ANAPLASTIC LYMPHOMA KINASE CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE
20230101046 · 2023-03-30
Assignee
Inventors
Cpc classification
C12Y207/10001
CHEMISTRY; METALLURGY
C07K14/705
CHEMISTRY; METALLURGY
A61K35/17
HUMAN NECESSITIES
C12N2740/10043
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
A61K2300/00
HUMAN NECESSITIES
C07K14/523
CHEMISTRY; METALLURGY
C07K14/535
CHEMISTRY; METALLURGY
A61K2300/00
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
A61K31/437
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A61K31/437
HUMAN NECESSITIES
C12N15/625
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
International classification
A61K31/675
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
C07K14/535
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
Abstract
Provided herein are anaplastic lymphoma kinase chimeric antigen receptors (ALK CARs). The invention also provides polynucleotides encoding ALK CARs, engineered immune cells comprising an ALK CAR, pharmaceutical compositions thereof, and kits for administering the same. Methods of treating a subject with a disease by administering the ALK CAR or engineered immune cell comprising an ALK CAR, or pharmaceutical compositions thereof, are also provided.
Claims
1. An anaplastic lymphoma kinase chimeric antigen receptor (ALK CAR) comprising: an extracellular binding domain comprising (a) or (b) (a) a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3) each comprising an amino acid sequence that is at least 80% identical to the HCDR1, HCDR2, and HCDR3 sequences of an anti-ALK antibody in Table 4, wherein the extracellular binding domain specifically binds to an anaplastic lymphoma kinase (ALK) polypeptide or antibody-binding fragment thereof; or (b) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to the VH of an anti-ALK antibody in Table 2, wherein the extracellular binding domain specifically binds to an anaplastic lymphoma kinase (ALK) polypeptide or antibody-binding fragment thereof; a transmembrane domain; and at least one signaling domain.
2. The ALK CAR of claim 1, wherein the extracellular binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of an anti-ALK antibody in Table 4 or the VH of an anti-ALK antibody in Table 2.
3. The ALK CAR of claim 1, wherein the extracellular binding domain further comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3) each comprising an amino acid sequence that is at least 80% identical to the LCDR1, LCDR2 and LCDR3 sequences of an anti-ALK antibody in Table 3.
4-15. (canceled)
16. The ALK CAR of claim 1, wherein the extracellular binding domain is an scFv.
17. The ALK CAR of claim 1, wherein the anti-ALK antibody comprises VH CDR amino acid sequences SYWMN, QIYPGDGDTNYNGKFKG, and YYYGSKAY, and VL CDR amino acid sequences RASENIYYSLA, NANSLED, KQAYDVPFT, VH CDR amino acid sequences TABLE-US-00170 SYWMH, RIDPNSGGTKYNEKFKS, and TABLE-US-00171 DYYGSSYRFAY, and VL CDR amino acid sequences TABLE-US-00172 SVSQGISNSLN, YTSSLHS and TABLE-US-00173 QQYSKLPLT; VH CDR amino acid sequences TABLE-US-00174 NYWMH, YINPSSGYTKYNQKFKD, and TABLE-US-00175 DYYGSSSWFAY, and VL CDR amino acid sequences TABLE-US-00176 KASQNVGTNVA, SASYRYS, and TABLE-US-00177 QQYNSYPYMYT; VH CDR amino acid sequences TABLE-US-00178 SYWVN, QIYPGDGDTNYNGKFKG, and TABLE-US-00179 SRGYFYGSTYDS, and VL CDR amino acid sequences TABLE-US-00180 RASESVDNYGISFMN, AASNQGS. and TABLE-US-00181 QQSKEVPWT; VH CDR amino acid sequences TABLE-US-00182 SYWMH, YIKPSSGYTKYNQKFKD, and TABLE-US-00183 DYYGSSSWFAY, and VL CDR amino acid sequences TABLE-US-00184 KASQNVGTNVA, SASYRYS, and TABLE-US-00185 QQYNSYPYMYT; VH CDR amino acid sequences TABLE-US-00186 SYAMS, YISSGGDYIYYADTVKG, and TABLE-US-00187 ERIWLRRFFDV, and VL CDR amino acid sequences TABLE-US-00188 KASQNVGTAVA, SASNRFT, and TABLE-US-00189 QQYSSYPLT; VH CDR amino acid sequences TABLE-US-00190 SYWMH, YINPSSGYTKYNQKFKD, and TABLE-US-00191 DYYGSSSWFAY. and VL CDR amino acid sequence TABLE-US-00192 KASQNVGTNVA, SASYRYS. and TABLE-US-00193 QRYNSYPYMFT.
18-23. (canceled)
24. The ALK CAR of claim 1, wherein the anti-ALK antibody comprises VH amino acid sequence TABLE-US-00194 QVQLQQSGAELVKPGASVKISCKASGYAFSSYWMNWV KQRPGKGLEWIGQIYPGDGDTNYNGKFKGKATLTADK SSSTAYMQLSSLTSEDSAVYFCASYYYGSKAYWGQGT LVTVSA, and VL amino acid sequence TABLE-US-00195 DIQMTQSPASLAASVGETVTITCRASENIYYSLAWYQQ KQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQYSMKIN SMQPEDTATYFCKQAYDVPFTFGSGTKLEIKR.
25. The ALK CAR of claim 1, wherein the anti-ALK antibody comprises VH amino acid sequence TABLE-US-00196 QVQLQQPGAEFVKPGASVKLSCKASGYTFTSYWMHWV KQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLTVDK PSSTAYMQLSSLTSEDSAVYYCARDYYGSSYRFAYWG QGTLVTVSA, and VL amino acid sequence TABLE-US-00197 AIQMTQTTSSLSASLGDRVTISCSVSQGIS NSLNWYQQKPDGTVKLLIYYTSSLHSGVPS RFSGSGSGTDYSLTISNLEPEDIATYYCQQ YSKLPLTFGAGTKLELKR; VH amino acid sequence TABLE-US-00198 QVQLQQSGAELAKPGASVKLSCKASGYT FTNYWMHWVKQRPGQGLEWIGYINPSSG YTKYNQKFKDKATLTADKSSSTAYMQLS SLTYEDSAVYYCARDYYGSSSWFAYWGQ GTLVTVSA, and VL amino acid sequence TABLE-US-00199 DIVMTQSQRFMSTSVGDRVSVTCKASQNV GTNVAWYQQKPGQSPKALIYSASYRYSGV PDRFTGSGSGTDFTLTVSNVQSEDLAEYF CQQYNSYPYMYTFGGGTKLEIKR; VH amino acid sequence TABLE-US-00200 QVQLQQSGAELVKPGASVKISCKASGYAF SSYWVNWVKQRPGKGLEWIGQIYPGDGDT NYNGKFKGKATLTADKSSSTAYMQLSSLT SEDSAVYFCARSRGYFYGSTYDSWGQGTT LTVSS, and VL amino acid sequence TABLE-US-00201 DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFM NWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTD FSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIK R; VH amino acid sequence TABLE-US-00202 QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMHWV KQRPGQGLEWIGYIKPSSGYTKYNQKFKDKATLTADK SSSTAYMQLSSLTYEDSAVYYCARDYYGSSSWFAYWG QGTLVTVSA, and VL amino acid sequence TABLE-US-00203 DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNVAWYQQ KPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTIS NVQSEDLAEYFCQQYNSYPYMYTFGGGTKLEIKR; VH amino acid sequence TABLE-US-00204 DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMSWVR QTPEKRLEWVTYISSGGDYIYYADTVKGRFTISRDNAR NTLYLQMSSLKSEDTAMYYCTRERIWLRRFFDVWGTGT TVTVSS. and VL amino acid sequence TABLE-US-00205 DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQL KPGQSPKLLIYSASNRFTGVPDRFTGSGSGTDFTLTIS NMQSEDLADYFCQQYSSYPLTFGSGTKLEIKR; or VH amino acid sequence TABLE-US-00206 QVQLQQSGAELAKPGASVKLSCKASGYT FTSYWMHWVKQRPGQGLEWIGYINPSSG YTKYNQKFKDKATLTADKSSSTAYMQLS SLTFEDSAVYYCARDYYGSSSWFAYWGQ GTLVTVSA, and VL amino acid sequence TABLE-US-00207 DIVMTQSQKFMSTSVGDRVSVTCKASQNV GTNVAWYQQKPGHSPKALIYSASYRYSGV PDRFTGSGSGTDFTLTISNVQSEDLAEYF CQRYNSYPYMFTFGGGTKLEIKR.
26-30. (canceled)
31. The ALK CAR of claim 1, wherein the transmembrane domain is selected from the group consisting of CD8, CD137 (4-1BB), and CD28.
32. (canceled)
33. The ALK CAR of claim 1, wherein the at least one signaling domain is selected from the group consisting of CD8, CD28, CD134 (OX40), CD137 (4-1BB), and CD3ζ.
34-41. (canceled)
42. A polynucleotide encoding the ALK CAR of claim 1.
43. A vector comprising the polynucleotide of claim 42.
44-46. (canceled)
47. An engineered immune cell expressing at the cell surface membrane an ALK CAR according to claim 1.
48. An engineered immune cell produced by transforming an immune cell with the polynucleotide of claim 42.
49-55. (canceled)
56. The engineered immune cell of claim 48, wherein the ALK-positive cancer has an ALK.sup.F1174L activating point mutation.
57. A method of engineering an immune cell comprising: providing an immune cell; and expressing at the surface of the immune cell at least one ALK CAR according to claim 1.
58. A method of engineering an immune cell comprising: providing an immune cell; introducing into the immune cell the polynucleotide according to claim 42; and expressing the polynucleotide in the immune cell.
59-60. (canceled)
61. A pharmaceutical composition comprising the ALK CAR of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.
62. (canceled)
63. A method of treating a subject with an ALK-positive cancer comprising administrating the pharmaceutical composition of claim 62 to the subject.
64. A method of treating a subject with an ALK-positive cancer comprising administering to the subject the ALK CAR of claim 1.
65. A method of treating a subject with an ALK-positive cancer, the method comprising: transforming immune cells with the vector of claim 43 to obtain an engineered immune cell; and administering an effective amount of the engineered immune cell to the subject.
66. The method of claim 65, wherein the immune cells are derived from the subject or a donor.
67-68. (canceled)
69. A method of treating a subject with an ALK-positive cancer, the method comprising administering an effective amount of an engineered immune cell comprising an ALK CAR and an effective amount of an ALK vaccine comprising at least one isolated ALK polypeptide or polynucleotide to the subject.
70-81. (canceled)
82. The method of claim 63, wherein the ALK-positive cancer has an ALK.sup.F1174L activating point mutation.
83. A kit comprising an agent for administration to a subject, wherein the agent comprises the ALK CAR of claim 1, and instructions for using the kit.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0266] As described below, the present invention features anaplastic lymphoma kinase chimeric antigen receptors (ALK CARs) and engineered immune cells comprising ALK CARs (e.g., ALK CAR-T cells). The ALK CARs of the present invention feature ALK antibody sequences that specifically bind to an ALK protein (e.g., ALK extracellular domain). The present invention also features polynucleotides encoding for ALK CARs. The ALK CAR, polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR may be used in methods to treat and/or reduce disease in a subject (e.g. ALK-positive cancer (e.g., neuroblastoma)).
[0267] The ALK CARs, polynucleotides encoding an ALK CARs, or engineered immune cells comprising ALK CARs described herein may also be used in pharmaceutical compositions that treat ALK-positive cancers (e.g., neuroblastoma) in a subject, particularly a human subject, to whom the pharmaceutical composition, is administered. ALK CARs, polynucleotides encoding an ALK CARs, or engineered immune cells comprising ALK CARs, and pharmaceutical compositions thereof, of the invention provide an additional treatment option for patients that have either become resistant to or have failed to respond to prior and traditional therapies for ALK-positive cancers.
Anaplastic Lymphoma Kinase Chimeric Antigen Receptors (ALK CARs) and CAR-T Cells
[0268] The invention provides anaplastic lymphoma kinase chimeric antigen receptors (ALK CARs) and immune effector cells that express ALK CARs. Immune effector cells expressing a chimeric antigen receptor (CAR) can enhance an immune effector cell's immunoreactive activity, wherein the CAR has an affinity for an epitope on an antigen (e.g., ALK), wherein the antigen is associated with an altered fitness of an organism. For example, the CAR can have an affinity for an epitope on a protein expressed in a neoplastic cell (e.g., ALK-positive cancer (e.g., neuroblastoma)). Because the CAR-T cells can act independently of major histocompatibility complex (MHC), activated CAR-T cells can kill the neoplastic cell expressing the antigen. The direct action of the CAR-T cell evades neoplastic cell defensive mechanisms that have evolved in response to WIC presentation of antigens to immune effector cells.
[0269] Some embodiments comprise autologous immune effector cell immunotherapy, wherein immune effector cells are obtained from a subject having a disease or altered fitness characterized by cancerous or otherwise altered cells expressing a surface marker (e.g., ALK-positive cancer (e.g., neuroblastoma)). The obtained immune effector cells are genetically modified to express a CAR and are effectively redirected against specific antigens (e.g., ALK). Thus, in some embodiments, immune effector cells are obtained from a subject in need of CAR-T immunotherapy. In some embodiments, these autologous immune effector cells are cultured and modified shortly after they are obtained from the subject. In other embodiments, the autologous cells are obtained and then stored for future use. This practice may be advisable for individuals who may be undergoing parallel treatment that will diminish immune effector cell counts in the future. In allogeneic immune effector cell immunotherapy, immune effector cells can be obtained from a donor other than the subject who will be receiving treatment. The immune effector cells, after modification to express a CAR, are administered to a subject for treating a neoplasia (e.g., ALK-positive cancer (e.g., neuroblastoma)). In some embodiments, immune effector cells to be modified to express a CAR can be obtained from pre-existing stock cultures of immune effector cells.
[0270] Immune effector cells can be isolated or purified from a sample collected from a subject or a donor using standard techniques known in the art. For example, immune effector cells can be isolated or purified from a whole blood sample by lysing red blood cells and removing peripheral mononuclear blood cells by centrifugation. The immune effector cells can be further isolated or purified using a selective purification method that isolates the immune effector cells based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO. Another technique for isolating or purifying immune effector cells is flow cytometry. In fluorescence activated cell sorting a fluorescently labelled antibody with affinity for an immune effector cell marker is used to label immune effector cells in a sample. A gating strategy appropriate for the cells expressing the marker is used to segregate the cells. For example, T lymphocytes can be separated from other cells in a sample by using, for example, a fluorescently labeled antibody specific for an immune effector cell marker (e.g., CD4, CD8, CD28, CD45) and corresponding gating strategy. In one embodiment, a CD45 gating strategy is employed. In some embodiments, a gating strategy for other markers specific to an immune effector cell is employed instead of, or in combination with, the CD45 gating strategy.
[0271] In some embodiments, the immune effector cells contemplated in the invention are effector T cells. In some embodiments, the effector T cell is a naïve CD8.sup.+ T cell, a cytotoxic T cell, a natural killer T (NKT) cell, or a regulatory T (Treg) cell. In some embodiments, the effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments the immune effector cell is a CD4.sup.+ CD8.sup.+ T cell or a CD4.sup.− CD8.sup.− T cell. In some embodiments the immune effector cell is a T helper cell. In some embodiments the T helper cell is a T helper 1 (Th1), a T helper 2 (Th2) cell, or a helper T cell expressing CD4 (CD4+ T cell). In some embodiments, the immune effector cell is any other subset of T cells. The modified immune effector cell may express, in addition to the CAR, an exogenous cytokine, a different chimeric receptor, or any other agent that would enhance immune effector cell signaling or function. For example, coexpression of the chimeric antigen receptor and a cytokine may enhance the CAR-T cell's ability to lyse a target cell. Nonlimiting examples of cytokines include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), the protein memory T-cell attractant “Regulated on Activation; Normal T Expressed and Secreted” (RANTES), granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), or interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α). In some embodiments, the cytokines are of human origin (e.g., hIL-1, hIL-2, hIL-4, hIL-6, hIL-7, hIL-12, hIL-15, hIL-21, hRANTES, hGM-CSF, hTNF-α, hTNF-α, hIFNγ or hMIP-1α).
[0272] Disclosed herein are ALK CARs that are artificially constructed chimeric proteins including an extracellular antigen binding domain (e.g., single chain variable fragment (scFv)) that specifically binds to ALK), linked to a transmembrane domain, linked to one or more intracellular T-cell signaling domains. Characteristics of the disclosed ALK CARs include their ability to redirect T-cell specificity and reactivity towards ALK expressing cells in a non-MHC-restricted manner. The non-MHC-restricted ALK recognition gives T cells expressing a disclosed CAR the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Binding of an antigen (e.g., ALK) to the extracellular binding domain can activate the CAR-T cell and generate an effector response, which includes CAR-T cell proliferation, cytokine production, and other processes that lead to the death of the antigen expressing cell.
[0273] In some embodiments, the ALK CAR further comprises a linker. In some embodiments, the ALK CAR further comprises a signal peptide. In some embodiments, the ALK CAR further comprises a reporter gene (e.g., green fluorescent protein (GFP)). In some embodiments, the ALK CAR further comprises a splice donor and/or splice acceptor sequences (e.g., CMV and/or HTLV splice acceptor and donor sequences). In some embodiments, the ALK CAR further comprises a packaging signal.
[0274] Provided herein are nucleic acids that encode the ALK CARs described herein. In some embodiments, the nucleic acid is isolated or purified. Delivery of the nucleic acids ex vivo can be accomplished using methods known in the art. For example, immune effector cells obtained from a subject (e.g., mammal) may be transformed with a nucleic acid vector encoding the CAR. The vector may then be used to transform recipient immune effector cells so that these cells will then express the CAR. Efficient means of transforming immune effector cells include transfection and transduction. Such methods are well known in the art. For example, applicable methods for delivery the nucleic acid molecule encoding the chimeric antigen receptor can be found in International Application No. PCT/US2009/040040 and U.S. Pat. Nos. 8,450,112; 9,132,153; and 9,669,058, each of which is incorporated herein in its entirety.
[0275] The ALK CARs can be of any length, i.e., can comprise any number of amino acids (or nucleotides encoding amino acids), provided that the CARs retain their biological activity, e.g., the ability to specifically bind to an antigen (e.g., ALK), detect diseased cells in a mammal, or treat or prevent disease (e.g. ALK-positive cancer (e.g., neuroblastoma)) in a subject (e.g., mammal). In some embodiments, the CAR is about 50 to about 5000 amino acids long. In some embodiments, the CAR is about 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.
