a cytotoxic receptor complex. In some embodiments, the vector is a retrovirus.

[0252] Furthermore, provided herein are engineered immune cells (e.g., NK cells and / or T cells) comprising the polynucleotides, vectors, or cytotoxic receptor complexes described herein. Furthermore, provided herein are compositions comprising mixtures of engineered immune cells (e.g., NK cells and / or engineered T cells), each population comprising the polynucleotides, vectors, or cytotoxic receptor complexes described herein.

[0253] The dose of immune cells such as NK cells or T cells can be easily determined for a given subject based on physique, disease type and condition, and the desired aggressiveness of the treatment, but depending on the embodiment, it can be about 10 5 cells / kg~about 10 12 cells / kg (e.g., 10 5 ~10 7 cells / kg, 10 7 ~10 10 cells / kg, 10 10 ~10 12 This is the range of cells / kg and the overlapping range within that range. In one embodiment, a dose-escalation regimen is used. In some embodiments, for example, about 1 × 10 6 cells / kg ~ approx. 1×10 8 NK cells are administered in the range of cells / kg. Various types of cancer or infectious diseases can be treated by certain embodiments.

[0254] Cancer type Some embodiments of the compositions and methods described herein relate to the administration of immune cells containing chimeric receptors, such as CD19-directed chimeric receptors, to subjects having cancer. Various embodiments provided herein include the treatment or prevention of the following non-exclusive exemplary cancers: Examples of cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendiceal cancer, central nervous system cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumors (including, but not limited to, astrocytoma, spinal cord tumor, brainstem glioma, craniopharyngioma, ependymoblastoma, ependymoldoma, medulloblastoma, medullary epithelioma), breast cancer, bronchial tumor, Burkitt lymphoma, cervical cancer, colon cancer, chronic lymphocytic leukemia (CLL), This includes, but is not limited to, chronic myeloid leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial carcinoma, esophageal cancer, gastric cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including, but not limited to, non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, colon cancer, lymphoma, melanoma, eye cancer, ovarian cancer, prostate cancer, pituitary cancer, uterine cancer and vaginal cancer.

[0255] cancer target Some embodiments of the compositions and methods described herein relate to immune cells containing chimeric receptors that target cancer antigens. Non-limiting examples of target antigens include CD5, CD19; CD123; CD22; CD30; CD171; CS1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); type C lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(ll)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells; glycosylated CD43 epitope expressed in non-hematopoietic carcinomas; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-IIR a); prostate stem cell antigen (PSCA); protease serine 21 (testicin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha (FRa or FR1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-related (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutation (ELF2M); ephrin B2; fibroblast-activating protein alpha (FAP);Insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropine) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-abl) consisting of a cleavage region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl); tyrosinase; ephrin type A receptor 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (a Neu5Ac(2-3)bDClalp(l-4)bDGlcp(ll)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading Frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide portion of globoH glycoceramide (GloboH); Mammary gland differentiation antigen (NY-BR-1); Uloplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenergic receptor beta 3 (ADRB3); Panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (or 51E2); TCR gamma alternative leading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ES0-1); cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma carcinoma testis antigen-1 (MAD-CT-1); melanoma carcinoma testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; tumor protein p53 (p53); p53 variant; prostain; survivor; telomerase;Prostate cancer tumor antigen-1 (PCT Al or galectin 8), melanoma antigen recognized by T cell 1 (Melan A or MARTI); rat sarcoma (Ras) variant; human telomerase; reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin Bl; v-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 IB 1 (CYPIB 1); CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cell 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); advanced glycation end product receptor (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutation (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);and immunoglobulin lambda-like polypeptide 1 (IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR alpha 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialyl Lewis antigen); fucosyl-GMl, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, ILl lRa, IL13Ra2, CD179b-IGLll, TCR gamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Timl- / HVCR1, CSF2RA (GM-CSFR-alpha), TGF beta R2, Lewis Ag, TCR-beta I chain, TCR-beta II chain, TCR-gamma chain, TCR-delta chain, FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, Influenza A hemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC), Autoantibody against desmoglein 3 (Dsg3), Autoantibody against desmoglein 1 (Dsgl), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras This includes, but is not limited to, antigens recognized by G12V, tissue factor 1 (TF1), AFP, GPRC5D, claudin 18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Livl, nectin-4, Cripto, gpA33, BST1 / CD157, low-conductance chloride channels, and TNT antibodies.

[0256] Furthermore, in some embodiments, immune cells are provided that contain a CD19-targeted chimeric antigen receptor and also express membrane-bound interleukin-15 (mbIL15), comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a heavy-chain variable (VH) domain and a light-chain variable (VL) domain, the VH domain comprising a VH domain having at least 95% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33, and the VL domain having at least 95% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32), a hinge and / or transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain comprises an OX40 subdomain). In some embodiments, the intracellular signaling domain further comprises a CD3 zeta subdomain. In some embodiments, the OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 6, and the CD3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the hinge domain comprises a CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the chimeric receptor further comprises the extracellular domain of the NKG2D receptor. In some embodiments, the extracellular domain of the NKG2D receptor comprises a functional fragment of NKG2D comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are T cells. In some embodiments, such immune cells are used to treat cancer by administering them to a subject in a manner that treats cancer or by other means, such as in the manufacture of cancer treatment pharmaceuticals. In some embodiments, cancer is acute lymphoblastic leukemia.

[0257] In some embodiments, a polynucleotide is provided that encodes a CD19-targeted chimeric antigen receptor, and also encodes membrane-bound interleukin-15 (mbIL15), comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a heavy-chain variable (VH) domain and a light-chain variable (VL) domain, the VH domain having at least 95% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33, and the VL domain having at least 95% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32), a hinge and / or transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain comprises an OX40 subdomain). In some embodiments, the intracellular signaling domain further comprises a CD3 zeta subdomain. In some embodiments, the encoded OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 16, and the encoded CD3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the hinge domain comprises a CD8a hinge domain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the encoded mbIL15 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the chimeric receptor further comprises the extracellular domain of the NKG2D receptor. In some embodiments, the extracellular domain of the encoded NKG2D receptor comprises a functional fragment of NKG2D containing the amino acid sequence of SEQ ID NO: 26.