[0276] In some embodiments, the CAR construct is derived from or comprises the m1928z CAR construct as provided in Davila et al., CD19 CAR-Targeted T Cells Induce Long-Term Remission and B Cell Aplasia in an Immunocompetent Mouse Model of B Cell Acute Lymphoblastic Leukemia, PLoS ONE (2013), which is incorporated by reference in its entirety herein.
Extracellular Binding Domain
[0277] The ALK CARs contemplated herein include an extracellular binding domain. The extracellular binding domain of an ALK CAR contemplated herein comprises an amino acid sequence of an antibody, or an antigen binding fragment thereof, that has an affinity for a specific antigen (e.g., ALK). In some embodiments the ALK CAR comprises an amino acid sequence of an ALK antibody. In some embodiments, the ALK CAR comprises the amino acid sequence of an antigen binding fragment of an ALK antibody. The ALK antibody (or fragment thereof) portion of the extracellular binding domain recognizes and binds to an epitope of an antigen (e.g., ALK). In some embodiments, the antibody fragment portion of an ALK CAR receptor is a single chain variable fragment (scFv). An scFv comprises the light and heavy variable domains of a monoclonal antibody. In other embodiments, the antibody fragment portion of an ALK CAR is a multichain variable fragment, which can comprise more than one extracellular binding domain and therefore bind to more than one antigen simultaneously. In a multiple chain variable fragment embodiment, a hinge region may separate the different variable fragments, providing necessary spatial arrangement and flexibility.
[0278] In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide (e.g., ALK protein). Antigens can be heterologous, such as those expressed in a pathogenic bacteria or virus. Antigens can also be synthetic; for example, some individuals have extreme allergies to synthetic latex and exposure to this antigen can result in an extreme immune reaction. In some embodiments, the antigen is autologous, and is expressed on a diseased or otherwise altered cell. For example, in some embodiments, the antigen (e.g., ALK protein) is expressed in a neoplastic cell (e.g., ALK-positive cancer (e.g., neuroblastoma)). In some embodiments, the neoplastic cell is an ALK-positive cancer. In some embodiments, the ALK-positive cancer is non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, and melanoma. In some embodiments, the ALK-positive cancer is neuroblastoma.
[0279] Antibody-antigen interactions are noncovalent interactions resulting from hydrogen bonding, electrostatic or hydrophobic interactions, or from van der Waals forces. The affinity of extracellular binding domain of the chimeric antigen receptor for an antigen can be calculated with the following formula:
K.sub.A=[Antibody−Antigen]/[Antibody][Antigen], wherein [0280] [Ab]=molar concentration of unoccupied binding sites on the antibody; [0281] [Ag]=molar concentration of unoccupied binding sites on the antigen; and [0282] [Ab−Ag]=molar concentration of the antibody-antigen complex.
The antibody-antigen interaction can also be characterized based on the dissociation of the antigen from the antibody. The dissociation constant (K.sub.D) is the ratio of the association rate to the dissociation rate and is inversely proportional to the affinity constant. Thus, K.sub.D=1/K.sub.A. Those skilled in the art will be familiar with these concepts and will know that traditional methods, such as ELISA assays, can be used to calculate these constants.
[0283] In some embodiments, the antibody portion of an ALK CAR comprises at least one heavy chain (H). In some embodiments, the antibody portion of an ALK CAR comprises at least one light chain (L). In some embodiments, the antibody portion of an ALK CAR comprises at least one heavy chain (H) and at least one light chain (L). In some embodiments, the antibody portion of an ALK CAR comprises two heavy chains, joined by disulfide bridges and two light chains, wherein the light chains are each joined to one of the heavy chains by disulfide bridges. In some embodiments, the light chain comprises a constant region (LC) and a variable region (VL). In some embodiments, the heavy chain comprises a constant region (HC) and a variable region (VH). Complementarity determining regions (CDRs) residing in the variable region of an antibody are responsible for the antibody's affinity for a particular antigen. Thus, antibodies that recognize different antigens comprise different CDRs. CDRs reside in the variable domains of the extracellular binding domain, and variable domains (i.e., the VH and VL) can be linked with a linker or, in some embodiments, with disulfide bridges.
[0284] In some embodiments, the extracellular binding domain of the ALK CAR includes sequences from an anti-ALK antibody. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody selected from ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7. In some embodiments, the extracellular binding domain includes VH and/or VL sequences from an anti-ALK antibody. In some embodiments, the extracellular binding domain includes VH and/or VL CDR sequences from an anti-ALK antibody. In some embodiments, the extracellular binding domain can include a VL and/or VH of an antibody selected from ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7 (e.g., as set forth in Table 1 and Table 2, respectively). In some embodiments, the extracellular binding domain can include the HCDR1, HCDR2, and HCDR3, and/or LCDR1, LCDR2, and LCDR3 of the VH and/or VL, respectively, of an antibody selected from ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7 (e.g., as set forth in Table 4 and Table 3, respectively).
[0285] In some embodiments, the ALK CAR comprises at least one linker. The at least one linker joins, or links, a variable heavy (VH) region to a constant heavy (CH) region of the extracellular binding domain of the CAR. Linkers can also link a variable light (VL) region to a variable constant (VC) region of the extracellular binding domain. In some embodiments, the linker is a flexible protein linker. In some embodiments, the linker is a (Gly.sub.4Ser).sub.n linker. In some embodiments, the linker is (Gly.sub.4Ser.sub.1).sub.3.
[0286] In some embodiments, the ALK CAR includes a signal peptide sequence, e.g., N-terminal to the antigen binding domain, that directs newly synthesized secretory or membrane proteins to and through membranes (e.g., the endoplasmic reticulum membrane). Signal peptide sequences typically contain three common structural features: N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region). The signal peptide sequence may comprise any suitable signal peptide sequence. While the signal peptide sequence may facilitate expression of the CAR on the surface of the cell, the presence of the signal peptide sequence in an expressed CAR is not necessary in order for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence may be cleaved off of the CAR. Accordingly, in some embodiments, the CAR lacks a signal peptide sequence. In some embodiments, the signal peptide sequence is approximately 16-30 amino acids in length. In one embodiment, the signal peptide sequence is mCD8. In one embodiment the leader peptide is CD8α. In one embodiment, the signal peptide sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence.
Transmembrane Domain
[0287] The ALK CARs contemplated herein include a transmembrane domain. The transmembrane domain of the ALK CARs described herein spans the CAR-T cells lipid bilayer cellular membrane and separates the extracellular binding domain and the intracellular signaling domain. The transmembrane domain may be derived either from a natural or from a synthetic source. In some embodiments, where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain may be derived from a non-human transmembrane domain and, in some embodiments, humanized (i.e., having the sequence of the nucleic acid encoding the transmembrane domain optimized such that it is more reliably or efficiently expressed in a human subject). In some embodiments, the transmembrane domain is derived from another transmembrane protein expressed in a human immune effector cell. Examples of such proteins include, but are not limited to, subunits of the T cell receptor (TCR) complex, PD1, or any of the Cluster of Differentiation proteins, or other proteins, that are expressed in the immune effector cell and that have a transmembrane domain. Transmembrane domains for use in the disclosed ALK CARs can include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain will be synthetic, and such sequences will comprise many hydrophobic residues.
[0288] In some embodiments, the ALK CAR transmembrane domain is fused to the extracellular domain. In some embodiments, the ALK CAR comprises a spacer between the transmembrane domain and the extracellular binding domain, the intracellular domain, or both. Such spacers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the spacer can be 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In still other embodiments the spacer can be between 100 and 500 amino acids in length. The spacer can be any polypeptide that links one domain to another and are used to position such linked domains to enhance or optimize CAR function. In some embodiments, the spacer domain can include an immunoglobulin domain, such as a human immunoglobulin sequence. In an embodiment, the immunoglobulin domain comprises an immunoglobulin CH2 and CH3 immunoglobulin G (IgG1) domain sequence (CH2CH3). The CH2CH3 domain extends the antigen binding domain of the CAR away from the membrane of CAR-expressing cells and may more accurately mimic the size and domain structure of a native TCR.
[0289] In some embodiments, a peptide linker, preferably between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular T cell signaling domain and/or T cell costimulatory domain of the ALK CAR. In one embodiment, the linker sequence includes one or more glycine-serine doublets. In some embodiments, the linker is a flexible protein linker. In some embodiments, the linker is a (Gly.sub.4Ser).sub.n linker. In some embodiments, the linker is (Gly.sub.4Ser.sub.1).sub.3.
[0290] In some embodiments, the transmembrane domain comprises the transmembrane domain of a T cell receptor, such as a CD8 transmembrane domain. In another embodiment, the transmembrane domain comprises the transmembrane domain of a T cell costimulatory molecule, such as CD137 (4-1BB) or CD28.
[0291] In some embodiments, the CD28 transmembrane domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00082 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGP SKPEWVLVVVGGVLACYSLLVTVAFIIEWVR
[0292] In some embodiments, the CD8 transmembrane domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the CD8 transmembrane domain of the m1928z CAR construct (see Davila et al., PlosOne 2013).
[0293] In some embodiments, the CD8 transmembrane domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00083 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC
Intracellular Signaling Domain
[0294] The ALK CARs contemplated herein comprise one or more T cell signaling domains that are capable of transducing a T cell effector function signal (e.g., an activation signal) and directing the T cell to perform a specialized function. T cell activation can be induced by a number of factors, including binding of cognate antigen to the T cell receptor on the surface of T cells and binding of cognate ligand to costimulatory molecules on the surface of the T cell. A T cell co-stimulatory molecule is a cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MEW class I molecule. Activation of a T cell leads to immune response, Such as T cell proliferation and differentiation (see, e.g., Smith-Garvin et al., Annu. Rev. Immunol., 27:591-619, 2009). Exemplary T cell signaling domains are known in the art. Non-limiting examples include the CD3ζ, CD8, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) signaling domains.
[0295] In some embodiments, the intracellular signaling domain of the ALK CAR contemplated herein comprises a primary signaling domain. In some embodiments, the chimeric antigen receptor comprises the primary signaling domain and a secondary, or co-stimulatory, signaling domain. In some embodiments, the primary signaling domain comprises one or more immunoreceptor tyrosine-based activation motifs or ITAMs. In some embodiments, the primary signaling domain comprises more than one ITAM. ITAMs incorporated into the chimeric antigen receptor may be derived from ITAMs from other cellular receptors. In some embodiments, the primary signaling domain comprising an ITAM may be derived from subunits of the TCR complex, such as CD3γ, CD3ε, CD3ζ, or CD3δ. In some embodiments, the primary signaling domain comprising an ITAM may be derived from FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, or CD66d. The secondary signaling domain, in some embodiments, is derived from CD28. In other embodiments, the secondary signaling domain is derived from CD2, CD4, CDS, CD8α, CD83, CD134, CD137, ICOS, or CD154.
[0296] In some embodiments, the ALK CAR can include a ON signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain or a combination of two or more thereof. In one embodiment, the cytoplasmic domain includes the signaling domain of CD3ζ and the signaling domain of CD28. In another embodiment, the cytoplasmic domain includes the signaling domain of CD3ζ and the signaling domain of CD137 (4-1BB). In yet another embodiment, the cytoplasmic domain includes the signaling domain of CD3-zeta and the signaling domain of CD28 and CD137. The order of the one or more T cell signaling domains on the CAR can be varied as needed by the person of ordinary skill in the art.
[0297] In some embodiments, the entire intracellular T cell signaling domain can be employed in an ALK CAR. In some embodiments, a truncated portion of the intracellular T cell signaling domain, which is still able to transduce T cell effector function, is used in an ALK CAR. In some embodiments, the cytoplasmic sequences of the T cell receptor (TCR) and co-stimulatory molecules that act in concert to initiate signal transduction following antigen receptor engagement are used in an ALK CAR.
[0298] In some embodiments, the CD3 ζ signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the CD3ζ signaling domain of the m1928z CAR construct (see Davila et al., PlosOne 2013).
[0299] In some embodiments, the CD3ζ signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00084 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR
[0300] In some embodiments, the CD8 signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the CD8 signaling domain of the m1928z CAR construct (see Davila et al., PlosOne 2013).
[0301] In some embodiments, the CD8 signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00085 FVPVFLPARPTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCNHRNR
[0302] In some embodiments, the CD28 signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the CD28 signaling domain of the m1928z CAR construct (see Davila et al., PlosOne 2013).
[0303] In some embodiments, the CD28 signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00086 SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
[0304] In some embodiments, the CD137 (4-1BB) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00087 KRGRKKLLYIFKQPEMRPVQTTQEEDGCSCRFPEEEEGGCEL
[0305] In some embodiments, the CD137 (4-1BB) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00088 RFSVVKRGRKKLLYIFKQPEMRPVQTTQEEDGCSCRFPEEEEGGCEL
[0306] In some embodiments, the CD134 (OX40) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00089 RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI
Anaplastic Lymphoma Kinase (ALK) Antibodies
[0307] The invention provides anaplastic lymphoma kinase chimeric antigen receptors (ALK CARs) that contain ALK antibody sequences that specifically bind to an ALK polypeptide or antibody-binding fragment thereof. The full-length ALK polypeptide includes an extracellular domain, a hydrophobic stretch corresponding to a single pass transmembrane region, and an intracellular kinase domain.
[0308] In some embodiments, the ALK polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a full-length ALK protein. In some embodiments, the ALK polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a full-length ALK protein in Homo Sapiens. In some embodiments, the ALK polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a full-length murine ALK protein. In some embodiments, the ALK polypeptide comprises an ALK extracellular domain. In some embodiments, the ALK polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an ALK extracellular domain in Homo Sapiens. In some embodiments, the ALK polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a murine ALK extracellular domain. In some embodiments, the ALK polypeptide comprises an ALK intracellular domain. In some embodiments, the ALK polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an ALK intracellular domain in Homo Sapiens. In some embodiments, the ALK polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a murine ALK intracellular domain.
[0309] In some embodiments, the ALK polypeptide comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an ALK amino acid sequence associated with GenBank™ Accession NOs.: BAD92714.1, ACY79563, NP_004295, ACI47591, or EDL38401.1). Human and murine ALK protein sequences are publicly available. One of ordinary skill in the art can identify additional ALK protein sequences, including ALK variants.
[0310] An exemplary ALK full-length amino acid sequence from Homo Sapiens is provided below (ALK cytoplasmic portion in bold font):
TABLE-US-00090 TASSGGMGAIGLLWLLPLLLSTAAVGSGMGTGQRA GSPAAGPPLQPREPLSYSRLQRKSLAVDEVVPSLF RVYARDLLLPPSSSELKAGRPEARGSLALDCAPLL RLLGPAPGVSWTAGSPAPAEARTLSRVLKGGSVRK LRRAKQLVLELGEEAILEGCVGPPGEAAVGLLQFN LSELFSWWIRQGEGRLRIRLMPEKKASEVGREGRL SAAIRASQPRLLFQIFGTGHSSLESPTNMPSPSPD YFTWNLTWIMKDSFPFLSHRSRYGLECSFDFPCEL EYSPPLHDLRNQSWSWRRIPSEEASQMDLLDGPGA ERSKEMPRGSFLLLNTSADSKHTILSPWMRSSSEH CTLAVSVHRHLQPSGRYIAQLLPHNEAAREILLMP TPGKHGWTVLQGRIGRPDNPFRVALEYISSGNRSL SAVDFFALKNCSEGTSPGSKMALQSSFTCWNGTVL QLGQACDFHQDCAQGEDESQMCRKLPVGFYCNFED GFCGWTQGTLSPHTPQWQVRTLKDARFQDHQDHAL LLSTTDVPASESATVTSATFPAPIKSSPCELRMSW LIRGVLRGNVSLVLVENKTGKEQGRMVWHVAAYEG LSLWQWMVLPLLDVSDRFWLQMVAWWGQGSRAIVA FDNISISLDCYLTISGEDKILQNTAPKSRNLFERN PNKELKPGENSPRQTPIFDPTVHWLFTTCGASGPH GPTQAQCNNAYQNSNLSVEVGSEGPLKGIQIWKVP ATDTYSISGYGAAGGKGGKNTMMRSHGVSVLGIFN LEKDDMLYILVGQQGEDACPSTNQLIQKVCIGENN VIEEEIRVNRSVHEWAGGGGGGGGATYVFKMKDGV PVPLIIAAGGGGRAYGAKTDTFHPERLENNSSVLG LNGNSGAAGGGGGWNDNTSLLWAGKSLQEGATGGH SCPQAMKKWGWETRGGFGGGGGGCSSGGGGGGYIG GNAASNNDPEMDGEDGVSFISPLGILYTPALKVME GHGEVNIKHYLNCSHCEVDECHMDPESHKVICFCD HGTVLAEDGVSCIVSPTPEPHLPLSLILSVVTSAL VAALVLAFSGIMIVYRRKHQELQAMQMELQSPEYK LSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRK NITLIRGLGHGAFGEVYEGQVSGMPNDPSPLQVAV KTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIG VSLQSLPRFILLELMAGGDLKS FLRETRPRPSQPSSIAMLDLLHVARDIACGCQYLE ENHFIHRDIAARNCLLTCPGPGRVAKIGDFGMARD IYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSKTD TWSFGVLLWEIFSLGYMPYPSKSNQEVLEFVTSGG RMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAIILE RIEYCTQDPDVINTALPIEYGPLVEEEEKVPVRPK DPEGVPPLLVSQQAKREEERSPAAPPPLPTTSSGK AAKKPTAAEISVRVPRGPAVEGGHVNMAFSQSNPP SELHKVHGSRNKPTSLWNPTYGSWFTEKPTKKNNP IAKKEPHDRGNLGLEGSCTVPPNVATGRLPGASLL LEPSSLTANMKEVPLFRLRHFPCGNVNYGYQQQGL PLEAATAPGAGHYEDTILKSKNSMNQPGP
[0311] An exemplary full-length ALK amino acid sequence from Homo Sapiens is provided below:
TABLE-US-00091 G A A A V V A A G T S R R L C S E G R G A P R C F P A A L W S A T Q S R G R * * W V R R G R Q D F G R P C P E R P Q L L P P G P L Q C L R T L R S R G A G E S K D A A N L R S A G A G I H A Q K F S R Q T V R S L P A A E R * L E G A Q D G S L R P R F P P R P G R R A W R S Q K E R K R R P G Q R A A A G S R R S Q P * K L Q R L E A A P R G D R P Q L R L R G A G E D G T Q L P P P F N H S S S S V P S A A S Y R R G R G T R R G E R E A Q G P S Q * A Q C A * V S L D S P L S F Q V C F I * T P A R L R A V G G K Q E T C A H A Q S S G D Q V E G A A G Y Q G L F R A S S H L G E S E G * G W A R R A V * T A S S G G M G A I G L L W L L P L L L S T A A V G S G M G T G Q R A G S P A A G P P L Q P R E P L S Y S R L Q R K S L A V D F V V P S L F R V Y A R D L L L P P S S S E L K A G R P E A R G S L A L D C A P L L R L L G P A P G V S W T A G S P A P A E A R T L S R V L K G G S V R K L R R A K Q L V L E L G E E A I L E G C V G P P G E A A V G L L Q F N L S E L F S W W I R Q G E G R L R I R L M P E K K A S E V G R E G R L S A A I R A S Q P R L L F Q I F G T G H S S L E S P T N M P S P S P D Y F T W N L T W I M K D S F P F L S H R S R Y G L E C S F D F P C E L E Y S P P L H D L R N Q S W S W R R I P S E E A S Q M D L L D G P G A E R S K E M P R G S F L L L N T S A D S K H T I L S P W M R S S S E H C T L A V S V H R H L Q P S G R Y I A Q L L P H N E A A R E I L L M P T P G K H G W T V L Q G R I G R P D N P F R V A L E Y I S S G N R S L S A V D F F A L K N C S E G T S P G S K M A L Q S S F T C W N G T V L Q L G Q A C D F H Q D C A Q G E D E S Q M C R K L P V G F Y C N F E D G F C G W T Q G T L S P H T P Q W Q V R T L K D A R F Q D H Q D H A L L L S T T D V P A S E S A T V T S A T F P A P I K S S P C E L R M S W L I R G V L R G N V S L V L V E N K T G K E Q G R M V W H V A A Y E G L S L W Q W M V L P L L D V S D R F W L Q M V A W W G Q G S R A I V A F D N I S I S L D C Y L T I S G E D K I L Q N T A P K S R N L F E R N P N K E L K P G E N S P R Q T P I F D P T V H W L F T T C G A S G P H G P T Q A Q C N N A Y Q N S N L S V E V G S E G P L K G I Q I W K V P A T D T Y S I S G Y G A A G G K G G K N T M M R S H G V S V L G I F N L E K D D M L Y I L V G Q Q G E D A C P S T N Q L I Q K V C I G E N N V I E E E I R V N R S V H E W A G G G G G G G G A T Y V F K M K D G V P V P L I I A A G G G G R A Y G A K T D T F H P E R L E N N S S V L G L N G N S G A A G G G G G W N D N T S L L W A G K S L Q E G A T G G H S C P Q A M K K W G W E T R G G F G G G G G G C S S G G G G G G Y I G G N A A S N N D P E M D G E D G V S F I S P L G I L Y T P A L K V M E G H G E V N I K H Y L N C S H C E V D E C H M D P E S H K V I C F C D H G T V L A E D G V S C I V S P T P E P H L P L S L I L S V V T S A L V A A L V L A F S G I M I V Y R R K H Q E L Q A M Q M E L Q S P E Y K L S K L R T S T I M T D Y N P N Y C F A G K T S S I S D L K E V P R K N I T L I R G L G H G A F G E V Y E G Q V S G M P N D P S P L Q V A V K T L P E V C S E Q D E L D F L M E A L I I S K F N H Q N I V R C I G V S L Q S L P R F I L L E L M A G G D L K S F L R E T R P R P S Q P S S L A M L D L L H V A R D I A C G C Q Y L E E N H F I H R D I A A R N C L L T C P G P G R V A K I G D F G M A R D I Y R A S Y Y R K G G C A M L P V K W M P P E A F M E G I F T S K T D T W S F G V L L W E I F S L G Y M P Y P S K S N Q E V L E F V T S G G R M D P P K N C P G P V Y R I M T Q C W Q H Q P E D R P N F A I I L E R I E Y C T Q D P D V I N T A L P I E Y G P L V E E E E K V P V R P K D P E G V P P L L V S Q Q A K R E E E R S P A A P P P L P T T S S G K A A K K P T A A E I S V R V P R G P A V E G G H V N M A F S Q S N P P S E L H K V H G S R N K P T S L W N P T Y G S W F T E K P T K K N N P I A K K E P H D R G N L G L E G S C T V P P N V A T G R L P G A S L L L E P S S L T A N M K E V P L F R L R H F P C G N V N Y G Y Q Q Q G L P L E A A T A P G A G H Y E D T I L K S K N S M N Q P G P * A R S H T H F S S L G S L R P W R R E R Q W L L H K P E T K C H V L F C A N L F * S T T K K A V F * K C F R K V L S M G S S Y S F E R R K Y H K N E * * I Q G P D V V A * G F Y A C L L Y T S L C F F Q I V C A L L Q C S Q N * L L L C F I V G V I D V S L P C * C G H E P F E G R G N G N K G V I C N D *
[0312] An exemplary Homo Sapiens ALK amino acid sequence from GenBank™ accession no. NP_004295 is provided below:
TABLE-US-00092 1 MGAIGLLWLL PLLLSTAAVG SGMGTGQRAG SPAAGPPLQP REPLSYSRLQ RKSLAVDFVV 61 PSLFRVYARD LLLPPSSSEL KAGRPEARGS LALDCAPLLR LLGPAPGVSW TAGSPAPAEA 121 RTLSRVLKGG SVRKLRRAKQ LVLELGEEAI LEGCVGPPGE AAVGLLQFNL SELFSWWIRQ 181 GEGRLRIRLM PEKKASEVGR EGRLSAAIRA SQPRLLFQIF GTGHSSLESP TNMPSPSPDY 241 FTWNLTWIMK DSFPFLSHRS RYGLECSFDF PCELEYSPPL HDLRNQSWSW RRIPSEEASQ 301 MDLLDGPGAE RSKEMPRGSF LLLNTSADSK HTILSPWMRS SSEHCTLAVS VHRHLQPSGR 361 YIAQLLPHNE AAREILLMPT PGKHGWTVLQ GRIGRPDNPF RVALEYISSG NRSLSAVDFF 421 ALKNCSEGTS PGSKMALQSS FTCWNGTVLQ LGQACDFHQD CAQGEDESQM CRKLPVGFYC 481 NFEDGFCGWT QGTLSPHTPQ WQVRTLKDAR FQDHQDHALL LSTTDVPASE SATVTSATFP 541 APIKSSPCEL RMSWLIRGVL RGNVSLVLVE NKTGKEQGRM VWHVAAYEGL SLWQWMVLPL 601 LDVSDRFWLQ MVAWWGQGSR AIVAFDNISI SLDCYLTISG EDKILQNTAP KSRNLFERNP 661 NKELKPGENS PRQTPIFDPT VHWLFTTCGA SGPHGPTQAQ CNNAYQNSNL SVEVGSEGPL 721 KGIQIWKVPA TDTYSISGYG AAGGKGGKNT MMRSHGVSVL GIFNLEKDDM LYILVGQQGE 781 DACPSTNQLI QKVCIGENNV IEEEIRVNRS VHEWAGGGGG GGGATYVFKM KDGVPVPLII 841 AAGGGGRAYG AKTDTFHPER LENNSSVLGL NGNSGAAGGG GGWNDNTSLL WAGKSLQEGA 901 TGGHSCPQAM KKWGWETRGG FGGGGGGCSS GGGGGGYIGG NAASNNDPEM DGEDGVSFIS 961 PLGILYTPAL KVMEGHGEVN IKHYLNCSHC EVDECHMDPE SHKVICFCDH GTVLAEDGVS 1021 CIVSPTPEPH LPLSLILSVV TSALVAALVL AFSGIMIVYR RKHQELQAMQ MELQSPEYKL 1081 SKLRTSTIMT DYNPNYCFAG KTSSISDLKE VPRKNITLIR GLGHGAFGEV YEGQVSGMPN 1141 DPSPLQVAVK TLPEVCSEQD ELDFLMEALI ISKFNHQNIV RCIGVSLQSL PRFILLELMA 1201 GGDLKSFLRE TRPRPSQPSS LAMLDLLHVA RDIACGCQYL EENHFIHRDI AARNCLLTCP 1261 GPGRVAKIGD FGMARDIYRA SYYRKGGCAM LPVKWMPPEA FMEGIFTSKT DTWSFGVLLW 1321 EIFSLGYMPY PSKSNQEVLE FVTSGGRMDP PKNCPGPVYR IMTQCWQHQP EDRPNFAIIL 1381 ERIEYCTQDP DVINTALPIE YGPLVEEEEK VPVRPKDPEG VPPLLVSQQA KREEERSPAA 1441 PPPLPTTSSG KAAKKPTAAE ISVRVPRGPA VEGGHVNMAF SQSNPPSELH KVHGSRNKPT 1501 SLWNPTYGSW ETEKPTKKNN PIAKKEPHDR GNLGLEGSCT VPPNVATGRL PGASLLLEPS 1561 SLTANMKEVP LFRLRHFPCG NVNYGYQQQG LPLEAATAPG AGHYEDTILK SKNSMNQPGP
[0313] An exemplary Homo Sapiens ALK polypeptide sequence from UniProt Accession No. Q9UM73 is provided below (extracellular domain (amino acids 19-1038) provided in bold font):
TABLE-US-00093 MGAIGLLWLLPLLLSTAAVGSGMGTGQRAGSPAAG PPLQPREPLSYSRLQRKSLAVDFVVPSLFRVYARD LLLPPSSSELKAGRPEARGSLALDCAPLLRLLGPA PGVSWTAGSPAPAEARTLSRVLKGGSVRKLRRAKQ LVLELGEEAILEGCVGPPGEAAVGLLQFNLSELFS WWIRQGEGRLRIRLMPEKKASEVGREGRLSAAIRA SQPRLLFQIFGTGHSSLESPTNMPSPSPDYFTWNL TWIMKDSFPFLSHRSRYGLECSFDFPCELEYSPPL HDLRNQSWSWRRIPSEEASQMDLLDGPGAERSKEM PRGSFLLLNTSADSKHTILSPWMRSSSEHCTLAVS VHRHLQPSGRYIAQLLPHNEAAREILLMPTPGKHG WTVLQGRIGRPDNPFRVALEYISSGNRSLSAVDFF ALKNCSEGTSPGSKMALQSSFTCWNGTVLQLGQAC DFHQDCAQGEDESQMCRKLPVGFYCNFEDGFCGWT QGTLSPHTPQWQVRTLKDARFQDHQDHALLLSTTD VPASESATVTSATFPAPIKSSPCELRMSWLIRGVL RGNVSLVLVENKTGKEQGRMVWHVAAYEGLSLWQW MVLPLLDVSDRFWLQMVAWWGQGSRAIVAFDNISI SLDCYLTISGEDKILQNTAPKSRNLFERNPNKELK PGENSPRQTPIFDPTVHWLFTTCGASGPHGPTQAQ CNNAYQNSNLSVEVGSEGPLKGIQIWKVPATDTYS ISGYGAAGGKGGKNTMMRSHGVSVLGIFNLEKDDM LYILVGQQGEDACPSTNQLIQKVCIGENNVIEEEI RVNRSVHEWAGGGGGGGGATYVFKMKDGVPVPLII AAGGGGRAYGAKTDTFHPERLENNSSVLGLNGNSG AAGGGGGWNDNTSLLWAGKSLQEGATGGHSCPQAM KKWGWETRGGFGGGGGGCSSGGGGGGYIGGNAASN NDPEMDGEDGVSFISPLGILYTPALKVMEGHGEVN IKHYLNCSHCEVDECHMDPESHKVICFCDHGTVLA EDGVSCIVSPTPEPHLPLSLILSVVTSALVAALVL AFSGIMIVYRRKHQELQAMQMELQSPEYKLSKLRT STIMTDYNPNYCFAGKTSSISDLKEVPRKNITLIR GLGHGAFGEVYEGQVSGMPNDPSPLQVAVKTLPEV CSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSL PRFILLELMAGGDLKSFLRETRPRPSQPSSLAMLD LLHVARDIACGCQYLEENHFIHRDIAARNCLLTCP GPGRVAKIGDFGMARDIYRASYYRKGGCAMLPVKW MPPEAFMEGIFTSKTDTWSFGVLLWEIFSLGYMPY PSKSNQEVLEFVTSGGRMDPPKNCPGPVYRIMTQC WQHQPEDRPNFAIILERIEYCTQDPDVINTALPIE YGPLVEEEEKVPVRPKDPEGVPPLLVSQQAKREEE RSPAAPPPLPTTSSGKAAKKPTAAEISVRVPRGPA VEGGHVNMAFSQSNPPSELHKVHGSRNKPTSLWNP TYGSWFTEKPTKKNNPIAKKEPHDRGNLGLEGSCT VPPNVATGRLPGASLLLEPSSLTANMKEVPLFRLR HFPCGNVNYGYQQQGLPLEAATAPGAGHYEDTILK SKNSMNQPGP
[0314] An exemplary ALK full-length amino acid sequence from Mus musculus is provided below:
TABLE-US-00094 1 MGQRQSILKR CPEGSFLLLN TSADSKHTIL SPWMRSSSDH CTLAVSVHRH LQPSGRYVAQ 61 LLPHNEAGRE ILLVPTPGKH GWTVLQGRVG RPANPFRVAL EYISSGNRSL SAVDFFALKN 121 CSEGTSPGSK MALQSSFTCW NGTVLQLGQA CDFHQDCAQG EDEGQLCSKL PAGFYCNFEN 181 GFCGWTQSPL SPHMPRWQVR TLRDAHSQGH QGRALLLSTT DILASEGATV TSATFPAPMK 241 NSPCELRMSW LIRGVLRGNV SLVLVENKTG KEQSRTVWHV ATDEGLSLWQ HTVLSLLDVT 301 DRFWLQIVTW WGPGSRATVG FDNISISLDC YLTISGEEKM SLNSVPKSRN LFEKNPNKES 361 KSWANISGPT PIFDPTVHWL FTTCGASGPH GPTQAQCNNA YQNSNLSVVV GSEGPLKGVQ 421 IWKVPATDTY SISGYGAAGG KGGKNTMMRS HGVSVLGIFN LEKGDTLYIL VGQQGEDACP 481 RANQLIQKVC VGENNVIEEE IRVNRSVHEW AGGGGGGGGA TYVFKMKDGV PVPLIIAAGG 541 GGRAYGAKTE TFHPERLESN SSVLGLNGNS GAAGGGGGWN DNTSLLWAGK SLLEGAAGGH 601 SCPQAMKKWG WETRGGFGGG GGGGAPQVEE AEDI
[0315] In some embodiments, the ALK antigen is isolated and/or purified. In some embodiments, the amino acid sequence of the antigen, e.g., the ALK protein, is reverse translated and optimized for expression in mammalian cells. As will be appreciated by a skilled practitioner in the art, optimization of the nucleic acid sequence includes optimization of the codons for expression of a sequence in mammalian cells and RNA optimization (such as RNA stability).
[0316] In some embodiments, the ALK polypeptide or antibody-binding fragment thereof (e.g., antigen or antigen protein) is encoded by a polynucleotide.
[0317] In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polynucleotide encoding full-length ALK protein. In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polynucleotide encoding full-length ALK protein in Homo Sapiens. In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polynucleotide encoding a full-length murine ALK protein. In some embodiments, the ALK polynucleotide encodes an ALK extracellular domain. In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polypeptide encoding an ALK extracellular domain in Homo Sapiens. In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polypeptide encoding a murine ALK extracellular domain. In some embodiments, the ALK polynucleotide encodes an ALK intracellular domain. In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polynucleotide encoding an ALK intracellular domain in Homo Sapiens. In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polynucleotide encoding a murine ALK intracellular domain. In some embodiments, the ALK polynucleotide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a polynucleotide encoding an ALK amino acid sequence associated with GenBank™ Accession Nos.: BAD92714.1, ACY79563, NP_004295, NM_007439.2, or ACI47591. Human and murine ALK polynucleotide sequences are publicly available. One of ordinary skill in the art can identify additional ALK polynucleotide sequences, including ALK variants.