[0258] Immune cells expressing a CD19-directed chimeric receptor comprising an extracellular anti-CD19 moiety, a hinge and / or a transmembrane domain and an intracellular signaling domain are also provided herein. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are T cells. In some embodiments, the hinge domain comprises a CD8a hinge domain or an Ig4 SH domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain, a CD28 transmembrane domain and / or a CD3 transmembrane domain. In some embodiments, the signaling domain comprises an OX40 signaling domain, a 4-1BB signaling domain, a CD28 signaling domain, an NKp80 signaling domain, a CD16 IC signaling domain, a CD3 zeta or CD3ζ ITAM signaling domain and / or an mRNA signaling domain. In some embodiments, the signaling domain comprises a 2A cleavage domain. In some embodiments, the mRNA signaling domain is separated from the rest or other portions of the CD19-directed chimeric receptor by the 2A cleavage domain. In some embodiments, such immune cells are administered to subjects with cancer for the treatment, prevention, or prevention of cancer progression.

[0259] Engineered NK cells or T cells are also provided herein that express a CD19-targeted chimeric antigen receptor comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprising a VH domain resulting from humanization of the VH domain amino acid sequence shown in SEQ ID NO: 33, and the VL domain comprising a VL domain resulting from humanization of the VL domain amino acid sequence shown in SEQ ID NO: 32), a hinge and / or transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain comprises an OX40 subdomain), and also express membrane-bound interleukin-15 (mbIL15).

[0260] A polynucleotide is provided herein that encodes a CD19-targeted chimeric antigen receptor, and also encodes membrane-bound interleukin-15 (mbIL15), comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a heavy-chain variable (VH) domain and a light-chain variable (VL) domain, the VH domain comprising a VH domain resulting from humanization of the VH domain amino acid sequence shown in SEQ ID NO: 33, and the VL domain comprising a VL domain resulting from humanization of the VL domain amino acid sequence shown in SEQ ID NO: 32), a hinge and / or transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain comprises an OX40 subdomain).

[0261] A polynucleotide encoding a CD19-targeted chimeric antigen receptor is provided herein, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety includes scFv), a hinge (wherein the hinge is a CD8 alpha hinge), a transmembrane domain, and an intracellular signaling domain (wherein the intracellular signaling domain includes CD3 zeta ITAM). In some embodiments, the transmembrane domain comprises a CD8 alpha transmembrane domain, an NKG2D transmembrane domain, and / or a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain comprises a CD28 signaling domain, a 4-1BB signaling domain, and / or an OX40 domain. In some embodiments, the intracellular signaling domain may also include domains selected from ICOS, CD70, CD161, CD40L, CD44, and combinations thereof.

[0262] Provided herein is a polynucleotide encoding a CD19-targeted chimeric antigen receptor comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a variable heavy chain or a variable light chain of scFv), a hinge (wherein the hinge is a CD8 alpha hinge), a transmembrane domain (wherein the transmembrane domain comprises a CD8 alpha transmembrane domain), and an intracellular signaling domain (wherein the intracellular signaling domain comprises a CD3 zeta ITAM). In some embodiments, the polynucleotide also encodes a cleaved epidermal growth factor receptor (EGFRt). In some embodiments, the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15). Provided herein are engineered NK cells or T cells expressing such a CD19-targeted chimeric antigen and methods for treating cancer by administration of such NK cells or T cells. Also provided is the use of such polynucleotides in the treatment of cancer, e.g., in the manufacture of cancer treatment pharmaceuticals.

[0263] A polynucleotide is provided herein that encodes a humanized CD19-targeted chimeric antigen receptor, and also encodes membrane-bound interleukin-15 (mbIL15), comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprising a VH domain selected from SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, and the VL domain comprising a VL domain selected from SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO: 119), a hinge and / or a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an OX40 subdomain, a CD28 subdomain, an iCOS subdomain, a CD28-41BB subdomain, a CD27 subdomain, a CD44 subdomain, or a combination thereof. In some embodiments, the chimeric antigen receptor comprises a hinge and a transmembrane domain, where the hinge is a CD8 alpha hinge and the transmembrane domain is a CD8 alpha or NKG2D transmembrane domain. In some embodiments, the intracellular signaling domain includes a CD3 zeta domain.

[0264] A polynucleotide encoding a humanized chimeric antigen receptor (CAR) is provided herein, wherein the CAR comprises a single-chain antibody or single-chain antibody fragment comprising a humanized anti-CD19 binding domain, a transmembrane domain, a primary intracellular signaling domain comprising a native intracellular signaling domain or functional fragment of CD3 zeta, and a costimulatory domain comprising a native intracellular signaling domain or functional fragment of a protein selected from the group consisting of OX40, CD27, CD28, ICOS, and 4-1BB, wherein the anti-CD19 binding domain comprises a light chain complementarity-determining region 1 (LC CDR1) of SEQ ID NO: 124, 127, or 130, a light chain complementarity-determining region 2 (LC CDR2) of SEQ ID NO: 125, 128, or 131, a light chain complementarity-determining region 3 (LC CDR3) of SEQ ID NO: 126, 129, or 132, and a heavy chain complementarity-determining region 1 (HC CDR3) of SEQ ID NO: 133, 136, 139, or 142 The polynucleotide includes CDR1), heavy chain complementarity determination region 2 (HC CDR2) of SEQ ID NOs. 134, 137, 140, or 143, and heavy chain complementarity determination region 3 (HC CDR3) of SEQ ID NOs. 135, 138, 141, or 144. In some embodiments, the polynucleotide further includes a region encoding membrane-bound interleukin 15 (mbIL15).

[0265] Provided herein is a polynucleotide encoding a humanized CD19-targeted chimeric antigen receptor, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the light chain variable (VL) domain of SEQ ID NO: 117), a hinge and / or transmembrane domain, and an intracellular signaling domain, and also encoding membrane-bound interleukin-15 (mbIL15). In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 161, SEQ ID NO: 167, SEQ ID NO: 173, SEQ ID NO: 179, SEQ ID NO: 185, SEQ ID NO: 191, SEQ ID NO: 197, or SEQ ID NO: 203.

[0266] A polynucleotide is provided herein that encodes a humanized CD19-targeted chimeric antigen receptor comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence containing the light chain variable (VL) domain of SEQ ID NO: 118), a hinge and / or transmembrane domain, and an intracellular signaling domain, and also encodes membrane-bound interleukin-15 (mbIL15). In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 163, SEQ ID NO: 169, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 187, SEQ ID NO: 193, SEQ ID NO: 199, or SEQ ID NO: 205.

[0267] A polynucleotide is provided that encodes a humanized CD19-targeted chimeric antigen receptor, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence containing the light chain variable (VL) domain of SEQ ID NO: 119), a hinge and / or transmembrane domain, and an intracellular signaling domain, and also encodes membrane-bound interleukin-15 (mbIL15). In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 165, SEQ ID NO: 171, SEQ ID NO: 177, SEQ ID NO: 183, SEQ ID NO: 189, SEQ ID NO: 195, SEQ ID NO: 201, or SEQ ID NO: 207.

[0268] A polynucleotide is provided that encodes a humanized CD19-targeted chimeric antigen receptor, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 120), a hinge and / or transmembrane domain, and an intracellular signaling domain, and also encodes membrane-bound interleukin-15 (mbIL15). In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 185, SEQ ID NO: 187, or SEQ ID NO: 189.

[0269] A polynucleotide is provided that encodes a humanized CD19-targeted chimeric antigen receptor, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 121), a hinge and / or transmembrane domain, and an intracellular signaling domain, and also encodes membrane-bound interleukin-15 (mbIL15). In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 191, SEQ ID NO: 193, or SEQ ID NO: 195.

[0270] A polynucleotide is provided that encodes a humanized CD19-targeted chimeric antigen receptor, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 122), a hinge and / or transmembrane domain, and an intracellular signaling domain, and also encodes membrane-bound interleukin-15 (mbIL15). In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 197, SEQ ID NO: 199, or SEQ ID NO: 201.

[0271] A polynucleotide is provided that encodes a humanized CD19-targeted chimeric antigen receptor, comprising an extracellular anti-CD19 binding moiety (wherein the anti-CD19 binding moiety comprises a humanized scFv sequence including the heavy chain variable (VH) domain of SEQ ID NO: 123), a hinge and / or transmembrane domain, and an intracellular signaling domain, and also encodes membrane-bound interleukin-15 (mbIL15). In some embodiments, the polynucleotide encodes the humanized chimeric antigen receptor of SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 203, SEQ ID NO: 205, or SEQ ID NO: 207.

[0272] In some embodiments, the provided polynucleotides do not encode sequence numbers 112, 113, 114, or 116.

[0273] A polynucleotide is provided which encodes a humanized CD19-targeted chimeric antigen receptor comprising an extracellular anti-CD19 binding moiety, a hinge and / or transmembrane domain, and an intracellular signaling domain, and which also encodes membrane-bound interleukin-15 (mbIL15), where the polynucleotide is selected from the group consisting of polynucleotides having at least 95% identity with SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 192, or SEQ ID NO: 200. In some embodiments, the polynucleotide has the sequence of SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 192, or SEQ ID NO: 200. In some embodiments, engineered NK cells or T cells expressing such a humanized CD19-targeted chimeric antigen receptor are provided. Also provided is a method for treating cancer in a subject, comprising administering such engineered NK cells or T cells to a subject having cancer. Uses of such polynucleotides in the treatment of cancer, such as in the manufacture of cancer treatment pharmaceuticals, are also provided. [Examples]

[0274] The materials and methods disclosed herein are non-limiting examples used in certain embodiments disclosed herein.

[0275] According to some embodiments, NK cells are isolated from peripheral blood mononuclear cells and expanded via the use of feeder cell lines. As further detailed below, in some embodiments, feeder cells are engineered to express certain stimulating molecules (e.g., interleukin, CD3, 4-1BBL, etc.) to promote immune cell expansion and activation. Engineered feeder cells are disclosed, for example, in international patent application PCT / SG2018 / 050138, which is incorporated herein by reference in whole. In some embodiments, stimulating molecules such as interleukin 12, 18, and / or 21 are added separately to the co-culture medium, for example, at various time points and in specific amounts, to allow for the expansion of a desired subpopulation of immune cells.

[0276] NK cells isolated from PBMCs were co-cultured with K562 cells expressing membrane-bound IL15 and 4-1BBL, with IL2 added to the culture medium. One group of manipulated NK cells was expanded in medium (day 0) supplemented with a combination of soluble IL12 and soluble IL18. The medium was refreshed with additional soluble IL12 and soluble IL18 on day 4. Further details of embodiments of such culture methods are disclosed in U.S. Provisional Patent Application 62 / 881311 (filed July 31, 2019), which is incorporated herein by reference in whole. Viral transduction with a CD19-targeted chimeric receptor construct was performed on day 7. The resulting manipulated NK cells were evaluated for a total culture period of 14 days or longer.

[0277] Example 1 Figure 5 describes a schematic experimental model for evaluating the antitumor efficacy of engineered NK cells produced by the method disclosed herein. NOD-scid IL2R gamma null On the mouse, 2x10 5 Nalm6 cells (a B-cell precursor leukemia cell line) were administered intravenously on day 0. On day 4, one group of mice had a cell count of 2.7 × 10⁶. 7One group received NK cells expressing NK19 CARs (see Figure 3A, although it should be noted that other CD19-targeted chimeric receptors may be used), while the other group received NK cells expanded using soluble IL12 / IL18 (specified as NK19-IL12 / 18 cells). Leukemia tumor burden was assessed by fluorescence angiography performed on days 3, 7, 11, 18, and 25. Figure 6A shows the contrast-enhanced results on days 3, 7, 11, and 18. Clearly, the tumor burden in mice received NK19 or NK19 IL12 / 18 was significantly lower than in control mice received untransduced NK cells or PBS. Figure 6B shows a line graph summarizing the contrast-enhanced data (larger flux (photons / second) values ​​indicate larger fluorescence signal detection and larger tumor burden). Both the NK19 and NK19 IL12 / 18 groups showed low tumor burden just 7 days after Nalm6 leukemia cell injection. This difference was more pronounced on day 11, at which point the tumors in the PBS and NT NK cell groups showed significant proliferation. Even on day 18, when tumor burden was widespread in the PBS and NT NK cell groups, the tumor burden in the NK19 and NK19 IL12 / 18 groups was significantly lower. Unexpectedly, the NK19 IL12 / 18 group showed a lower tumor burden than those that received the NK19 construct. This suggests that NK19 cells are quite effective in preventing leukemia cell proliferation, and therefore, the further development of this effect is not only unexpected, but also surprising that the upstream cell proliferation method affects not only the cell number itself but also the activity level of the expanding cells.