[0318] An exemplary Homo Sapiens ALK amino acid sequence from GenBank′ accession no. NM_004304 is provided below:
TABLE-US-00095 1 agatgcgatc cagcggctct gggggcggca gcggtggtag cagctggtac ctcccgccgc 61 ctctgttcgg agggtcgcgg ggcaccgagg tgctttccgg ccgccctctg gtcggccacc 121 caaagccgcg ggcgctgatg atgggtgagg agggggcggc aagatttcgg gcgcccctgc 181 cctgaacgcc ctcagctgct gccgccgggg ccgctccagt gcctgcgaac tctgaggagc 241 cgaggcgccg gtgagagcaa ggacgctgca aacttgcgca gcgcgggggc tgggattcac 301 gcccagaagt tcagcaggca gacagtccga agccttcccg cagcggagag atagcttgag 361 ggtgcgcaag acggcagcct ccgccctcgg ttcccgccca gaccgggcag aagagcttgg 421 aggagccaaa aggaacgcaa aaggcggcca ggacagcgtg cagcagctgg gagccgccgt 481 tctcagcctt aaaagttgca gagattggag gctgccccga gaggggacag accccagctc 541 cgactgcggg gggcaggaga ggacggtacc caactgccac ctcccttcaa ccatagtagt 601 tcctctgtac cgagcgcagc gagctacaga cgggggcgcg gcactcggcg cggagagcgg 661 gaggctcaag gtcccagcca gtgagcccag tgtgcttgag tgtctctgga ctcgcccctg 721 agcttccagg tctgtttcat ttagactcct gctcgcctcc gtgcagttgg gggaaagcaa 781 gagacttgcg cgcacgcaca gtcctctgga gatcaggtgg aaggagccgc tgggtaccaa 841 ggactgttca gagcctcttc ccatctcggg gagagcgaag ggtgaggctg ggcccggaga 901 gcagtgtaaa cggcctcctc cggcgggatg ggagccatcg ggctcctgtg gctcctgccg 961 ctgctgcttt ccacggcagc tgtgggctcc gggatgggga ccggccagcg cgcgggctcc 1021 ccagctgcgg ggccgccgct gcagccccgg gagccactca gctactcgcg cctgcagagg 1081 aagagtctgg cagttgactt cgtggtgccc tcgctcttcc gtgtctacgc ccgggaccta 1141 ctgctgccac catcctcctc ggagctgaag gctggcaggc ccgaggcccg cggctcgcta 1201 gctctggact gcgccccgct gctcaggttg ctggggccgg cgccgggggt ctcctggacc 1261 gccggttcac cagccccggc agaggcccgg acgctgtcca gggtgctgaa gggcggctcc 1321 gtgcgcaagc tccggcgtgc caagcagttg gtgctggagc tgggcgagga ggcgatcttg 1381 gagggttgcg tcgggccccc cggggaggcg gctgtggggc tgctccagtt caatctcagc 1441 gagctgttca gttggtggat tcgccaaggc gaagggcgac tgaggatccg cctgatgccc 1501 gagaagaagg cgtcggaagt gggcagagag ggaaggctgt ccgcggcaat tcgcgcctcc 1561 cagccccgcc ttctcttcca gatcttcggg actggtcata gctccttgga atcaccaaca 1621 aacatgcctt ctccttctcc tgattatttt acatggaatc tcacctggat aatgaaagac 1681 tccttccctt tcctgtctca tcgcagccga tatggtctgg agtgcagctt tgacttcccc 1741 tgtgagctgg agtattcccc tccactgcat gacctcagga accagagctg gtcctggcgc 1801 cgcatcccct ccgaggaggc ctcccagatg gacttgctgg atgggcctgg ggcagagcgt 1861 tctaaggaga tgcccagagg ctcctttctc cttctcaaca cctcagctga ctccaagcac 1921 accatcctga gtccgtggat gaggagcagc agtgagcact gcacactggc cgtctcggtg 1981 cacaggcacc tgcagccctc tggaaggtac attgcccagc tgctgcccca caacgaggct 2041 gcaagagaga tcctcctgat gcccactcca gggaagcatg gttggacagt gctccaggga 2101 agaatcgggc gtccagacaa cccatttcga gtggccctgg aatacatctc cagtggaaac 2161 cgcagcttgt ctgcagtgga cttctttgcc ctgaagaact gcagtgaagg aacatcccca 2221 ggctccaaga tggccctgca gagctccttc acttgttgga atgggacagt cctccagctt 2281 gggcaggcct gtgacttcca ccaggactgt gcccagggag aagatgagag ccagatgtgc 2341 cggaaactgc ctgtgggttt ttactgcaac tttgaagatg gcttctgtgg ctggacccaa 2401 ggcacactgt caccccacac tcctcaatgg caggtcagga ccctaaagga tgcccggttc 2461 caggaccacc aagaccatgc tctattgctc agtaccactg atgtccccgc ttctgaaagt 2521 gctacagtga ccagtgctac gtttcctgca ccgatcaaga gctctccatg tgagctccga 2581 atgtcctggc tcattcgtgg agtcttgagg ggaaacgtgt ccttggtgct agtggagaac 2641 aaaaccggga aggagcaagg caggatggtc tggcatgtcg ccgcctatga aggcttgagc 2701 ctgtggcagt ggatggtgtt gcctctcctc gatgtgtctg acaggttctg gctgcagatg 2761 gtcgcatggt ggggacaagg atccagagcc atcgtggctt ttgacaatat ctccatcagc 2821 ctggactgct acctcaccat tagcggagag gacaagatcc tgcagaatac agcacccaaa 2881 tcaagaaacc tgtttgagag aaacccaaac aaggagctga aacccgggga aaattcacca 2941 agacagaccc ccatctttga ccctacagtt cattggctgt tcaccacatg tggggccagc 3001 gggccccatg gccccaccca ggcacagtgc aacaacgcct accagaactc caacctgagc 3061 gtggaggtgg ggagcgaggg ccccctgaaa ggcatccaga tctggaaggt gccagccacc 3121 gacacctaca gcatctcggg ctacggagct gctggcggga aaggcgggaa gaacaccatg 3181 atgcggtccc acggcgtgtc tgtgctgggc atcttcaacc tggagaagga tgacatgctg 3241 tacatcctgg ttgggcagca gggagaggac gcctgcccca gtacaaacca gttaatccag 3301 aaagtctgca ttggagagaa caatgtgata gaagaagaaa tccgtgtgaa cagaagcgtg 3361 catgagtggg caggaggcgg aggaggaggg ggtggagcca cctacgtatt taagatgaag 3421 gatggagtgc cggtgcccct gatcattgca gccggaggtg gtggcagggc ctacggggcc 3481 aagacagaca cgttccaccc agagagactg gagaataact cctcggttct agggctaaac 3541 ggcaattccg gagccgcagg tggtggaggt ggctggaatg ataacacttc cttgctctgg 3601 gccggaaaat ctttgcagga gggtgccacc ggaggacatt cctgccccca ggccatgaag 3661 aagtgggggt gggagacaag agggggtttc ggagggggtg gaggggggtg ctcctcaggt 3721 ggaggaggcg gaggatatat aggcggcaat gcagcctcaa acaatgaccc cgaaatggat 3781 ggggaagatg gggtttcctt catcagtcca ctgggcatcc tgtacacccc agctttaaaa 3841 gtgatggaag gccacgggga agtgaatatt aagcattatc taaactgcag tcactgtgag 3901 gtagacgaat gtcacatgga ccctgaaagc cacaaggtca tctgcttctg tgaccacggg 3961 acggtgctgg ctgaggatgg cgtctcctgc attgtgtcac ccaccccgga gccacacctg 4021 ccactctcgc tgatcctctc tgtggtgacc tctgccctcg tggccgccct ggtcctggct 4081 ttctccggca tcatgattgt gtaccgccgg aagcaccagg agctgcaagc catgcagatg 4141 gagctgcaga gccctgagta caagctgagc aagctccgca cctcgaccat catgaccgac 4201 tacaacccca actactgctt tgctggcaag acctcctcca tcagtgacct gaaggaggtg 4261 ccgcggaaaa acatcaccct cattcggggt ctgggccatg gcgcctttgg ggaggtgtat 4321 gaaggccagg tgtccggaat gcccaacgac ccaagccccc tgcaagtggc tgtgaagacg 4381 ctgcctgaag tgtgctctga acaggacgaa ctggatttcc tcatggaagc cctgatcatc 4441 agcaaattca accaccagaa cattgttcgc tgcattgggg tgagcctgca atccctgccc 4501 cggttcatcc tgctggagct catggcgggg ggagacctca agtccttcct ccgagagacc 4561 cgccctcgcc cgagccagcc ctcctccctg gccatgctgg accttctgca cgtggctcgg 4621 gacattgcct gtggctgtca gtatttggag gaaaaccact tcatccaccg agacattgct 4681 gccagaaact gcctcttgac ctgtccaggc cctggaagag tggccaagat tggagacttc 4741 gggatggccc gagacatcta cagggcgagc tactatagaa agggaggctg tgccatgctg 4801 ccagttaagt ggatgccccc agaggccttc atggaaggaa tattcacttc taaaacagac 4861 acatggtcct ttggagtgct gctatgggaa atcttttctc ttggatatat gccatacccc 4921 agcaaaagca accaggaagt tctggagttt gtcaccagtg gaggccggat ggacccaccc 4981 aagaactgcc ctgggcctgt ataccggata atgactcagt gctggcaaca tcagcctgaa 5041 gacaggccca actttgccat cattttggag aggattgaat actgcaccca ggacccggat 5101 gtaatcaaca ccgctttgcc gatagaatat ggtccacttg tggaagagga agagaaagtg 5161 cctgtgaggc ccaaggaccc tgagggggtt cctcctctcc tggtctctca acaggcaaaa 5221 cgggaggagg agcgcagccc agctgcccca ccacctctgc ctaccacctc ctctggcaag 5281 gctgcaaaga aacccacagc tgcagagatc tctgttcgag tccctagagg gccggccgtg 5341 gaagggggac acgtgaatat ggcattctct cagtccaacc ctccttcgga gttgcacaag 5401 gtccacggat ccagaaacaa gcccaccagc ttgtggaacc caacgtacgg ctcctggttt 5461 acagagaaac ccaccaaaaa gaataatcct atagcaaaga aggagccaca cgacaggggt 5521 aacctggggc tggagggaag ctgtactgtc ccacctaacg ttgcaactgg gagacttccg 5581 ggggcctcac tgctcctaga gccctcttcg ctgactgcca atatgaagga ggtacctctg 5641 ttcaggctac gtcacttccc ttgtgggaat gtcaattacg gctaccagca acagggcttg 5701 cccttagaag ccgctactgc ccctggagct ggtcattacg aggataccat tctgaaaagc 5761 aagaatagca tgaaccagcc tgggccctga gctcggtcgc acactcactt ctcttccttg 5821 ggatccctaa gaccgtggag gagagagagg caatggctcc ttcacaaacc agagaccaaa 5881 tgtcacgttt tgttttgtgc caacctattt tgaagtacca ccaaaaaagc tgtattttga 5941 aaatgcttta gaaaggtttt gagcatgggt tcatcctatt ctttcgaaag aagaaaatat 6001 cataaaaatg agtgataaat acaaggccca gatgtggttg cataaggttt ttatgcatgt 6061 ttgttgtata cttccttatg cttctttcaa attgtgtgtg ctctgcttca atgtagtcag 6121 aattagctgc ttctatgttt catagttggg gtcatagatg tttccttgcc ttgttgatgt 6181 ggacatgagc catttgaggg gagagggaac ggaaataaag gagttatttg taatgactaa
[0319] An exemplary full-length ALK nucleic acid sequence from Homo Sapiens is provided below:
TABLE-US-00096 2 ggggcggcagcggtggtagcagctggtacctcccgccgcctctgttcggagggtcgcggg 61 62 gcaccgaggtgctttccggccgccctctggtcggccacccaaagccgcgggcgctgatga 121 122 tgggtgaggagggggcggcaagatttcgggcgcccctgccctgaacgccctcagctgctg 181 182 ccgccggggccgctccagtgcctgcgaactctgaggagccgaggcgccggtgagagcaag 241 242 gacgctgcaaacttgcgcagcgcgggggctgggattcacgcccagaagttcagcaggcag 301 302 acagtccgaagccttcccgcagcggagagatagcttgagggtgcgcaagacggcagcctc 361 362 cgccctcggttcccgcccagaccgggcagaagagcttggaggagccaaaaggaacgcaaa 421 422 aggcggccaggacagcgtgcagcagctgggagccgccgttctcagccttaaaagttgcag 481 482 agattggaggctgccccgagaggggacagaccccagctccgactgcggggggcaggagag 541 542 gacggtacccaactgccacctcccttcaaccatagtagttcctctgtaccgagcgcagcg 601 602 agctacagacgggggcgcggcactcggcgcggagagcgggaggctcaaggtcccagccag 661 662 tgagcccagtgtgcttgagtgtctctggactcgcccctgagcttccaggtctgtttcatt 721 722 tagactcctgctcgcctccgtgcagttgggggaaagcaagagacttgcgcgcacgcacag 781 782 tcctctggagatcaggtggaaggagccgctgggtaccaaggactgttcagagcctcttcc 841 842 catctcggggagagcgaagggtgaggctgggcccggagagcagtgtaaacggcctcctcc 901 902 ggcgggatgggagccatcgggctcctgtggctcctgccgctgctgctttccacggcagct 961 962 gtgggctccgggatggggaccggccagcgcgcgggctccccagctgcggggccgccgctg 1021 1022 cagccccgggagccactcagctactcgcgcctgcagaggaagagtctggcagttgacttc 1081 1082 gtggtgccctcgctcttccgtgtctacgcccgggacctactgctgccaccatcctcctcg 1141 1142 gagctgaaggctggcaggcccgaggcccgcggctcgctagctctggactgcgccccgctg 1201 1202 ctcaggttgctggggccggcgccgggggtctcctggaccgccggttcaccagccccggca 1261 1262 gaggcccggacgctgtccagggtgctgaagggcggctccgtgcgcaagctccggcgtgcc 1321 1322 aagcagttggtgctggagctgggcgaggaggcgatcttggagggttgcgtcgggcccccc 1381 1382 ggggaggcggctgtggggctgctccagttcaatctcagcgagctgttcagttggtggatt 1441 1442 cgccaaggcgaagggcgactgaggatccgcctgatgcccgagaagaaggcgtcggaagtg 1501 1502 ggcagagagggaaggctgtccgcggcaattcgcgcctcccagccccgccttctcttccag 1561 1562 atcttcgggactggtcatagctccttggaatcaccaacaaacatgccttctccttctcct 1621 1622 gattattttacatggaatctcacctggataatgaaagactccttccctttcctgtctcat 1681 1682 cgcagccgatatggtctggagtgcagctttgacttcccctgtgagctggagtattcccct 1741 1742 ccactgcatgacctcaggaaccagagctggtcctggcgccgcatcccctccgaggaggcc 1801 1802 tcccagatggacttgctggatgggcctggggcagagcgttctaaggagatgcccagaggc 1861 1862 tcctttctccttctcaacacctcagctgactccaagcacaccatcctgagtccgtggatg 1921 1922 aggagcagcagtgagcactgcacactggccgtctcggtgcacaggcacctgcagccctct 1981 1982 ggaaggtacattgcccagctgctgccccacaacgaggctgcaagagagatcctcctgatg 2041 2042 cccactccagggaagcatggttggacagtgctccagggaagaatcgggcgtccagacaac 2101 2102 ccatttcgagtggccctggaatacatctccagtggaaaccgcagcttgtctgcagtggac 2161 2162 ttctttgccctgaagaactgcagtgaaggaacatccccaggctccaagatggccctgcag 2221 2222 agctccttcacttgttggaatgggacagtcctccagcttgggcaggcctgtgacttccac 2281 2282 caggactgtgcccagggagaagatgagagccagatgtgccggaaactgcctgtgggtttt 2341 2342 tactgcaactttgaagatggcttctgtggctggacccaaggcacactgtcaccccacact 2401 2402 cctcaatggcaggtcaggaccctaaaggatgcccggttccaggaccaccaagaccatgct 2461 2462 ctattgctcagtaccactgatgtccccgcttctgaaagtgctacagtgaccagtgctacg 2521 2522 tttcctgcaccgatcaagagctctccatgtgagctccgaatgtcctggctcattcgtgga 2581 2582 gtcttgaggggaaacgtgtccttggtgctagtggagaacaaaaccgggaaggagcaaggc 2641 2642 aggatggtctggcatgtcgccgcctatgaaggcttgagcctgtggcagtggatggtgttg 2701 2702 cctctcctcgatgtgtctgacaggttctggctgcagatggtcgcatggtggggacaagga 2761 2762 tccagagccatcgtggcttttgacaatatctccatcagcctggactgctacctcaccatt 2821 2822 agcggagaggacaagatcctgcagaatacagcacccaaatcaagaaacctgtttgagaga 2881 2882 aacccaaacaaggagctgaaacccggggaaaattcaccaagacagacccccatctttgac 2941 2942 cctacagttcattggctgttcaccacatgtggggccagcgggccccatggccccacccag 3001 3002 gcacagtgcaacaacgcctaccagaactccaacctgagcgtggaggtggggagcgagggc 3061 3062 cccctgaaaggcatccagatctggaaggtgccagccaccgacacctacagcatctcgggc 3121 3122 tacggagctgctggcgggaaaggcgggaagaacaccatgatgcggtcccacggcgtgtct 3181 3182 gtgctgggcatcttcaacctggagaaggatgacatgctgtacatcctggttgggcagcag 3241 3242 ggagaggacgcctgccccagtacaaaccagttaatccagaaagtctgcattggagagaac 3301 3302 aatgtgatagaagaagaaatccgtgtgaacagaagcgtgcatgagtgggcaggaggcgga 3361 3362 ggaggagggggtggagccacctacgtatttaagatgaaggatggagtgccggtgcccctg 3421 3422 atcattgcagccggaggtggtggcagggcctacggggccaagacagacacgttccaccca 3481 3482 gagagactggagaataactcctcggttctagggctaaacggcaattccggagccgcaggt 3541 3542 ggtggaggtggctggaatgataacacttccttgctctgggccggaaaatctttgcaggag 3601 3602 ggtgccaccggaggacattcctgcccccaggccatgaagaagtgggggtgggagacaaga 3661 3662 gggggtttcggagggggtggaggggggtgctcctcaggtggaggaggcggaggatatata 3721 3722 ggcggcaatgcagcctcaaacaatgaccccgaaatggatggggaagatggggtttccttc 3781 3782 atcagtccactgggcatcctgtacaccccagctttaaaagtgatggaaggccacggggaa 3841 3842 gtgaatattaagcattatctaaactgcagtcactgtgaggtagacgaatgtcacatggac 3901 3902 cctgaaagccacaaggtcatctgcttctgtgaccacgggacggtgctggctgaggatggc 3961 3962 gtctcctgcattgtgtcacccaccccggagccacacctgccactctcgctgatcctctct 4021 4022 gtggtgacctctgccctcgtggccgccctggtcctggctttctccggcatcatgattgtg 4081 4082 taccgccggaagcaccaggagctgcaagccatgcagatggagctgcagagccctgagtac 4141 4142 aagctgagcaagctccgcacctcgaccatcatgaccgactacaaccccaactactgcttt 4201 4202 gctggcaagacctcctccatcagtgacctgaaggaggtgccgcggaaaaacatcaccctc 4261 4262 attcggggtctgggccatggcgcctttggggaggtgtatgaaggccaggtgtccggaatg 4321 4322 cccaacgacccaagccccctgcaagtggctgtgaagacgctgcctgaagtgtgctctgaa 4381 