[0278] Example 2 Experiments were conducted to determine whether certain stimulating domains used in CARs (also referred to as co-stimulatory domains, considering that many constructs use multiple “signaling” domains in tandem, triple, or other multiple forms) affect expression (and activity). NK cells were produced by transduction with viruses encoding various CARs, as shown in Figures 3A–3C (although other constructs are used in some embodiments). As a non-limiting example, NK cells were produced by transduction with a bicistronic virus encoding anti-CD19 scFv, an intracellular OX40 co-stimulatory domain, a CD3ζ signaling domain, and membrane-bound IL-15, which support long-term cell survival and proliferation (NK19, see Figure 3A). Other CAR constructs tested, NK19-1, 2, 3, 4, 5, 8, 9, 10, 11, 12, and NK19-13, were transduced into NK cells in a similar manner. For constructs that use a Flag domain to determine expression, it is recognized that in some embodiments, similar constructs without the Flag (or other tag domain) are provided.

[0279] Figure 7 summarizes the expression data for NK cells 19-1 to NK5 and NK8 to NK19-13, as evidenced by CD19 Flag detection. The data are presented as the percentage of CD19-Flag expressing NK cells relative to the total number of NK cells present. Data were obtained four days after transduction with the corresponding virus encoding the NK19-"X" CAR. As is evident from the expression data, all constructs were expressed by at least 55% of NK cells. In fact, expression was detected in approximately 75% or more of the total NK cells for 8 of the 11 constructs for which data was created, and several constructs were expressed with efficiencies exceeding 80%. This expression data suggests that, according to some embodiments, the selection of specific stimulation domains may enable more efficient expression of CARs by NK cells. This is advantageous because, in some embodiments, a significant portion of a given NK cell preparation is clinically useful (e.g., fewer inserted NK cells are needed to produce a clinically relevant manipulated NK cell dose).

[0280] As described herein, various co-stimulatory domains can be used in chimeric antigen receptors targeting CD19 (or other tumor markers). Figure 8A shows data on the expression of CD19-targeting CARs using various co-stimulatory domains. Non-limiting examples of co-stimulatory domains may include, but are not limited to, OX40, CD28, iCOS, CD28 / 41BB, CS27, and CD44. Figure 8A shows mean fluorescence intensity data representing the expression of the indicated CAR constructs by NK cells. As evidenced by the low MFI detected against the GFP control, these data indicate that these constructs are (a) expressed by NK cells and (b) expressed relatively stably by NK cells for 4 weeks after transduction. Figure 8B shows the expression efficiency of CARs with the indicated co-stimulatory domains. Expression efficiency varied somewhat, in the range of approximately 60% to 80%, but each of the indicated CARs with the indicated co-stimulatory domains was shown to be well expressed and relatively constant for at least 4 weeks.

[0281] Data regarding the cytotoxic efficacy of NK cells expressing CD19-directed CARs utilizing various co-stimulatory domains are shown in Figures 9A-9F. Cultured Nalm6 cells or Raji cells were exposed to engineered NK cells expressing the indicated NK19 construct and co-cultured for the number of days indicated for exposure to NK19-X expressing NK cells (X axis represents days). Figure 9A shows the cytotoxic effect of the indicated construct on Nalm6 cells 7 days after transduction of NK cells from the first donor with the indicated construct. The effector cell to target cell ratio in this experiment was 1:1. Each of the NK19-10, NK19-8, NK19-11, NK19-5, and NK19-12, indicated by the arrows, increased the number of detectable Nalm6 cells to a level equivalent to that of GFP-only expressing NK cells as control. However, each of NK19-3, NK19-9, NK19-4, NK19-13, NK19-1, and NK19-2 showed significantly lower Nalm6 cell number increases (i.e., stronger cytotoxicity). In some embodiments, such constructs are expressed in NK cells and used to treat B-cell leukemia (or other tumor types). In some embodiments, one or more stimulating domains from one construct are manipulated and inserted into other constructs along with various stimulating domains, resulting in advantageously synergistic signaling and further increased cytotoxicity. As a non-limiting example, the NK19-1 construct with an OX40 stimulating domain is further manipulated in some embodiments to express a CD44 stimulating domain in addition to OX40. As a further non-limiting example, the NK19-1 construct with an OX40 stimulating domain is further manipulated in some embodiments to express CD44 and CD17 stimulating domains in addition to OX40.

[0282] Figure 9B shows the corresponding cytotoxicity data of engineered NK cells from a second donor against Raji B-cell leukemia cells 7 days after transduction. The effector cell to target cell ratio here was also 1:1. As shown in the figure, several constructs increased the number of Raji cells, as did the activity of the engineered constructs against Nalm6 cells. However, each of NK19-13, NK19-4, NK19-2, NK19-3, NK19-1, and NK19-9 inhibited Raji cell proliferation to a considerable extent, and some constructs hardly promoted Raji cell proliferation at all. In some embodiments, such constructs are therefore expressed in NK cells and used to treat B-cell leukemia (or other tumor types).

[0283] Figure 9C shows data on NK cells from donor 1 against Nalm6 cells 14 days after transduction. The effector-to-target cell ratio is 1:1. As shown, even 2 weeks after transduction, NK cells expressing NK19-13, NK19-4, NK19-3, NK19-2, NK19-1, and NK19-9 substantially inhibited the proliferation of all Nalm6 cells and showed a high degree of cytotoxicity against tumor cells. In some embodiments, such constructs are therefore expressed in NK cells and used to treat B-cell leukemia (or other tumor types). As described above, in some embodiments, constructs are produced using combinations of two, three, or more stimulating domains that result in synergistic NK cell stimulation and enhanced cytotoxicity.

[0284] Figure 9D shows data on NK cells from donor 2 against Raji cells 14 days after transduction. The effector cell to target cell ratio was 1:2. As shown, NK cells expressing only GFP allowed essentially the same level of Raji cell proliferation as untreated Raji cells. In contrast, NK cells expressing NK19-3, NK19-1, NK19-4, NK19-13, NK19-2, and NK19-9, respectively, significantly delayed Raji cell proliferation, and several constructs allowed little to no Raji cell proliferation. In some embodiments, such constructs are therefore expressed in NK cells and used to treat B-cell leukemia (or other tumor types). As described above, in some embodiments, constructs are produced using combinations of two, three, or more stimulating domains that result in synergistic NK cell stimulation and enhanced cytotoxicity.

[0285] Figure 9E shows summary data of cytotoxicity against Nalm6 cells 7 days after transduction at E:T ratios of 1:1 and 1:2. At an E:T ratio of 1:2, all but one of the NK19 constructs showed cytotoxicity equal to or greater than that of NK cells expressing GFP alone. In fact, even at the 1:2 ratio, six of the constructs achieved approximately 50% or more cytotoxicity. When tested at E:T 1:1, seven of the constructs achieved approximately 50% or more cytotoxicity; however, five of the constructs (NK19-13, NK19-2, NK19-9, NK19-3, and NK19-1) achieved cytotoxicity exceeding 70%. Figure 9F shows the corresponding data for Raji cells. All constructs tested showed enhanced cytotoxicity beyond that of GFP-expressing NK cells at both 1:2 and 1:1 E:T ratios. At an E:T ratio of 1:2, four constructs exceeded 40% cytotoxicity, while at 1:1, seven constructs exceeded their mortality rate. Furthermore, at 1:1 E:T, three constructs produced 60% or greater cytotoxicity, with the most effective construct achieving nearly 90% cytotoxicity. In summary, these data indicate that various CD19-targeted CAR constructs can not only be expressed, but are also stably expressed and effective in inducing cytotoxicity in multiple cancer cell types, exceeding 80% mortality rates in some cases. According to further embodiments, CD19-targeting constructs using combinations of two, three or more stimulating domains can be produced, which further enhance the cytotoxicity of NK cells expressing them. In some embodiments, CD19-targeted constructs can synergistically interact with NK cells expressing receptors for other tumor markers, such as ligands for NKG2D (such chimeric receptor-carrying NK cells are described in PCT / US2018 / 024650, which is incorporated herein by reference in whole). For example, a chimeric receptor comprising a ligand for NKG2D, an OX40 stimulating domain, and a binding domain that binds to the CD3 zeta signaling domain can be used with any of the CD19 targeting constructs disclosed herein. In some embodiments, such a chimeric receptor has a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 145. In some embodiments, such a chimeric receptor has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 146.

[0286] Further experiments were conducted to evaluate the cytotoxicity of selected CD19-directed CAR constructs. NK cells isolated from three different donors (all three donors were in the 14-day experiment) were transduced with vectors encoding the following constructs: NK19-1 (OX40 co-stimulatory domain); NK19-2 (CD28 co-stimulatory domain); NK19-3 (ICOS co-stimulatory domain); NK19-4 (CD28-41BB co-stimulatory domain); NK19-9 (CD27 co-stimulatory domain); and NK19-13 (CD44 co-stimulatory domain). After 7 days (n=3) or 14 days (n=2) of transduction, these manipulated NK cells were co-cultured with Nalm6 cells or Raji cells in an E:T ratio of 1:1. The results are shown in Figure 10A (expressed as enhanced cytotoxicity percentage against GFP-expressing NK cells). Consistent with the data in Figure 9, each of the tested constructs resulted in enhanced cytotoxicity against Nalm6 cells, ranging from an average 40% increase in NK19-4 to an overall average of approximately 50% increase in the other five constructs. Figure 10B shows the corresponding data for Raji cells. Similarly, each construct was superior to GFP-only expressing NK cells, with the average increase in cytotoxicity over GFP NK cells ranging from approximately 40% to approximately 50%. Using cells from two donors 14 days after transduction, NK19-1, NK19-9, and NK19-13 were tested in Nalm6 and Raji cells. Figure 10C shows the enhanced cytotoxicity of these constructs over calculated GFP-expressing NK cells, with nearly 80% increases observed in NK19-9 expressing NK cells, approximately 75% in NK19-1 expressing cells, and over 60% in NK19-13 expressing cells. Similar results were observed with Raji cells – NK19-13-expressing cells showed a 20% improvement in cytotoxicity, NK19-1-expressing cells showed approximately 60% enhanced activity, and NK19-9-expressing cells showed nearly 70% or more enhanced cytotoxic activity against Raji cells. These results further support the use of the embodiments disclosed herein, in which engineered NK cells expressing CD19-targeted CARs are provided, as a method of cancer immunotherapy resulting in enhanced cytotoxicity against targeted tumor cells.

[0287] Figures 11A–11E show data on cytokine release profiles from NK cells cultured with Nalm6 cells, linking them to mechanisms by which NK cells control tumor and virus-infected cells through the release of cytotoxic granules and pro-inflammatory cytokines. Figure 11A shows that all NK cells expressing NK19-1, NK19-9, or NK19-13 express high concentrations of granzyme B, a serine protease present in granules released by NK cells, compared to control or GFP-expressing NK cells. These CD19-targeted CAR-expressing NK cells released approximately four times more granzyme B than control GFP-expressing NK cells. Figure 11B shows data related to increased perforin release by engineered NK cells. Interestingly, perforin levels did not substantially exceed the concentrations derived from GFP-expressing NK cells (however, perforin concentrations were higher than control). Perforin works in conjunction with granzyme B (and other granzymes) to create pores in the cell membrane, allowing granzymes to pass through, and then exert their protease effect on intracellular protein targets. This data suggests that perforin release may be nearly the same or reduced in relation to certain constructs, but actually more efficient at pore formation, and therefore lead to a similar degree of pore formation. Alternatively, if perforin released from NK19-expressing NK cells is not highly efficient at pore formation, this is offset by an increase in granzyme B (and / or other granzymes). Thus, enhanced cytotoxicity is still achieved.