4382 caggacgaactggatttcctcatggaagccctgatcatcagcaaattcaaccaccagaac 4441 4442 attgttcgctgcattggggtgagcctgcaatccctgccccggttcatcctgctggagctc 4501 4502 atggcggggggagacctcaagtccttcctccgagagacccgccctcgcccgagccagccc 4561 4562 tcctccctggccatgctggaccttctgcacgtggctcgggacattgcctgtggctgtcag 4621 4622 tatttggaggaaaaccacttcatccaccgagacattgctgccagaaactgcctcttgacc 4681 4682 tgtccaggccctggaagagtggccaagattggagacttcgggatggcccgagacatctac 4741 4742 agggcgagctactatagaaagggaggctgtgccatgctgccagttaagtggatgccccca 4801 4802 gaggccttcatggaaggaatattcacttctaaaacagacacatggtcctttggagtgctg 4861 4862 ctatgggaaatcttttctcttggatatatgccataccccagcaaaagcaaccaggaagtt 4921 4922 ctggagtttgtcaccagtggaggccggatggacccacccaagaactgccctgggcctgta 4981 4982 taccggataatgactcagtgctggcaacatcagcctgaagacaggcccaactttgccatc 5041 5042 attttggagaggattgaatactgcacccaggacccggatgtaatcaacaccgctttgccg 5101 5102 atagaatatggtccacttgtggaagaggaagagaaagtgcctgtgaggcccaaggaccct 5161 5162 gagggggttcctcctctcctggtctctcaacaggcaaaacgggaggaggagcgcagccca 5221 5222 gctgccccaccacctctgcctaccacctcctctggcaaggctgcaaagaaacccacagct 5281 5282 gcagagatctctgttcgagtccctagagggccggccgtggaagggggacacgtgaatatg 5341 5342 gcattctctcagtccaaccctccttcggagttgcacaaggtccacggatccagaaacaag 5401 5402 cccaccagcttgtggaacccaacgtacggctcctggtttacagagaaacccaccaaaaag 5461 5462 aataatcctatagcaaagaaggagccacacgacaggggtaacctggggctggagggaagc 5521 5522 tgtactgtcccacctaacgttgcaactgggagacttccgggggcctcactgctcctagag 5581 5582 ccctcttcgctgactgccaatatgaaggaggtacctctgttcaggctacgtcacttccct 5641 5642 tgtgggaatgtcaattacggctaccagcaacagggcttgcccttagaagccgctactgcc 5701 5702 cctggagctggtcattacgaggataccattctgaaaagcaagaatagcatgaaccagcct 5761 5762 gggccctgagctcggtcgcacactcacttctcttccttgggatccctaagaccgtggagg 5821 5822 agagagaggcaatggctccttcacaaaccagagaccaaatgtcacgttttgttttgtgcc 5881 5882 aacctattttgaagtaccaccaaaaaagctgtattttgaaaatgctttagaaaggttttg 5941 5942 agcatgggttcatcctattctttcgaaagaagaaaatatcataaaaatgagtgataaata 6001 6002 caaggcccagatgtggttgcataaggtttttatgcatgtttgttgtatacttccttatgc 6061 6062 ttctttcaaattgtgtgtgctctgcttcaatgtagtcagaattagctgcttctatgtttc 6121 6122 atagttggggtcatagatgtttccttgccttgttgatgtggacatgagccatttgagggg 6181 6182 agagggaacggaaataaaggagttatttgtaatgactaaaa 6222
[0320] An exemplary Mus musculus ALK nucleic acid sequence from GenBank™ accession no. NM_007439.2 is provided below:
TABLE-US-00097 1 gtgttcacgc ccagaagttc agcgggcagg gtgatcgatc cgaagacttc ctgcagcgga 61 ggtcacttga gggggcgcta gaaagcagcc ccctccggtg gtccttgcct agacctggga 121 aggagcgcag aggaggtgac aggagcggag gacgtgggca agacagtgac cgactcggag 181 ccacggttca cagcctggaa agttgcagaa gattggaagc taagaggaga gctctggtcg 241 ccgagggctc cttgaacggt acctaattgc cacctccctg gtccctgagc aaaggcctct 301 acaaatgggg cgcagcacgg cgagaggcgc aggatccagc tgttgagccc agggtgtctc 361 actgtctccg aactaccccc tgactttgtc ttccgttttg ctgagaaccc ttctcgcctc 421 cttgtagctt gggaaaagca agggcgctct atagtgtaca cacagtccct gagatctagt 481 ggaaggagcc attcaggacc aaggactatt tggagccctt tcctgtttgg gggagagtga 541 agggcgaggc tggaccagca agggaaggga gactagtgta aactcgccct ccagcgggat 601 gggagctgct gggttcctgt ggctgctgcc tccactgctt ttggcagcag cctcgtactc 661 cggagctgca accgatcagc gcgcgggttc cccagcctca gggcctcctc tgcagccccg 721 ggagccgctc agttattcgc gcctgcagag gaagagtctg gcagtggact tcgttgtacc 781 ctcgctcttc cgcgtctatg cccgagacct gctgctaccg cagccacggt ccccctcgga 841 gcccgaggct ggcgggctgg aggcgcgggg atcactggcc ctggattgtg agcctctgct 901 caggctgctg gggccactgc ctggaatctc ctgggcagat ggagccagtt ctcctagtcc 961 cgaggcgggt ccgacgctgt ccagggtgct gaagggaggc tcggtgcgca agctcaggcg 1021 tgccaaacag ctggtgctgg agctgggcga ggagacgatt cttgaaggct gtattggtcc 1081 cccagaggag gtagcggctg tggggatact ccagttcaac ctcagcgagc tgttcagctg 1141 gtggattctc cacggcgaag ggaggctgag gatccgcctg atgcctgaga agaaggcatc 1201 ggaagtgggc agggagggaa ggctatccag tgcgatccga gcctcccagc cccgccttct 1261 cttccagatc ttcgggacgg gacacagctc catggagtca ccctcagaaa cgccttctcc 1321 tcctggtacc ttcatgtgga atctgacctg gacgatgaaa gactccttcc ctttcctttc 1381 ccaccgcagt cgatatggtc tggagtgcag ctttgacttc ccctgtgagc tggaatattc 1441 tcctcccctg cacaaccacg ggaatcagag ctggtcctgg cgccatgtgc cctccgagga 1501 ggcctcgagg atgaacttgc tggatgggcc agaggcagag cattctcaag agatgcccag 1561 aggctccttc ctcctcctga acacctctgc agattccaag cataccattc tgagcccatg 1621 gatgaggagc agtagtgatc actgcacact ggctgtctcc gtgcacagac atctacagcc 1681 ttcggggaga tatgttgccc agctcctacc ccacaacgaa gctggaagag agattctttt 1741 ggtgcccacc ccagggaagc atggctggac agtgctgcag gggagagtcg ggcgcccagc 1801 aaacccattt cgagtggctc tggaatacat ctccagtggc aaccggagct tgtcggcggt 1861 ggatttcttt gccctgaaga actgcagtga agggacatcc ccaggctcca agatggcatt 1921 gcagagttcc ttcacttgtt ggaatgggac cgtcctccag ctcgggcaag cctgtgattt 1981 ccaccaggac tgtgcccaag gagaagatga gggccagctg tgcagtaaac ttcctgctgg 2041 attttactgt aactttgaaa atggcttctg tggctggacc caaagtccac tctcacccca 2101 tatgccccgg tggcaagtga ggaccctaag agatgcccat tcccagggcc accaaggccg 2161 tgccctgttg ctcagcacca ctgacatcct cgcttctgaa ggtgcaacag tgaccagtgc 2221 caccttccct gcaccaatga aaaattctcc ttgtgagctc cgcatgtcct ggctcatccg 2281 cggggttttg agaggaaacg tatctctggt gctggtggag aacaaaaccg gaaaggagca 2341 aagccggact gtctggcatg tcgccactga cgaaggctta agcctgtggc agcatacagt 2401 gctgtccctc ctcgatgtga ctgacaggtt ctggctgcag atagtcacat ggtggggtcc 2461 aggatccagg gcaaccgtgg gatttgacaa catttccatc agcctcgact gctaccttac 2521 catcagtgga gaggagaaaa tgtccctgaa ttcagtaccc aaatctagaa atctgtttga 2581 gaaaaaccca aacaaggagt caaaatcctg ggcaaacata tcaggaccaa ctcccatctt 2641 cgaccctaca gttcactggc tgttcaccac gtgtggggcc agtggacctc atggccccac 2701 ccaggcacag tgcaacaacg cctaccagaa ttccaacttg agcgtggtgg tgggaagtga 2761 agggcccttg aagggagtcc agatttggaa agtgccagct actgacacct acagtatctc 2821 gggctacgga gcagctggcg ggaaaggtgg gaaaaacacc atgatgcggt cccatggcgt 2881 gtctgtcctg ggcatcttca atctggagaa aggtgacaca ctctacatcc ttgtcggtca 2941 gcaaggggag gatgcctgtc ccagggcaaa ccaactaatc cagaaagtct gtgtgggtga 3001 gaacaatgtc atagaagaag agatccgagt gaacagaagc gtgcacgagt gggcaggagg 3061 aggaggaggt gggggtggag ccacctacgt gtttaagatg aaagatggcg tgcctgtacc 3121 cctgatcatt gcagctggtg gtggtggcag ggcctatggg gccaagacag aaacgttcca 3181 cccagagaga ctggagagta actcctcggt tctagggctg aacggcaatt ccggagccgc 3241 aggtggtgga ggcggctgga atgataacac ttccttgctc tgggccggaa agtctttgct 3301 ggagggtgcc gccggaggac attcctgccc ccaggccatg aagaagtggg ggtgggagac 3361 aagagggggt ttcggagggg gtggaggggg gtgctcctca ggtggaggag gcggaggata 3421 tataggtggc aacgcagcat caaacaatga ccccgaaatg gatggggaag atggggtttc 3481 cttcatcagt ccattgggta tcctgtacac cccggcctta aaagtgatgg agggccacgg 3541 ggaagtgaat atcaagcatt atctaaactg cagtcactgc gaggtagacg aatgtcacat 3601 ggaccccgag agccacaaag tcatctgctt ctgtgatcat gggaccgtgc tggctgatga 3661 tggtgtctcc tgcattgtgt cacccacccc ggagccccac ctgccgctct cattgatcct 3721 ctccgtcgtg acctctgccc tggtggctgc ccttgttctg gcattctccg gcatcatgat 3781 tgtgtaccgt cggaagcacc aggagttgca ggctatgcag atggaactgc agagccccga 3841 gtataagctg agcaagctac ggacctcgac catcatgacc gactacaacc ccaactactg 3901 cttcgctggc aagacttcct ccatcagtga cctgaaagaa gtgccacgga aaaacatcac 3961 actcatccgg ggcctaggcc atggcgcatt tggggaggtg tatgaaggcc aggtgtctgg 4021 aatgcccaat gacccaagcc ctctacaagt ggctgtaaag acgctgccag aagtgtgttc 4081 agaacaagat gagctggact ttctcatgga agctctgatc atcagcaaat tcaaccacca 4141 gaatattgtt cgctgcatcg gggtgagtct acaagccctg ccccgcttca tcctgctgga 4201 actcatggct ggcggagacc tcaagtcctt cctcagggag acacgccctc gcccgaacca 4261 acccacctcc ctggccatgc tggaccttct gcatgtggct cgggacattg cctgtggctg 4321 tcagtaccta gaggagaatc actttatcca ccgggatatt gctgctagaa actgtctgtt 4381 gacctgccca ggagctggaa gaatagcaaa gattggagac tttgggatgg cccgagatat 4441 ctacagggcc agctactacc gaaagggagg ctgcgccatg ctgccggtca agtggatgcc 4501 ccctgaagcc ttcatggaag ggatatttac ctctaaaaca gacacatggt cttttggagt 4561 gttgctatgg gaaatatttt ctcttggata tatgccgtac cccagcaaga gcaaccagga 4621 agttctggag tttgtcacca gcggaggacg gatggacccg cctaagaact gccccgggcc 4681 tgtataccgg ataatgacgc agtgctggca gcatcagcct gaagacagac ccaacttcgc 4741 catcattttg gagaggatcg aatactgcac ccaggacccc gatgtgatca acacagctct 4801 gcccatcgaa tacggtccag tagtagaaga ggaggagaaa gtgcccatgc gccccaaaga 4861 ccccgagggg atgccacctt tgctggtgtc tccccagcct gcgaagcacg aggaggcgtc 4921 cgcagctccc cagcccgcag ccctgacggc accaggccca tcggtgaaga agcccccggg 4981 tgcgggtgcg ggcgcgggcg cgggtgcggg tgccggcccg gtgccccgag gtgcggccga 5041 tcggggccac gtgaacatgg ctttctctca gcccaaccct cccccggagc tgcacaaagg 5101 cccgggatcc agaaacaagc cgaccagcct gtggaacccc acctacggct cgtggttcac 5161 cgagaagcct gccaaaaaga cccatcctcc gccaggcgcc gagccgcagg cgcgggcagg 5221 agcggccgag ggtggctgga ccgggccggg cgcggggccc cgcagagccg aggcagcgct 5281 gctgctagag ccatcggcgc tgagcgccac catgaaggag gtgccgctgt tcaggctgcg 5341 ccacttcccc tgcggcaatg tcaactatgg ttaccagcaa cagggtctcc ccttggaagc 5401 cacagccgcg ccaggggaca ccatgctgaa aagcaagaat aaggtcaccc agccggggcc 5461 ctgagccctg tactccacta gcttctcctc ctggcggagc cggagcccac ccagagggag 5521 atggacagga tggctccacc acaaacccaa gaccaaaact ttcatttttg tgccaacttg 5581 ttttgaagtg ccacatttta aaaaaaggaa acttgtgttt ttaagatgtg ttagaaggtt 5641 ttttgagcat gggttcatct atcctctcaa aagaagaaaa tgccattctt taaaaaagaa 5701 aaaaaagcaa tcagtgcaag gcccagattg gttgcgccaa gttttcgtgc atggtctgct 5761 gtacagtccc ctaaggcttc tttccgattt ttgtgtgcgc tctgcttccg cgtagtcaga 5821 aatagctgct tccatgtctc atagggggag tcctaggtgt ttcctttgcc ttatgaatat 5881 gaaccactcg aggggcgggc gagggaacag aaataaag
[0321] In some embodiments, fusion proteins comprising the ALK antigen polypeptides are described herein. In some embodiments, the ALK polypeptide can be fused to any heterologous amino acid sequence to form a fusion protein. For example, a fusion protein includes an ALK protein fused to a heterologous protein. In some embodiments, the fusion protein is an ALK protein fused to a nucleophosmin (NPM) protein. In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a NPM-ALK fusion protein in Homo Sapiens. In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary NPM-ALK fusion protein amino acid sequence from Homo Sapiens as provided below (ALK cytoplasmic portion in bold font):
TABLE-US-00098 MEDSMDMDMSPLRPQNYLFGCELKADKDYHFKVDN DENEHQLSLRTVSLGAGAKDELHIVEAEAMNYEGS PIKVTLATLKMSVQPTVSLGGFEITPPVVLRLKCG SGPVHISGQHLVVYRRKHQELQAMOMELQSPEYKL SKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKN NTLIRGLGHGAFGEVYEGQVSGMPNDPSPLQVAVK TLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGV SLQSLPRFILLELMAGGDLKSFLRETRPRPSQPSS IAMLDLLHVARDIACGCQYLEENHFIHRDIAARNC LLTCPGPGRVAKIGDFGMARDIYRASYYRKGGCAM LPVKWMPPEAFMEGIFTSKTDTWSFGVLLWEIFS LGYMPYPSKSNQEVLEFVTSGGRMDPPKNCPGP VYRIMTQCWQHQPEDRPNFAIILERIEYCTQDPDV INTALPIEYGPLVEEEEKVPVRPKDPEGVPPLLVS QQAKREEERSPAAPPPLPTTSSGKAAKKPTAAEVS VRVPRGPAVEGGHVNMAFSQSNPPSELHRVHGSRN KPTSLWNPTYGSWFTEKPTKKNNPIAKKEPHERGN LGLEGSCTVPPNVATGRLPGASLLLEPSSLTANMK EVPLFRLRHFPCGNVNYGYOOQGLPLEAATAPGAG HYEDTILKSKNSMNQPGP
[0322] In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary NPM-ALK fusion protein amino acid sequence from Homo Sapiens (GenBank: AAA58698.1) as provided below:
TABLE-US-00099 1 MEDSMDMDMS PLRPQNYLFG CELKADKDYH FKVDNDENEH QLSLRTVSLG AGAKDELHIV 61 EAEAMNYEGS PIKVTLATLK MSVQPTVSLG GFEITPPVVL RLKCGSGPVH ISGQHLVVYR 121 RKHQELQAMQ MELQSPEYKL SKLRTSTIMT DYNPNYCFAG KTSSISDLKE VPRKNITLIR 181 GLGHGAFGEV YEGQVSGMPN DPSPLQVAVK TLPEVCSEQD ELDFLMEALI ISKFNHQNIV 241 RCIGVSLQSL PRFILLELMA GGDLKSFLRE TRPRPSQPSS LAMLDLLHVA RDIACGCQYL 301 EENHFIHRDI AARNCLLTCP GPGRVAKIGD FGMARDIYRA SYYRKGGCAM LPVKWMPPEA 361 FMEGIFTSKT DTWSFGVLLW EIFSLGYMPY PSKSNQEVLE FVTSGGRMDP PKNCPGPVYR 421 IMTQCWQHQP EDRPNFAIIL ERIEYCTQDP DVINTALPIE YGPLVEEEEK VPVRPKDPEG 481 VPPLLVSQQA KREEERSPAA PPPLPTTSSG KAAKKPTAAE VSVRVPRGPA VEGGHVNMAF 541 SQSNPPSELH KVHGSRNKPT SLWNPTYGSW FTEKPTKKNN PIAKKEPHDR GNLGLEGSCT 601 VPPNVATGRL PGASLLLEPS SLTANMKEVP LFRLRHFPCG NVNYGYQQQG LPLEAATAPG 661 AGHYEDTILK SKNSMNQPGP
[0323] In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary NPM-ALK fusion protein amino acid sequence from Homo Sapiens as provided below:
TABLE-US-00100 M E D S M D M D M S P L R P Q N Y L F G C E L K A D K D Y H F K V D N D E N E H Q L S L R T V S L G A G A K D E L H I V E A E A M N Y E G S P I K V T L A T L K M S V Q P T V S L G G F E I T P P V V L R L K C G S G P V H I S G Q H L V V Y R R K H Q E L Q A M Q M E L Q S P E Y K L S K L R T S T I M T D Y N P N Y C F A G K T S S I S D L K E V P R K N I T L I R G L G H G A F G E V Y E G Q V S G M P N D P S P L Q V A V K T L P E V C S E Q D E L D F L M E A L I I S K F N H Q N I V R C I G V S L Q S L P R F I L L E L M A G G D L K S F L R E T R P R P S Q P S S L A M L D L L H V A R D I A C G C Q Y L E E N H F I H R D I A A R N C L L T C P G P G R V A K I G D F G M A R D I Y R A S Y Y R K G G C A M L P V K W M P P E A F M E G I F T S K T D T W S F G V L L W E I F S L G Y M P Y P S K S N Q E V L E F V T S G G R M D P P K N C P G P V Y R I M T Q C W Q H Q P E D R P N F A I I L E R I E Y C T Q D P D V I N T A L P I E Y G P L V E E E E K V P V R P K D P E G V P P L L V S Q Q A K R E E E R S P A A P P P L P T T S S G K A A K K P T A A E V S V R V P R G P A V E G G H V N M A F S Q S N P P S E L H K V H G S R N K P T S L W N P T Y G S W F T E K P T K K N N P I A K K E P H D R G N L G L E G S C T V P P N V A T G R L P G A S L L L E P S S L T A N M K E V P L F R L R H F P C G N V N Y G Y Q Q Q G L P L E A A T A P G A G H Y E D T I L K S K N S M N Q P G P
[0324] In some embodiments, the NPM-ALK fusion protein is encoded by a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the exemplary nucleic acid sequence from Homo Sapiens as provided below:
TABLE-US-00101 1 atggaagattcgatggacatggacatgagccccctgaggccccagaactatcttttcggt 60 61 tgtgaactaaaggccgacaaagattatcactttaaggtggataatgatgaaaatgagcac 120 121 cagttatctttaagaacggtcagtttaggggctggtgcaaaggatgagttgcacattgtt 180 181 gaagcagaggcaatgaattacgaaggcagtccaattaaagtaacactggcaactttgaaa 240 241 atgtctgtacagccaacggtttcccttgggggctttgaaataacaccaccagtggtctta 300 301 aggttgaagtgtggttcagggccagtgcatattagtggacagcacttagtagtgtaccgc 360 361 cggaagcaccaggagctgcaagccatgcagatggagctgcagagccctgagtacaagctg 420 421 agcaagctccgcacctcgaccatcatgaccgactacaaccccaactactgctttgctggc 480 481 aagacctcctccatcagtgacctgaaggaggtgccgcggaaaaacatcaccctcattcgg 540 541 ggtctgggccatggcgcctttggggaggtgtatgaaggccaggtgtccggaatgcccaac 600 601 gacccaagccccctgcaagtggctgtgaagacgctgcctgaagtgtgctctgaacaggac 660 661 gaactggatttcctcatggaagccctgatcatcagcaaattcaaccaccagaacattgtt 720 721 cgctgcattggggtgagcctgcaatccctgccccggttcatcctgctggagctcatggcg 780 781 gggggagacctcaagtccttcctccgagagacccgccctcgcccgagccagccctcctcc 840 841 ctggccatgctggaccttctgcacgtggctcgggacattgcctgtggctgtcagtatttg 900 901 gaggaaaaccacttcatccaccgagacattgctgccagaaactgcctcttgacctgtcca 960 961 ggccctggaagagtggccaagattggagacttcgggatggcccgagacatctacagggcg 1020 1021 agctactatagaaagggaggctgtgccatgctgccagttaagtggatgcccccagaggcc 1080 1081 ttcatggaaggaatattcacttctaaaacagacacatggtcctttggagtgctgctatgg 1140 1141 gaaatcttttctcttggatatatgccataccccagcaaaagcaaccaggaagttctggag 1200 1201 tttgtcaccagtggaggccggatggacccacccaagaactgccctgggcctgtataccgg 1260 1261 ataatgactcagtgctggcaacatcagcctgaagacaggcccaactttgccatcattttg 1320 1321 gagaggattgaatactgcacccaggacccggatgtaatcaacaccgctttgccgatagaa 1380 1381 tatggtccacttgtggaagaggaagagaaagtgcctgtgaggcccaaggaccctgagggg 1440 1441 gttcctcctctcctggtctctcaacaggcaaaacgggaggaggagcgcagcccagctgcc 1500 1501 ccaccacctctgcctaccacctcctctggcaaggctgcaaagaaacccacagctgcagag 1560 1561 gtctctgttcgagtccctagagggccggccgtggaagggggacacgtgaatatggcattc 1620 1621 tctcagtccaaccctccttcggagttgcacaaggtccacggatccagaaacaagcccacc 1680 1681 agcttgtggaacccaacgtacggctcctggtttacagagaaacccaccaaaaagaataat 1740 1741 cctatagcaaagaaggagccacacgacaggggtaacctggggctggagggaagctgtact 1800 1801 gtcccacctaacgttgcaactgggagacttccgggggcctcactgctcctagagccctct 1860 1861 tcgctgactgccaatatgaaggaggtacctctgttcaggctacgtcacttcccttgtggg 1920 1921 aatgtcaattacggctaccagcaacagggcttgcccttagaagccgctactgcccctgga 1980 1981 gctggtcattacgaggataccattctgaaaagcaagaatagcatgaaccagcctgggccc 2040 2041 tga 2043
[0325] In some embodiments, the fusion protein is an ALK protein fused to an echinoderm microtubule-associated protein-like 4 (EML4) protein. In some embodiments, the ELM4-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a ELM4-ALK fusion protein in Homo Sapiens or a variant thereof. In some embodiments, the ELM4-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary ELM4-ALK fusion protein amino acid sequence from Homo Sapiens (GenBank: BAM37627.1) as provided below:
TABLE-US-00102 1 MDGFAGSLDD SISAASTSDV QDRLSALESR VQQQEDEITV LKAALADVLR RLAISEDHVA 61 SVKKSVSSKG QPSPRAVIPM SCITNGSGAN RKPSHTSAVS IAGKETLSSA AKSGTEKKKE 121 KPQGQREKKE ESHSNDQSPQ IRASPSPQPS SQPLQIHRQT PESKNATPTK SIKRPSPAEK 181 SHNSWENSDD SRNKLSKIPS TPKLIPKVTK TADKHKDVII NQEGEYIKMF MRGRPITMFI 241 PSDVDNYDDI RTELPPEKLK LEWAYGYRGK DCRANVYLLP TGEIVYFIAS VVVLFNYEER 301 TQRHYLGHTD CVKCLAIHPD KIRIATGQIA GVDKDGRPLQ PHVRVWDSVT LSTLQIIGLG 361 TFERGVGCLD FSKADSGVHL CVIDDSNEHM LTVWDWQRKA KGAEIKTTNE VVLAVEFHPT 421 DANTIITCGK SHIFFWTWSG NSLTRKQGIF GKYEKPKFVQ CLAFLGNGDV LTGDSGGVML 481 IWSKTTVEPT PGKGPKGVYQ ISKQIKAHDG SVFTLCQMRN GMLLTGGGKD RKIILWDHDL 541 NPEREIEFSA SRARLPGHVA ADHPPAVYRR KHQELQAMQM ELQSPEYKLS KLRTSTIMTD 601 YNPNYCFAGK TSSISDLKEV PRKNITLIRG LGHGAFGEVY EGQVSGMPND PSPLQVAVKT 661 LPEVCSEQDE LDFLMEALII SKFNHQNIVR CIGVSLQSLP RFILLELMAG GDLKSFLRET 721 RPRPSQPSSL AMLDLLHVAR DIACGCQYLE ENHFIHRDIA ARNCLLTCPG PGRVAKIGDF 781 GMARDIYRAS YYRKGGCAML PVKWMPPEAF MEGIFTSKTD TWSFGVLLWE IFSLGYMPYP 841 SKSNQEVLEF VTSGGRMDPP KNCPGPVYRI MTQCWQHQPE DRPNFAIILE RIEYCTQDPD 901 VINTALPIEY GPLVEEEEKV PVRPKDPEGV PPLLVSQQAK REEERSPAAP PPLPTTSSGK 961 AAKKPTAAEI SVRVPRGPAV EGGHVNMAFS QSNPPSELHK VHGSRNKPTS LWNPTYGSWF 1021 TEKPTKKNNP IAKKEPHDRG NLGLEGSCTV PPNVATGRLP GASLLLEPSS LTANMKEVPL 1081 FRLRHFPCGN VNYGYOOOGL PLEAATAPGA GHYEDTILKS KNSMNQPGP
[0326] In some embodiments, the ELM4-ALK fusion protein is encoded by a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence from Homo Sapiens (GenBank: AB274722.1) as provided below:
TABLE-US-00103 1 ggcggcgcgg cgcggcgctc gcggctgctg cctgggaggg aggccgggca ggcggctgag 61 cggcgcggct ctcaacgtga cggggaagtg gttcgggcgg ccgcggctta ctaccccagg 121 gcgaacggac ggacgacgga ggcgggagcc ggtagccgag ccgggcgacc tagagaacga 181 gcgggtcagg ctcagcgtcg gccactctgt cggtccgctg aatgaagtgc ccgcccctct 241 gagcccggag cccggcgctt tccccgcaag atggacggtt tcgccggcag tctcgatgat 301 agtatttctg ctgcaagtac ttctgatgtt caagatcgcc tgtcagctct tgagtcacga 361 gttcagcaac aagaagatga aatcactgtg ctaaaggcgg ctttggctga tgttttgagg 421 cgtcttgcaa tctctgaaga tcatgtggcc tcagtgaaaa aatcagtctc aagtaaaggc 481 caaccaagcc ctcgagcagt tattcccatg tcctgtataa ccaatggaag tggtgcaaac 541 agaaaaccaa gtcataccag tgctgtctca attgcaggaa aagaaactct ttcatctgct 601 gctaaaagtg gtacagaaaa aaagaaagaa aaaccacaag gacagagaga aaaaaaagag 661 gaatctcatt ctaatgatca aagtccacaa attcgagcat caccttctcc ccagccctct 721 tcacaacctc tccaaataca cagacaaact ccagaaagca agaatgctac tcccaccaaa 781 agcataaaac gaccatcacc agctgaaaag tcacataatt cttgggaaaa ttcagatgat 841 agccgtaata aattgtcgaa aataccttca acacccaaat taataccaaa agttaccaaa 901 actgcagaca agcataaaga tgtcatcatc aaccaagaag gagaatatat taaaatgttt 961 atgcgcggtc ggccaattac catgttcatt ccttccgatg ttgacaacta tgatgacatc 1021 agaacggaac tgcctcctga gaagctcaaa ctggagtggg catatggtta tcgaggaaag 1081 gactgtagag ctaatgttta ccttcttccg accggggaaa tagtttattt cattgcatca 1141 gtagtagtac tatttaatta tgaggagaga actcagcgac actacctggg ccatacagac 1201 tgtgtgaaat gccttgctat acatcctgac aaaattagga ttgcaactgg acagatagct 1261 ggcgtggata aagatggaag gcctctacaa ccccacgtca gagtgtggga ttctgttact 1321 ctatccacac tgcagattat tggacttggc acttttgagc gtggagtagg atgcctggat 1381 ttttcaaaag cagattcagg tgttcattta tgtgttattg atgactccaa tgagcatatg 1441 cttactgtat gggactggca gaagaaagca aaaggagcag aaataaagac aacaaatgaa 1501 gttgttttgg ctgtggagtt tcacccaaca gatgcaaata ccataattac atgcggtaaa 1561 tctcatattt tcttctggac ctggagcggc aattcactaa caagaaaaca gggaattttt 1621 gggaaatatg aaaagccaaa atttgtgcag tgtttagcat tcttggggaa tggagatgtt 1681 cttactggag actcaggtgg agtcatgctt atatggagca aaactactgt agagcccaca 1741 cctgggaaag gacctaaagt gtaccgccgg aagcaccagg agctgcaagc catgcagatg 1801 gagctgcaga gccctgagta caagctgagc aagctccgca cctcgaccat catgaccgac 1861 tacaacccca actactgctt tgctggcaag acctcctcca tcagtgacct gaaggaggtg 1921 ccgcggaaaa acatcaccct cattcggggt ctgggccatg gagcctttgg ggaggtgtat 1981 gaaggccagg tgtccggaat gcccaacgac ccaagccccc tgcaagtggc tgtgaagacg 2041 ctgcctgaag tgtgctctga acaggacgaa ctggatttcc tcatggaagc cctgatcatc 2101 agcaaattca accaccagaa cattgttcgc tgcattgggg tgagcctgca atccctgccc 2161 cggttcatcc tgctggagct catggcgggg ggagacctca agtccttcct ccgagagacc 2221 cgccctcgcc cgagccagcc ctcctccctg gccatgctgg accttctgca cgtggctcgg 2281 gacattgcct gtggctgtca gtatttggag gaaaaccact tcatccaccg agacattgct 2341 gccagaaact gcctcttgac ctgtccaggc cctggaagag tggccaagat tggagacttc 2401 gggatggccc gagacatcta cagggcgagc tactatagaa agggaggctg tgccatgctg 2461 ccagttaagt ggatgccccc agaggccttc atggaaggaa tattcacttc taaaacagac 2521 acatggtcct ttggagtgct gctatgggaa atcttttctc ttggatatat gccatacccc 2581 agcaaaagca accaggaagt tctggagttt gtcaccagtg gaggccggat ggacccaccc 2641 aagaactgcc ctgggcctgt ataccggata atgactcagt gctggcaaca tcagcctgaa 2701 gacaggccca actttgccat cattttggag aggattgaat actgcaccca ggacccggat 2761 gtaatcaaca ccgctttgcc gatagaatat ggtccacttg tggaagagga agagaaagtg 2821 cctgtgaggc ccaaggaccc tgagggggtt cctcctctcc tggtctctca acaggcaaaa 2881 cgggaggagg agcgcagccc agctgcccca ccacctctgc ctaccacctc ctctggcaag 2941 gctgcaaaga aacccacagc tgcagaggtc tctgttcgag tccctagagg gccggccgtg 3001 gaagggggac acgtgaatat ggcattctct cagtccaacc ctccttcgga gttgcacagg 3061 gtccacggat ccagaaacaa gcccaccagc ttgtggaacc caacgtacgg ctcctggttt 3121 acagagaaac ccaccaaaaa gaataatcct atagcaaaga aggagccaca cgagaggggt 3181 aacctggggc tggagggaag ctgtactgtc ccacctaacg ttgcaactgg gagacttccg 3241 ggggcctcac tgctcctaga gccctcttcg ctgactgcca atatgaagga ggtacctctg 3301 ttcaggctac gtcacttccc ttgtgggaat gtcaattacg gctaccagca acagggcttg 3361 cccttagaag ccgctactgc ccctggagct ggtcattacg aggataccat tctgaaaagc 3421 aagaatagca tgaaccagcc tgggccctga gctcggtcac acactcactt ctcttccttg 3481 ggatccctaa gaccgtggag gagagagagg caatcaatgg ctccttcaca aaccagagac 3541 caaatgtcac gttttgtttt gtgccaacct attttgaagt accaccaaaa aagctgtatt 3601 ttgaaaatgc tttagaaagg ttttgagcat gggttcatcc tattctttcg aaagaagaaa 3661 atatcataaa aatgagtgat aaatacaagg cccagatgtg gttgcataag gtttttatgc 3721 atgtttgttg tatacttcct tatgcttctt ttaaattgtg tgtgctctgc ttcaatgtag 3781 tcagaattag ctgcttctat gtttcatagt tggggtcata gatgtttcct tgccttgttg 3841 atgtggacat gagccatttg aggggagagg gaacggaaat aaaggagtta tttgtaatga 3901 aaaaaaaaaa aaaaaaaaaa aaaaaa
[0327] In some embodiments, the ELM4-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary ELM4-ALK variant 1 fusion protein amino acid sequence from Homo Sapiens (GenBank: BAF73611.1) as provided below:
TABLE-US-00104 1 MDGFAGSLDD SISAASTSDV QDRLSALESR VQQQEDEITV LKAALADVLR RLAISEDHVA 61 SVKKSVSSKG QPSPRAVIPM SCITNGSGAN RKPSHTSAVS IAGKETLSSA AKSGTEKKKE 121 KPQGQREKKE ESHSNDQSPQ IRASPSPQPS SQPLQIHRQT PESKNATPTK SIKRPSPAEK 181 SHNSWENSDD SRNKLSKIPS TPKLIPKVTK TADKHKDVII NQEGEYIKMF MRGRPITMFI 241 PSDVDNYDDI RTELPPEKLK LEWAYGYRGK DCRANVYLLP TGEIVYFIAS VWVVFNYEER 301 TQRHYLGHTD CVKCLAIHPD KIRIATGQIA GVDKDGRPLQ PHVRVWDSVT LSTLQIIGLG 361 TFERGVGCLD FSKADSGVHL CVIDDSNEHM LTVWDWQKKA KGAEIKTTNE VVLAVEFHPT 421 DANTIITCGK SHIFFWTWSG NSLTRKQGIF GKYEKPKFVQ CLAFLGNGDV LTGDSGGVML 481 IWSKTTVEPT PGKGPKVYRR KHQELQAMQM ELQSPEYKLS KLRTSTIMTD YNPNYCFAGK 541 TSSISDLKEV PRKNITLIRG LGHGAFGEVY EGQVSGMPND PSPLQVAVKT LPEVCSEQDE 601 LDFLMEALII SKFNHQNIVR CIGVSLQSLP RFILLELMAG GDLKSFLRET RPRPSQPSSL 661 AMLDLLHVAR DIACGCQYLE ENHFIHRDIA ARNCLLTCPG PGRVAKIGDF GMARDIYRAS 721 YYRKGGCAML PVKWMPPEAF MEGIFTSKTD TWSFGVLLWE IFSLGYMPYP SKSNQEVLEF 781 VTSGGRMDPP KNCPGPVYRI MTQCWQHQPE DRPNFAIILE RIEYCTQDPD VINTALPIEY 841 GPLVEEEEKV PVRPKDPEGV PPLLVSQQAK REEERSPAAP PPLPTTSSGK AAKKPTAAEV 901 SVRVPRGPAV EGGHVNMAFS QSNPPSELHR VHGSRNKPTS LWNPTYGSWF TEKPTKKNNP 961 IAKKEPHERG NLGLEGSCTV PPNVATGRLP GASLLLEPSS LTANMKEVPL FRLRHFPCGN 1021 VNYGYOOOGL PLEAATAPGA GHYEDTILKS KNSMNQPGP
[0328] In some embodiments, the ALK CAR of the present invention includes sequences from an anti-ALK antibody that specifically binds to a mammalian ALK protein or antigen. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that binds to a murine ALK protein or an antibody-binding portion thereof. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that binds to a human ALK protein or an antibody-binding portion thereof. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that binds to a portion of the extracellular domain of the ALK receptor. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that binds to a portion of the extracellular domain of a murine ALK receptor. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that binds to a portion of the extracellular domain of a human ALK receptor. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that is a murine antibody. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that is a human antibody. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that is a humanized antibody. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that is a chimeric antibody.
[0329] In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that modulates ALK activity (e.g., ALK signaling) and/or ALK expression. In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that inhibits ALK signaling and/or ALK expression (e.g., inhibits ALK phosphorylation). In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that activates ALK signaling and/or ALK expression (e.g., agonist of ALK phosphorylation).