[0288] Figure 11C shows that NK cells expressing NK19-1, NK19-9, or NK19-13 all release high concentrations of the inflammatory cytokine TNF-alpha compared to control or GFP-expressing NK cells. Figure 11D shows similar data regarding GM-CSF release by NK19-expressing NK cells, and Figure 11E shows similar data regarding interferon-gamma release. Similarly, when tested with Raji cells, similar release patterns are obtained, as shown in Figures 12A-12E. These data indicate that NK19-expressing cells exert cytotoxic effects, at least in part, through increased release of inflammatory cytokines and / or cytotoxic granules. As described above, in some embodiments, the engineered CARs are designed to have two, three, or more costimulatory domain combinations, resulting in synergistic increases in cytokine / granule release and cytotoxicity against the target cancer.

[0289] As further evidence of the increased effect of the NK19 construct on tumor progression, 1 × 10⁶ mice were given NK19 constructs on day 0 in NSG mice. 5Nalm6 cells (expressing a fluorescent reporter) were injected intravenously. On day 3, mice received either a PBS control injection, untransduced NK cells ("NTNK", 10M), NK19-2 expressing NK cells (10 million cells), NK19-9 expressing cells (10 million cells), NK19-1 expressing cells (10 million cells), or NK19-1 expressing cells (30 million cells). Fluorescence contrast imaging for Nalm6 cell detection was performed on days 3, 8, 11, 18, and 25 (contrast data are not shown). This data is shown in Figure 13. As shown, injection of NK cells expressing any of the NK19 variants reduced the progression of Nalm6 proliferation. NK19-2 expressing NK cells showed slight Nalm6 proliferation on day 8, further Nalm6 proliferation by day 11, and significant proliferation by day 18 (but less than that of NTNK cells). Neither NK19-9 nor NK19-1 (at any dose) expressed NK cells showed a contrast-enhanced tumor burden on day 8. Mice receiving NK19-9 expressed NK cells showed a slight increase in Nalm6 cell proliferation by day 11, which progressed further by day 18. On day 11, mice receiving either dose of NK19-1 expressed cells showed no Nalm6 cell proliferation. By day 18, mice receiving 10 million NK19-9 cells showed some tumor proliferation. However, mice receiving 30 million NK19-9 expressed NK cells showed only slight Nalm6 cell proliferation.

[0290] Figure 14A shows a line graph of bioluminescence intensity detected in mice, as indicated (e.g., the fluorescence signal shown in Figure 13; note that Figure 13 does not show contrast-enhanced data for day 25). Matching the image in Figure 13, the line graph in Figure 14A shows an increase in Nalm6 cell numbers across all groups at day 25 (indicated by an increase in BLI), with significant cell increases detected in the PBS and NTNK groups, and somewhat less in the NK19-2, NK19-1(10M), and NK19-9 groups. NK19-1(30M) showed the smallest increase, representing the construct's ability to reduce the rate of Nalm6 progression due to its cytotoxic effect on Nalm6 cells. While each construct ultimately allowed some Nalm6 proliferation, the tested NK19 constructs delayed the onset of proliferation, even slightly, as evidenced by the horizontal line up to day 11 of the NK19 curve. To better illustrate this aspect, the data were replotted on a logarithmic scale Y-axis, separating the curves, in Figure 14B. As illustrated, the NTNK and PBS curves show an upward trend from day 3 onwards, indicating Nalm6 proliferation almost immediately. In contrast, the NK19-9, NK19-2, and both NK19-1 curves are either declining or showing a slight upward trend until day 7. On day 11, consistent with the image in Figure 13, Nalm6 cell proliferation is detected in the NK19-9, NK19-2, and NK19-1 (10M) groups. In contrast, the NK19-1 (30M) group is near baseline, reflecting the absence of any significant Nalm6 cell proliferation. Cell proliferation shows an upward trend in the NK19-9 (30M) group on day 18. Although administration of NK19 constructs increases the number of tumor cells, the delayed onset of proliferation is advantageous in some embodiments. For example, this provides an opportunity to readmit the patient to a further dose of engineered NK cells expressing a CD19-targeted construct. In some embodiments, subsequent doses (as possible with the initial dose) may include NK cells edited to reduce allogenicity, for example, by gene editing, as desired. Thus, in some embodiments, CD19-directed CAR-expressing NK cells are administered two, three, four or more times.This delay in tumor cell proliferation provides an opportunity to sequentially (or simultaneously) administer NK cells expressing chimeric constructs for various tumor markers and / or some other diverse anti-cancer therapies (e.g., checkpoint inhibitors, antibody therapy, chemotherapy, etc.). Figure 14C shows data on CD3 expression in cells transduced with various constructs in blood samples taken from mice treated as shown on the X axis. CD3 is a T cell marker. As shown, however, CD3 expression was negligible for T cells engineered to express the NK19-1 construct and for mixed populations of NK cells and T cells. Figure 14D shows data on CD56 expression, a marker for human NK cells. There is slight background staining, but the blood samples from mice treated with NK cells expressing the shown constructs show relatively low expression (as can be seen considering (i) the blood is a mouse blood sample, mouse cells make up the majority of the sample, and (ii) CD56 is only detected in human NK cells (e.g., administered)). This data is consistent in that the 30M NK19-1 treated group shows significantly higher CD56 expression than the other groups and the T cell / NK+ T cell group expressing CD56 at background levels. Figure 14E shows data on GFP expression by tumor cells. Blood samples were collected approximately 3 weeks into the in vivo experiment. As shown (consistent with image / BLI data), the control PBS and NTNK groups show high GFP expression percentages (e.g., the majority of total viable blood cells in the sample are tumor cells). Each of the engineered constructs shows significantly lower GFP expression based on the control / reduction of tumor cell proliferation by the engineered construct. Figure 14F shows data similar to Figure 14D, but with CD19 expression measured across all viable cells in a mouse blood sample. Similar to GFP, the PBS and NKNT groups show approximately 10-12% CD19 expression, indicating that 10-12% of viable cells in the blood sample are tumor cells, with the remainder being mouse blood cells. As shown in other experimental groups, the significantly lower CD19 expression reflects the proliferation of tumor cells in the manipulated CAR construct.These data are consistent with the embodiments disclosed herein, in which engineered NK cells expressing CD19-directed CARs are highly cytotoxic and enable treatment of cancerous tumors.

[0291] Further evaluation of humanized structures As detailed above, in some embodiments, the CARs disclosed herein in some embodiments include the use of a humanized sequence, such as in the extracellular binding portion. In some embodiments, one or more aspects of that region are subjected to a humanized campaign. In some embodiments, one or more of the heavy and / or light chains of the antibody are humanized, which may provide benefits including, but are not limited to, reduced immunogenicity, increased stability, long-term efficacy, and increased potency (as described above).

[0292] Example 3 Figure 15 shows schematic diagrams of a series of humanized constructs according to several embodiments disclosed herein. Such constructs are designated by the identification noun "H". For illustrative purposes, NK19H-1 is a humanized anti-CD19 CAR using an scFv consisting of the first light chain and first heavy chain ('L1H1'), while NK19H-3 uses an scFv consisting of the third light chain and first heavy chain ('L3H1'). Figure 15 also shows data on the stability and aggregation of various heavy and light chain combinations after transient expression and secretion from 293T cells. The data are shown in relation to the mean fluorescence intensity detected by flow cytometry when each antibody was heated to 70°C and cooled to room temperature ('heated') versus when the same variant was maintained on ice ('unheated'). After heat treatment, the ScFv variants were used in flow cytometry protocols at various concentrations (0.5, 0.25, and 0.125 μg / mL). Loss of fluorescence intensity indicates that the ScFv has lost its structural integrity or aggregated due to heat treatment. ScFv exhibiting the best thermal stability, as shown by MFI (Multi-Focused Inspection) comparable under both conditions, is preferable for further development in certain embodiments.

[0293] After evaluating the stability of the constructs, the selected anti-CD19 CAR constructs were further evaluated. Figure 16 shows summary data of the expression of the selected constructs by NK cells from three donors (#140, #9, and #20). As shown in the figure, expression was evaluated by the detection of CD19 Flag. The data is expressed as the percentage of CD19-Flag expressing NK cells relative to the total number of NK cells present. This data was collected 4 days after transduction with a suitable virus encoding an NK19H-"X" CAR. As shown in the expression data, each of the selected constructs expressed NK cells to varying degrees. Expression levels ranged from approximately 20% expression of total NK cells for NK19H-2, NK19H-11, and NK19H-12. Most of the other constructs were sufficiently expressed up to approximately 40%–60% of NK cells, comparable to the expression of the unhumanized NK19-1 construct. The NK19H-5 construct was expressed by over 80% of NK cells. While some constructs may be expressed more efficiently, those with lower expression levels were also evaluated because, in some embodiments, such constructs still exhibited significant cytotoxicity to target cells. According to some embodiments, however, higher expression efficiency may be advantageous, for example, because a significant portion of a given NK cell preparation is clinically useful (e.g., fewer inserted NK cells are needed to produce a clinically relevant manipulated NK cell dose).

[0294] Figures 17A–17E describe this cytotoxicity data. Figure 17A shows the cytotoxicity of NK cells derived from donor 20 and transduced with the shown construct against a CD19-positive Nalm6 leukemia cell line (E:T 1:1; 20K cells / well). This data indicates that, despite varying expression levels, most NK19H constructs were able to exert a cytotoxic effect on Nalm6 target cells. NK19H-11 showed minimal efficacy, allowing Nalm6 cell proliferation to just below the control level. In contrast, NK19H-1 and NK19H-3 showed cytotoxicity comparable to the unhumanized NK19-1 construct, allowing some cell proliferation at subsequent points in co-culture. Notably, NK19H-4 and NK19H-5 showed significant cytotoxicity, exhibiting only very limited Nalm6 proliferation throughout the experiment.

[0295] Figure 17B shows NK cells from donor 20 tested against the CD19-positive Burkitt lymphoma cell line Raji. Similar to Nalm6 cells, various humanized anti-CD19 constructs exhibited varying levels of cytotoxicity against the target cells. As with the data in Figure 17A, the NK19H-11 construct showed limited cytotoxicity, while all other constructs showed promising cytotoxicity against the target cells. NK19H-1 functioned comparably to the unhumanized NK19-1 construct, while each of the NK19H-3, 19H-4, and 19H-5 constructs showed high levels of cytotoxicity, limiting Raji proliferation to subsequent stages of co-culture (NK19H-5 allowed only very limited Raji cell proliferation). Figure 17C shows the corresponding Nalm6 data from donor 140. Here, a similar pattern of efficacy was detected, with NK19H-11 allowing Nalm6 proliferation similar to that of the negative control. However, each of NK19H-1, 19H-3, and 19H-4 showed at least the same level of cytotoxicity as unhumanized NK19-1. Similarly, NK19H-5 showed significant cytotoxicity and limited Nalm6 proliferation throughout the experiment. Figure 17D shows data from donor 9 against Raji cells. Only NK19H-11 allowed some substantial Raji cell proliferation. In contrast, NK cells from this donor expressing unhumanized NK19-1 or any of the humanized NK19H-1, 19H-3, 19H-4, or 19H-5 constructs completely suppressed Raji cell proliferation via the induction of cytotoxic effects. Figure 17E shows data from NK cells from donor 9 against Nalm6 cells. In this experiment, the cytotoxicity of the three humanized constructs (NK19H-11, 19H-1, and 19H-3) was limited. There was some inter-donor variability, as expression of any of these constructs induced cytotoxicity in NK cells from other donors. The NK19H-4 and NK19H-5 constructs showed significant cytotoxic effects, limiting Nalm6 proliferation to nearly zero throughout the experiment. In summary, these data are consistent with the expression data and, in several embodiments, demonstrate that humanized CARs targeting CD19 are effective in killing tumor cells, even with variability in expression efficiency. Furthermore, in some embodiments, expression efficiency does not correlate with cytotoxicity, and constructs with limited expression efficiency can still exhibit significant cytotoxicity.