[0330] In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody that is selected from ALK Antibody #1 (ALK #1), ALK Antibody #2 (ALK #2), ALK Antibody #3 (ALK #3), ALK Antibody #4 (ALK #4), ALK Antibody #5 (ALK #5), ALK Antibody #6 (ALK #6), or ALK Antibody #7 (ALK #7). In some embodiments, the ALK CAR includes sequences from ALK #1. In some embodiments, the ALK CAR includes sequences from ALK #2. In some embodiments, the ALK CAR includes sequences from ALK #3. In some embodiments, the ALK CAR includes sequences from ALK #4. In some embodiments, the ALK CAR includes sequences from ALK #5. In some embodiments, the ALK CAR includes sequences from ALK #6. In some embodiments, the ALK CAR includes sequences from ALK #7.
[0331] In some embodiments, the ALK CAR includes sequences from an anti-ALK antibody or an antigen binding fragment thereof comprising a VL region selected from ALK Antibody #1 (ALK #1), ALK Antibody #2 (ALK #2), ALK Antibody #3 (ALK #3), ALK Antibody #4 (ALK #4), ALK Antibody #5 (ALK #5), ALK Antibody #6 (ALK #6), or ALK Antibody #7 (ALK #7) (see Table 1). In some embodiments, the ALK CAR includes sequences from the VL region of ALK #1. In some embodiments, the ALK CAR includes sequences from the VL region of ALK #2. In some embodiments, the ALK CAR includes sequences from the VL region of ALK #3. In some embodiments, the ALK CAR includes sequences from the VL region of ALK #4. In some embodiments, the ALK CAR includes sequences from the VL region of ALK #5. In some embodiments, the ALK CAR includes sequences from the VL region of ALK #6. In some embodiments, the ALK CAR includes sequences from the VL region of ALK #7.
TABLE-US-00105 TABLE 1 Variable Light Chain (VL) ALK Antibody Sequences ALK Antibody VL Amino Acid Sequence VL Nucleic Acid Sequence ALK#1 DIQMTQSPASLAASVGETVTITCRA gacatccaga tgactcagtc tccagcctcc SENIYYSLAWYQQKQGKSPQLLIYN ctggctgcat ctgtgggaga aactgtcacc ANSLEDGVPSRFSGSGSGTQYSMKI atcacatgtc gagcaagtga gaacatttac NSMQPEDTATYFCKQAYDVPFTFGS tacagtttag catggtatca gcagaagcaa GTKLEIKR gggaaatctc ctcagctcct gatctataat gcaaacagct tggaagatgg tgtcccatcg aggttcagtg gcagtggatc tgggacacag tattctatga agatcaacag catgcagcct gaagataccg caacttattt ctgtaaacag gcttatgacg ttccattcac gttcggctcg gggacaaagt tggaaataaa acgg ALK#2 AIQMTQTTSSLSASLGDRVTISCSV gctatccaga tgacacagac tacatcctcc SQGISNSLNWYQQKPDGTVKLLIYY ctgtctgcct ctctgggaga cagagtcacc TSSLHSGVPSRFSGSGSGTDYSLTI atcagttgca gtgtaagtca gggcattagc SNLEPEDIATYYCQQYSKLPLTFGA aattctttaa actggtatca gcagaaacca GTKLELKR gatggaactg ttaaactcct gatctattac acatcaagtt tacactcagg agtcccatca aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcagcaa cctggaacct gaagatattg ccacttacta ttgtcagcag tatagtaagc ttccgctcac gttcggtgct gggaccaagc tggagctgaa acgg ALK#3 DIVMTQSQRFMSTSVGDRVSVT gacattgtga tgacccagtc tcaaagattc CKASQNVGTNVAWYQQKPGQS atgtccacat cagtaggaga cagggtcagc PKALIYSASYRYSGVPDRFTGS gtcacctgca aggccagtca gaatgtgggt GSGTDFTLTVSNVQSEDLAEYF actaatgtag cctggtatca acagaaacca CQQYNSYPYMYTFGGGTKLEIK gggcaatctc ctaaagcact gatttactcg R gcatcctacc ggtacagtgg agtccctgat cgcttcacag gcagtggatc tgggacagat ttcactctca ccgtcagcaa tgtgcagtct gaagacttgg cagagtattt ctgtcagcaa tataacagct atccgtacat gtacacgttc ggagggggga ccaagctgga aataaaacgg ALK#4 DIVLTQSPASLAVSLGQRATISCRA gacattgtgc tgacccaatc tccagcttct SESVDNYGISFMNWFQQKPGQPP ttggctgtgt ctctagggca gagggccacc KLLIYAASNQGSGVPARFSGSGSG atctcctgca gagccagcga aagtgttgat TDFSLNIHPMEEDDTAMYFCQQSK aattatggca ttagttttat gaactggttc EVPWTFGGGTKLEIKR caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg acgttcggtg gaggcaccaa gctggaaatc aaacgg ALK#5 DIVMTQSQRFMSTSVGDRVSVTCKA gacattgtga tgacccagtc tcaaagattc SQNVGTNVAWYQQKPGQSPKALIYS atgtccacat cagtaggaga cagggtcagc ASYRYSGVPDRFTGSGSGTDFTLTI gtcacctgca aggccagtca gaatgtgggt SNVQSEDLAEYFCQQYNSYPYMYTF actaatgtag cctggtatca acagaaacca GGGTKLEIKR gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct gaagacttgg cagagtattt ctgtcaacaa tataacagct atccgtacat gtacacgttc ggagggggga ccaagctgga aataaaacgg ALK#6 DIVMTQSQKFMSTSVGDRVSITCKA gacattgtga tgacccagtc tcaaaaattc SQNVGTAVAWYQLKPGQSPKLLIY atgtccacat cagtaggaga cagggtcagc SASNRFTGVPDRFTGSGSGTDFTL atcacctgta aggccagtca gaatgtgggt TISNMQSEDLADYFCQQYSSYPLTF actgctgtag cctggtatca actgaaacca GSGTKLEIKR ggacaatctc ctaaactact gatttactcg gcatccaatc ggttcactgg agtccctgat cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tatgcagtct gaagacctgg cagattattt ctgccagcaa tatagcagct atcctctcac gttcggctcg gggacaaagt tggaaataaa acgg ALK#7 DIVMTQSQKFMSTSVGDRVSVTCK gacattgtga tgacccagtc tcaaaaattc ASQNVGTNVAWYQQKPGHSPKALI atgtccacat cagtaggaga cagggtcagc YSASYRYSGVPDRFTGSGSGTDFT gtcacctgca aggccagtca gaatgtgggt LTISNVQSEDLAEYFCQRYNSYPY actaatgtag cctggtatca acagaaacca MFTFGGGTKLEIKR gggcactctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct gaagacttgg cagagtattt ctgtcagcga tataacagct atccgtacat gttcacgttc ggagggggga ccaagctgga aataaaacgg
[0332] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VL amino acid sequence as provided below:
TABLE-US-00106 DIQMTQSPASLAASVGETVTITCRASENIYYSLAW YQQKQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQ YSMKINSMQPEDTATYFCKQAYDVPFTFGSGTKLE IKR
[0333] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VL amino acid sequence as provided below:
TABLE-US-00107 AIQMTQTTSSLSASLGDRVTISCSVSQGISNSLNW YQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTD YSLTISNLEPEDIATYYCQQYSKLPLTFGAGTKLE LKR
[0334] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VL amino acid sequence as provided below:
TABLE-US-00108 DIVMTQSQRFMSTSVGDRVSVTCKASQNVGTNVAW YQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTD FTLTVSNVQSEDLAEYFCQQYNSYPYMYTFGGGTK LEIKR
[0335] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VL amino acid sequence as provided below:
TABLE-US-00109 DIVLTQSPASLAVSLGQRATISCRASESVDNYGIS FMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSG SGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGG TKLEIKR
[0336] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VL amino acid sequence as provided below:
TABLE-US-00110 DIVMTQSQREMSTSVGDRVSVTCKASQNVGTNVAW YQQKPGQSPKALIYSASYRYSGVPDRETGSGSGTD FTLTISNVQSEDLAEYFCQQYNSYPYMYTFGGGTK LEIKR
[0337] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VL amino acid sequence as provided below:
TABLE-US-00111 DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAW YQLKPGQSPKLLIYSASNRFTGVPDRFTGSGSGTD FTLTISNMQSEDLADYFCQQYSSYPLTFGSGTKLE IKR
[0338] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VL amino acid sequence as provided below:
TABLE-US-00112 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAW YQQKPGHSPKALIYSASYRYSGVPDRFTGSGSGTD FTLTISNVQSEDLAEYFCQRYNSYPYMFTFGGGTK LEIKR
[0339] In some embodiments, the ALK CAR includes an anti-ALK antibody VL region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00113 gacatccaga tgactcagtc tccagcctcc ctggctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gaacatttac tacagtttag catggtatca gcagaagcaa 120 gggaaatctc ctcagctcct gatctataat gcaaacagct tggaagatgg tgtcccatcg 180 aggttcagtg gcagtggatc tgggacacag tattctatga agatcaacag catgcagcct 240 gaagataccg caacttattt ctgtaaacag gcttatgacg ttccattcac gttcggctcg 300 gggacaaagt tggaaataaa acgg 324
[0340] In some embodiments, the ALK CAR includes an anti-ALK antibody VL region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00114 gctatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gtgtaagtca gggcattagc aattctttaa actggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctattac acatcaagtt tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcagcaa cctggaacct 240 gaagatattg ccacttacta ttgtcagcag tatagtaagc ttccgctcac gttcggtgct 300 gggaccaagc tggagctgaa acgg 324
[0341] In some embodiments, the ALK CAR includes an anti-ALK antibody VL region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00115 gacattgtga tgacccagtc tcaaagattc atgtccacat cagtaggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120 gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccgtcagcaa tgtgcagtct 240 gaagacttgg cagagtattt ctgtcagcaa tataacagct atccgtacat gtacacgttc 300 ggagggggga ccaagctgga aataaaacgg 330
[0342] In some embodiments, the ALK CAR includes an anti-ALK antibody VL region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00116 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gaactggttc 120 caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc 180 ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240 cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg 300 acgttcggtg gaggcaccaa gctggaaatc aaacgg 336
[0343] In some embodiments, the ALK CAR includes an anti-ALK antibody VL region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00117 gacattgtga tgacccagtc tcaaagattc atgtccacat cagtaggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120 gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240 gaagacttgg cagagtattt ctgtcaacaa tataacagct atccgtacat gtacacgttc 300 ggagggggga ccaagctgga aataaaacgg 330
[0344] In some embodiments, the ALK CAR includes an anti-ALK antibody VL region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00118 gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgta aggccagtca gaatgtgggt actgctgtag cctggtatca actgaaacca 120 ggacaatctc ctaaactact gatttactcg gcatccaatc ggttcactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tatgcagtct 240 gaagacctgg cagattattt ctgccagcaa tatagcagct atcctctcac gttcggctcg 300 gggacaaagt tggaaataaa acgg 324
[0345] In some embodiments, the ALK CAR includes an anti-ALK antibody VL region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00119 gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120 gggcactctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240 gaagacttgg cagagtattt ctgtcagcga tataacagct atccgtacat gttcacgttc 300 ggagggggga ccaagctgga aataaaacgg 330
[0346] In some embodiments, the ALK CAR includes an anti-ALK antibody or an antigen binding fragment thereof comprising a VH region selected from ALK Antibody #1 (ALK #1), ALK Antibody #2 (ALK #2), ALK Antibody #3 (ALK #3), ALK Antibody #4 (ALK #4), ALK Antibody #5 (ALK #5), ALK Antibody #6 (ALK #6), or ALK Antibody #7 (ALK #7) (see Table 2). In some embodiments, the ALK CAR includes the VH region selected from ALK #1. In some embodiments, the ALK CAR includes the VH region selected from ALK #2. In some embodiments, the ALK CAR includes the VH region selected from ALK #3. In some embodiments, the ALK CAR includes the VH region selected from ALK #4. In some embodiments, the ALK CAR includes the VH region selected from ALK #5. In some embodiments, the ALK CAR includes the VH region selected from ALK #6. In some embodiments, the ALK CAR includes the VH region selected from ALK #7.
TABLE-US-00120 TABLE 2 Variable Heavy Chain (VH) ALK Antibody Sequences ALK Antibody VH Amino Acid Sequence VH Nucleic Acid Sequence ALK#1 QVQLQQSGAELVKPGASVKISC caggttcagc tgcagcagtc tggggctgag KASGYAFSSYWMNWWKQRPG ctggtgaagc ctggggcctc agtgaagatt KGLEWIGQIYPGDGDTNYNGKF tcctgcaaag cttctggcta cgcattcagt KGKATLTADKSSSTAYMQLSSL agctactgga tgaactgggt gaagcagagg TSEDSAVYFCASYYYGSKAYW cctggaaagg gtcttgagtg gattggacag GQGTLVTVSA atttatcctg gagatggtga tactaactac aacggaaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac atgcagctca gcagcctgac ctctgaggac tctgcggtct atttctgtgc ctcttattac tacggtagta aggcttactg gggccaaggg actctggtca ctgtctctgc a ALK#2 QVQLQQPGAEFVKPGASVKLS caggtccaac tgcagcagcc tggggctgag CKASGYTFTSYWMHWWKQRP tttgtgaagc ctggggcttc agtgaagctg GRGLEWIGRIDPNSGGTKYNEK tcctgcaagg cttctggcta caccttcacc FKSKATLTVDKPSSTAYMQLSS agctactgga tgcactgggt gaagcagagg LTSEDSAVYYCARDYYGSSYRF cctggacgag gccttgagtg gattggaagg AYWGQGTLVTVSA attgatccta atagtggtgg tactaagtac aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aagagattac tacggtagta gctaccggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca ALK#3 QVQLQQSGAELAKPGASVKLS caggtccagc tgcagcagtc tggggctgaa CKASGYTFTNYWM H WWKQRP ctggcaaaac ctggggcctc agtgaagctg GQGLEWIGYINPSSGYTKYNQK tcctgcaagg cttctggcta cacctttact FKDKATLTADKSSSTAYMQLSS aactactgga tgcactgggt aaaacagagg LTYEDSAVYYCARDYYGSSSW cctggacagg gtctggaatg gattggatac FAYWGQGTLVTVSA attaatccta gcagtggtta tactaagtac aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac atgcagctga gcagcctgac atatgaggac tctgcagtct attactgtgc aagagattac tacggtagta gctcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca ALK#4 QVQLQQSGAELVKPGASVKISC caggttcagc tgcaacagtc tggggctgag KASGYAFSSYWVNWVKQRPGK ctggtgaagc ctggggcctc agtgaagatt GLEWIGQIYPGDGDTNYNGKFK tcctgcaaag cttctggcta cgcattcagt GKATLTADKSSSTAYMQLSSLT agctactggg tgaactgggt gaagcagagg SEDSAVYFCARSRGYFYGSTY cctggaaagg gtcttgagtg gattggacag DSWGQGTTLTVSS atttatcctg gagatggtga tactaactac aacggaaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac atgcagctca gcagcctgac ctctgaggac tctgcggtct atttctgtgc aagatcaaga gggtatttct acggtagtac ctacgactcc tggggccaag gcaccactct cacagtctcc tca ALK#5 QVQLQQSGAELAKPGASVKLS caggtccagc tgcagcagtc tggggctgaa CKASGYTFTSYWMHWVKQRP ctggcaaaac ctggggcctc agtgaagctg GQGLEWIGYIKPSSGYTKYNQK tcctgcaagg cttctggcta cacctttact FKDKATLTADKSSSTAYMQLSS agctactgga tgcactgggt aaaacagagg LTYEDSAVYYCARDYYGSSSW cctggacagg gtctggaatg gattggatac FAYWGQGTLVTVSA attaagccta gcagtggtta tactaagtac aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac atgcagctga gcagcctgac atatgaggac tctgcagtct attactgtgc aagagattac tacggtagta gctcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca ALK#6 DVKLVESGEGLVKPGGSLKLSC gacgtgaagc tggtggagtc tggggaaggc AASGFTFSSYAMSWVRQTPEK ttagtgaagc ctggagggtc cctgaaactc RLEWVTYISSGGDYIYYADTVK tcctgtgcag cctctggatt cactttcagt GRFTISRDNARNTLYLQMSSLK agctatgcca tgtcttgggt tcgccagact SEDTAMYYCTRERIWLRRFFDV ccagagaaga ggctggagtg ggtcacatac WGTGTTVTVSS attagtagtg gtggtgatta catctactat gcagacactg tgaagggccg attcaccatc tccagagaca atgccaggaa caccctgtac ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtac aagagagcgg atatggttac gacggttctt cgatgtctgg ggcacaggga ccacggtcac cgtctcctca ALK#7 QVQLQQSGAELAKPGASVKLS caggtccagc tgcagcagtc tggggctgaa CKASGYTFTSYWMHVWKQRP ctggcaaaac ctggggcctc agtgaagctg GQGLEWIGYINPSSGYTKYNQK tcctgcaagg cttctggcta cacctttact FKDKATLTADKSSSTAYMQLSS agctactgga tgcactgggt aaaacagagg LTFEDSAVYYCARDYYGSSSW cctggacagg gtctggaatg gattggatac FAYWGQGTLVTVSA attaatccta gcagtggtta tactaagtac aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac atgcagctga gcagcctgac atttgaggac tctgcagtct attactgtgc aagagattac tacggtagta gctcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca
[0347] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VH amino acid sequence as provided below:
TABLE-US-00121 QVQLQQSGAELVKPGASVKISCKASGYAFSSYWMN WKQRPGKGLEWIGQIYPGDGDTNYNGKFKGKATLT ADKSSSTAYMQLSSLTSEDSAVYFCASYYYGSKAY WGQGTLVTVSA
[0348] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VH amino acid sequence as provided below:
TABLE-US-00122 QVQLQQPGAEFVKPGASVKLSCKASGYTFTSYWMH WKQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLT VDKPSSTAYMQLSSLTSEDSAVYYCARDYYGSSYR FAYWGQGTLVTVSA
[0349] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VH amino acid sequence as provided below:
TABLE-US-00123 QVQLQQSGAELAKPGASVKLSCKASGYTFTNYWMH WKQRPGQGLEWIGYINPSSGYTKYNQKFKDKATLT ADKSSSTAYMQLSSLTYEDSAVYYCARDYYGSSSW FAYWGQGTLVTVSA
[0350] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VH amino acid sequence as provided below:
TABLE-US-00124 QVQLQQSGAELVKPGASVKISCKASGYAFSSYWVN WKQRPGKGLEWIGQIYPGDGDTNYNGKFKGKATLT ADKSSSTAYMQLSSLTSEDSAVYFCARSRGYFYGS TYDSWGQGTTLTVSS
[0351] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VH amino acid sequence as provided below:
TABLE-US-00125 QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMH WVKQRPGQGLEWIGYIKPSSGYTKYNQKFKDKATL TADKSSSTAYMQLSSLTYEDSAVYYCARDYYGSSS WFAYWGQGTLVTVSA
[0352] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VH amino acid sequence as provided below:
TABLE-US-00126 DVKLVESGEGLVKPGGSLKLSCAASGFTFSSYAMS WRQTPEKRLEWTYISSGGDYIYYADTVKGRFTISR DNARNTLYLQMSSLKSEDTAMYYCTRERIWLRRFF DVWGTGTTVTVSS
[0353] In some embodiments, the ALK CAR includes a sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary anti-ALK antibody VH amino acid sequence as provided below:
TABLE-US-00127 QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMH WVKQRPGQGLEWIGYINPSSGYTKYNQKFKDKATL TADKSSSTAYMQLSSLTFEDSAVYYCARDYYGSSS WFAYWGQGTLVTVSA
[0354] In some embodiments, the ALK CAR includes an anti-ALK antibody VH region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00128 caggttcagc tgcagcagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatt 60 tcctgcaaag cttctggcta cgcattcagt agctactgga tgaactgggt gaagcagagg 120 cctggaaagg gtcttgagtg gattggacag atttatcctg gagatggtga tactaactac 180 aacggaaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240 atgcagctca gcagcctgac ctctgaggac tctgcggtct atttctgtgc ctcttattac 300 tacggtagta aggcttactg gggccaaggg actctggtca ctgtctctgc a 351
[0355] In some embodiments, the ALK CAR includes an anti-ALK antibody VH region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00129 caggtccaac tgcagcagcc tggggctgag tttgtgaagc ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120 cctggacgag gccttgagtg gattggaagg attgatccta atagtggtgg tactaagtac 180 aatgagaagt tcaagagcaa ggccacactg actgtagaca aaccctccag cacagcctac 240 atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtgc aagagattac 300 tacggtagta gctaccggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360
[0356] In some embodiments, the ALK CAR includes an anti-ALK antibody VH region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00130 caggtccagc tgcagcagtc tggggctgaa ctggcaaaac ctggggcctc agtgaagctg 60 tcctgcaagg cttctggcta cacctttact aactactgga tgcactgggt aaaacagagg 120 cctggacagg gtctggaatg gattggatac attaatccta gcagtggtta tactaagtac 180 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 atgcagctga gcagcctgac atatgaggac tctgcagtct attactgtgc aagagattac 300 tacggtagta gctcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360
[0357] In some embodiments, the ALK CAR includes an anti-ALK antibody VH region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00131 caggttcagc tgcaacagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatt 60 tcctgcaaag cttctggcta cgcattcagt agctactggg tgaactgggt gaagcagagg 120 cctggaaagg gtcttgagtg gattggacag atttatcctg gagatggtga tactaactac 180 aacggaaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240 atgcagctca gcagcctgac ctctgaggac tctgcggtct atttctgtgc aagatcaaga 300 gggtatttct acggtagtac ctacgactcc tggggccaag gcaccactct cacagtctcc 360 tca 363
[0358] In some embodiments, the ALK CAR includes an anti-ALK antibody VH region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00132 caggtccagc tgcagcagtc tggggctgaa ctggcaaaac ctggggcctc agtgaagctg 60 tcctgcaagg cttctggcta cacctttact agctactgga tgcactgggt aaaacagagg 120 cctggacagg gtctggaatg gattggatac attaagccta gcagtggtta tactaagtac 180 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 atgcagctga gcagcctgac atatgaggac tctgcagtct attactgtgc aagagattac 300 tacggtagta gctcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360
[0359] In some embodiments, the ALK CAR includes an anti-ALK antibody VH region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00133 gacgtgaagc tggtggagtc tggggaaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatgcca tgtcttgggt tcgccagact 120 ccagagaaga ggctggagtg ggtcacatac attagtagtg gtggtgatta catctactat 180 gcagacactg tgaagggccg attcaccatc tccagagaca atgccaggaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtac aagagagcgg 300 atatggttac gacggttctt cgatgtctgg ggcacaggga ccacggtcac cgtctcctca 360
[0360] In some embodiments, the ALK CAR includes an anti-ALK antibody VH region that is encoded by a polynucleotide that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary nucleic acid sequence as provided below:
TABLE-US-00134 caggtccagc tgcagcagtc tggggctgaa ctggcaaaac ctggggcctc agtgaagctg 60 tcctgcaagg cttctggcta cacctttact agctactgga tgcactgggt aaaacagagg 120 cctggacagg gtctggaatg gattggatac attaatccta gcagtggtta tactaagtac 180 aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240 atgcagctga gcagcctgac atttgaggac tctgcagtct attactgtgc aagagattac 300 tacggtagta gctcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360
[0361] In some embodiments, the ALK CAR includes an anti-ALK antibody provided herein, or an antigen-binding fragment thereof, comprising a VL region and a VH region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the VL and VH amino acid sequences of any one of antibodies ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7. In some embodiments, the ALK CAR includes an anti-ALK antibody provided herein, or an antigen-binding fragment thereof, comprising a VL region and a VH region of any one of antibodies ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7.