[0296] Example 4 Further experiments were conducted in parallel with the above using NK cells from additional donors. Figure 18 shows the construct expression efficiency from three donors (#945, 137, and 138) for the constructs shown. As with the previous experiments, the data shown represent the number of CD19-Flag positive cells out of the total number of NK cells evaluated. The data for these donors showed high overall expression efficiency for all but one humanized construct. Only NK19H-3 was expressed less than the unhumanized NK19-1 construct. In these donor NK cells, expression of the humanized construct was detected in approximately 70% to 80% of the NK cells. As described above, following the evaluation of expression, cytotoxicity against CD19-expressing target cells was evaluated. Figure 19A shows data for NK cells from donor (#137) that expressed the construct shown and were co-cultured with Raji cells. In this experiment, NK cells expressing the manipulated construct shown were exposed to tumor cells at two points: 7 days after transduction and 14 days after the addition of a further large dose of tumor cells. The arrows indicate the second administration of tumor cells. As shown, untreated Raji cells expanded throughout the experiment. NK cells expressing GFP or non-humanized NK19-1 induced some cytotoxicity compared to controls, as indicated by the reduction in Raji cell proliferation. NK cells expressing NKH19-3 (the humanized construct with the lowest expression efficiency) were also able to reduce Raji proliferation. Each of the other humanized NK constructs was able to reduce Raji cell proliferation compared to controls, even 14 days after transduction, demonstrating the sustained enhancement of engineered NK cells disclosed here. Figure 19B shows the corresponding data for donor 137 NK cells against Nalm6 cells, with engineered NK cells added similarly at 7 days post-transduction (experiment day 0) and 14 days post-transduction (experiment day approximately 7). The cytotoxicity of the constructs shown was more variable in this particular experiment. However, several humanized anti-CD19 constructs were able to produce significant cytotoxicity, reducing Nalm6 cell proliferation compared to controls. These data suggest that, according to some embodiments, more frequent dosing schedules (e.g., every 2 days, every 3 days, every 4 days, every 5 days, etc.) may be beneficial for certain subjects. Advantageously, in some embodiments, the manipulated NK cells described herein are homogeneous and can be readily used in more frequent dosing regimens.Figure 19C shows the corresponding data for donor 138 against Raji cells. Here, most of the humanized constructs still exhibited significant cytotoxic effects on Raji cells even after a second administration 14 days after transduction. Six out of seven humanized constructs showed this behavior, outperforming the unhumanized NK19-1 constructs. Figure 19D shows the corresponding data for donor 138 against Nalm6 cells. Further high doses of Nalm6 increased Nalm6 proliferation in the final stages of the experiment, but almost all of the humanized constructs performed better than the control. In fact, NK19H-5-carrying NK cells were able to limit Nalm6 proliferation until the last few days of the experiment (after the second administration). As described above, these data demonstrate that humanized anti-CD19 CAR constructs can not only be expressed by NK cells but can also exert cytotoxic activity against target cells with enhanced persistence. In some embodiments, this allows for the drug to be divided into multiple doses over a long period, which is advantageous for cancer treatment, while limiting potential immunogenicity (at least by partial humanization and / or reduced administration frequency).

[0297] Example 5 Further data on various humanized constructs were obtained from additional donors. Figures 20A–20B show expression data for various anti-CD19 CAR constructs in NK cells from two additional donors. Figure 20A shows the mean fluorescence intensity of NK cells 10 days after transduction. As shown, consistent with several embodiments disclosed herein, each of the humanized anti-CD19 CAR constructs showed enhanced overall expression compared to the non-humanized anti-CD19 CAR. Figure 20B shows expression data for CD19-Flag positive NK cells as a percentage of the total number of NK cells analyzed. As shown, each of the humanized anti-CD19 constructs was expressed more efficiently than the non-humanized construct (which was already expressed in approximately 80% of NK cells). Humanized CARs were expressed in approximately 85%–95% of NK cells by the construct. Figure 21A shows cytotoxicity data of engineered NK cells from donor 703 (one of the two donors in Figure 20) against Raji cells, co-cultured again with NK cells 7 and 14 days after transduction. As ...

Claims

1. An anti-CD19 antibody or its antigen-binding fragment comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 120 and the VL domain comprises the amino acid sequence of SEQ ID NO:

118.

2. The anti-CD19 antibody or its antigen-binding fragment according to claim 1, which is a single-stranded variable fragment (scFv).

3. A chimeric antigen receptor (CAR) comprising an anti-CD19 antibody or its antigen-binding fragment, a transmembrane domain, and an intracellular signaling domain as described in claim 1 or 2.

4. An anti-CD19 antibody or its antigen-binding fragment according to claim 1 or 2, or a polynucleotide encoding a CAR according to claim 3.

5. A vector comprising the polynucleotide described in claim 4.

6. The vector according to claim 5, which is a retrovirus vector.

7. An anti-CD19 antibody or its antigen-binding fragment according to claim 1 or 2, or an immune cell expressing the CAR according to claim 3.

8. An immune cell comprising the polynucleotide described in claim 4, or the vector described in claim 5 or 6.

9. The immune cell according to claim 7 or 8, which is a natural killer (NK) cell or a T cell.

10. Natural killer (NK) cells expressing the anti-CD19 antibody or its antigen-binding fragment according to claim 1 or 2.

11. Natural killer (NK) cells expressing the CAR described in claim 3

12. A pharmaceutical composition comprising an anti-CD19 antibody or its antigen-binding fragment according to claim 1 or 2, or NK cells according to a plurality of claims 10 or 11.

13. A pharmaceutical composition comprising the polynucleotide described in claim 4, or the vector described in claim 5 or 6.

14. A pharmaceutical composition according to claim 12 for the treatment of cancer.

15. A pharmaceutical composition according to claim 13 for the treatment of cancer.

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