[0362] The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence positions of a given CDR can be readily determined using any methods known in the art, including those described by Kabat et al. (“Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; “Kabat” numbering scheme), Al-Lazikani et al., (JMB 273,927-948, 1997: “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.” Dev. Comp. Immunol., 27:55-77, 2003: “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, herein a VH-CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, and a VL-CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are referred herein as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are referred herein as HCDR1, HCDR2, and HCDR3.
[0363] In some embodiments, the ALK CAR includes CDRs of an anti-ALK antibody that specifically binds ALK (e.g., human ALK). In some embodiments, the ALK CAR includes the CDRs of an anti-ALK antibody that specifically binds the ECD of ALK (e.g., human ALK ECD). In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK Antibody #1 (ALK #1), ALK Antibody #2 (ALK #2), ALK Antibody #3 (ALK #3), ALK Antibody #4 (ALK #4), ALK Antibody #5 (ALK #5), ALK Antibody #6 (ALK #6), or ALK Antibody #7 (ALK #7) (see Table 3). In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK #1. In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK #2. In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK #3. In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK #4. In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK #5. In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK #6. In some embodiments, the ALK CAR includes one or more CDRs of a VL region selected from ALK #7.
TABLE-US-00135 TABLE 3 Variable Light Chain (VL) Complementary Determining Region (CDR) ALK Antibody Sequences (Kabat) ALK VL CDR Amino Acid Antibody CDR Sequence ALK#1 LCDR1 RASENIYYSLA LCDR2 NANSLED LCDR3 KQAYDVPFT ALK#2 LCDR1 SVSQGISNSLN LCDR2 YTSSLHS LCDR3 QQYSKLPLT ALK#3 LCDR1 KASQNVGTNVA LCDR2 SASYRYS LCDR3 QQYNSYPYMYT ALK#4 LCDR1 RASESVDNYGISFMN LCDR2 AASNQGS LCDR3 QQSKEVPWT ALK#5 LCDR1 KASQNVGTNVA LCDR2 SASYRYS LCDR3 QQYNSYPYMYT ALK#6 LCDR1 KASQNVGTAVA LCDR2 SASNRFT LCDR3 QQYSSYPLT ALK#7 LCDR1 KASQNVGTNVA LCDR2 SASYRYS LCDR3 QRYNSYPYMFT
[0364] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00136 RASENIYYSLA
[0365] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00137 NANSLED
[0366] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00138 KQAYDVPFT
[0367] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00139 SVSQGISNSLN
[0368] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00140 YTSSLHS
[0369] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as provided below:
TABLE-US-00141 QQYSKLPLT
[0370] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00142 KASQNVGTNVA
[0371] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00143 SASYRYS
[0372] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00144 QQYNSYPYMYT
[0373] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00145 RASESVDNYGISFMN
[0374] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00146 AASNQGS
[0375] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00147 QQSKEVPWT
[0376] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00148 KASQNVGTAVA
[0377] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00149 SASNRFT
[0378] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00150 QQYSSYPLT
[0379] In some embodiments, the ALK CAR includes an anti-ALK antibody LCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00151 QRYNSYPYMFT
[0380] In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK Antibody #1 (ALK #1), ALK Antibody #2 (ALK #2), ALK Antibody #3 (ALK #3), ALK Antibody #4 (ALK #4), ALK Antibody #5 (ALK #5), ALK Antibody #6 (ALK #6), or ALK Antibody #7 (ALK #7) (see Table 4). In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK #1. In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK #2. In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK #3. In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK #4. In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK #5. In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK #6. In some embodiments, the ALK CAR includes one or more CDRs of a VH region selected from ALK #7.
TABLE-US-00152 TABLE 4 Variable Heavy Chain (VH) Complementary Determining Region (CDR) ALK Antibody Sequences (Kabat) ALK VH CDR Amino Acid Antibody CDR Sequence ALK#1 HCDR1 SYWMN HCDR2 QIYPGDGDTNYNGKFKG HCDR3 YYYGSKAY ALK#2 HCDR1 SYWMH HCDR2 RIDPNSGGTKYNEKFKS HCDR3 DYYGSSYRFAY ALK#3 HCDR1 NYWMH HCDR2 YINPSSGYTKYNQKFKD HCDR3 DYYGSSSWFAY ALK#4 HCDR1 SYWVN HCDR2 QIYPGDGDTNYNGKFKG HCDR3 SRGYFYGSTYDS ALK#5 HCDR1 SYWMH HCDR2 YIKPSSGYTKYNQKFKD HCDR3 DYYGSSSWFAY ALK#6 HCDR1 SYAMS HCDR2 YISSGGDYIYYADTVKG HCDR3 ERIWLRRFFDV ALK#7 HCDR1 SYWMH HCDR2 YINPSSGYTKYNQKFKD HCDR3 DYYGSSSWFAY
[0381] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
[0382] SYWMN
[0383] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00153 QIYPGDGDTNYNGKFKG
[0384] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00154 YYYGSKAY
[0385] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00155 SYWMH
[0386] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00156 RIDPNSGGTKYNEKFKS
[0387] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00157 DYYGSSYRFAY
[0388] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00158 NYWMH
[0389] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00159 YINPSSGYTKYNQKFKD
[0390] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00160 DYYGSSSWFAY
[0391] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00161 SYWVN
[0392] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00162 SRGYFYGSTYDS
[0393] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00163 SYWMH
[0394] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00164 YIKPSSGYTKYNQKFKD
[0395] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
[0396] SYAMS
[0397] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR2 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00165 YISSGGDYIYYADTVKG
[0398] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR3 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00166 ERIWLRRFFDV
[0399] In some embodiments, the ALK CAR includes an anti-ALK antibody HCDR1 that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:
TABLE-US-00167 SYWMH
[0400] In some embodiments, the ALK CAR comprises one or more CDRs from a VL region and one or more CDRs from a VH region that are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the CDRs of the VL and VH amino acid sequences of any one of antibodies ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7. In some embodiments, the ALK CAR comprises one or more CDRs from a VL region and one or more CDRs from a VH region of any one of antibodies ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7. In some embodiments, the ALK CAR comprises three CDRs from a VL region of any one of antibodies ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7. In some embodiments, the ALK CAR comprises three CDRs from a VH region of any one of antibodies ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7. In some embodiments, the ALK CAR comprises three CDRs from a VL region and three CDRs from a VH region of any one of antibodies ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7.
Vectors
[0401] CAR-T cells may be produced by using genome-integrating vectors, including but not limited to viral vectors, including retrovirus, lentivirus or transposon, or non-genome-integrating (episomal) DNA/RNA vectors, such as plasmids or mRNA. Production of CARs and CAR-T cells is known in the art (see e.g., U.S. Pat. Nos. 7,446,190, 7,741,465, 9,181,527; Kalos et al. Sci Transl Med. 2011, 3(95):95ra73, Milone et al. Mol Ther. 2009, 17(8):1453-64, and Maude et al. N Engl J Med. 2014, 371(16):1507-17, which are incorporated herein in their entirety).
[0402] Vectors containing a nucleotide sequence encoding an ALK CAR are provided. The vectors used to express an ALK CAR as described herein may be any suitable expression vector known and used in the art. In some embodiments, the vector is a prokaryotic or eukaryotic vector. In some embodiments, the vector is an expression vector, such as a eukaryotic (e.g., mammalian) expression vector. In another embodiment, the vector is a plasmid (prokaryotic or bacterial) vector. In another embodiment, the vector is a viral vector (e.g., lentiviral vector). In some embodiments, the vector further includes a promoter operably linked to the nucleotide sequence encoding the ALK CAR. In a particular embodiment, the promoter is a cytomegalovirus (CMV) promoter.
[0403] In some embodiments, the vectors comprise a nucleotide sequence encoding a VH and/or VL amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a VH and/or VL amino acid sequence of ALK Antibody #1 (ALK #1), ALK Antibody #2 (ALK #2), ALK Antibody #3 (ALK #3), ALK Antibody #4 (ALK #4), ALK Antibody #5 (ALK #5), ALK Antibody #6 (ALK #6), or ALK Antibody #7 (ALK #7). In some embodiments, the vectors comprise a nucleotide sequence encoding the VH and VL of ALK #1. In some embodiments, the vectors comprise a nucleotide sequence encoding the VH and VL of ALK #2. In some embodiments, the vectors comprise a nucleotide sequence encoding the VH and VL of ALK #3. In some embodiments, the vectors comprise a nucleotide sequence encoding the VH and VL of ALK #4. In some embodiments, the vectors comprise a nucleotide sequence encoding the VH and VL of ALK #5. In some embodiments, the vectors comprise a nucleotide sequence encoding the VH and VL of ALK #6. In some embodiments, the vectors comprise a nucleotide sequence encoding the VH and VL of ALK #7.
[0404] In some embodiments, the vectors comprise a nucleotide sequence encoding one or more CDRs of a VH and/or VL amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CDR amino acid sequences of ALK Antibody #1 (ALK #1), ALK Antibody #2 (ALK #2), ALK Antibody #3 (ALK #3), ALK Antibody #4 (ALK #4), ALK Antibody #5 (ALK #5), ALK Antibody #6 (ALK #6), or ALK Antibody #7 (ALK #7). In some embodiments, the vectors comprise a nucleotide sequence encoding the CDRs of ALK #1. In some embodiments, the vectors comprise a nucleotide sequence encoding the CDRs of ALK #2. In some embodiments, the vectors comprise a nucleotide sequence encoding the CDRs of ALK #3. In some embodiments, the vectors comprise a nucleotide sequence encoding the CDRs of ALK #4. In some embodiments, the vectors comprise a nucleotide sequence encoding the CDRs of ALK #5. In some embodiments, the vectors comprise a nucleotide sequence encoding the CDRs of ALK #6. In some embodiments, the vectors comprise a nucleotide sequence encoding the CDRs of ALK #7.
[0405] Provided herein are ALK CAR-T cells produced by transfecting a host cell (e.g., T cell, natural killer (NK) cell, cytotoxic T lymphocyte (CTL) cell, or regulatory T cell) with an expression vector containing a polynucleotide encoding an ALK CAR, as described herein, as known and used in the art under conditions sufficient to allow for expression of the ALK CAR, thereby producing the CAR-T cell. Isolated cells (e.g., T cells, NK cells, CTL cells, or regulatory T cells) containing the vectors are also provided. Collections of plasmids (vectors) are also contemplated. In certain embodiments, the collection of plasmids includes plasmid encoding an ALK CAR as described herein.
[0406] Methods of generating chimeric antigen receptors and T cells including such receptors are known in the art and further described herein (see, e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online Feb. 23, 2010, pages 1-9; Till et al., 2008, Blood, 1 12:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; Tumaini et al., Cytotherapy, 15, 1406-1417, 2013; Haso et al., (2013) Blood, 121, 1165-1174; PCT Pubs. WO2012/079000, WO2013/126726; and U.S. Pub. 2012/0213783, each of which is incorporated by reference herein in its entirety).
Pharmaceutical Compositions
[0407] Compositions comprising at least one ALK CAR, polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, as described herein are provided. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In some embodiments, an adjuvant (a pharmacological or immunological agent that modifies or boosts an immune response, e.g., to produce more antibodies that are longer-lasting) is also employed. For example, without limitation, the adjuvant can be an inorganic compound, such as alum, aluminum hydroxide, or aluminum phosphate; mineral or paraffin oil; squalene; detergents such as Quil A; plant saponins; Freund's complete or incomplete adjuvant, a biological adjuvant (e.g., cytokines such as IL-1, IL-2, IL-12, or IL-15); bacterial products such as killed Bordetella pertussis, or toxoids; or immunostimulatory oligonucleotides (such as CpG oligonucleotides).
[0408] Compositions and preparations (e.g., physiologically or pharmaceutically acceptable compositions) containing ALK CARs, polynucleotides encoding an ALK CAR, or engineered immune cells comprising an ALK CARs for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Nonlimiting examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and canola oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present in such compositions and preparations, such as, for example, antimicrobials, antioxidants, chelating agents, colorants, stabilizers, inert gases and the like.
[0409] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl and aryl amines and substituted ethanolamines.
[0410] Provided herein are pharmaceutical compositions which include a therapeutically effective amount of an ALK CAR, polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, alone, or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid or aqueous solution, suspension, emulsion, dispersion, tablet, pill, capsule, powder, or sustained release formulation. A liquid or aqueous composition can be lyophilized and reconstituted with a solution or buffer prior to use. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the commonly known pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Other media that can be used in the compositions and administration methods as described are normal saline and sesame oil.
Methods of Treatment, Administration and Delivery
[0411] Methods of treating a disease (e.g., ALK-positive cancers (e.g., neuroblastoma)), or symptoms thereof, are provided. The methods comprise administering a therapeutically effective amount of an ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, as described herein, or a pharmaceutical composition thereof, as described herein, to a subject (e.g., a mammal), in particular, a human subject. The invention provides methods of treating a subject suffering from, or at risk of, or susceptible to disease, or a symptom thereof, or delaying the progression of a disease (e.g., ALK-positive cancer (e.g., neuroblastoma)). In some embodiments, the method includes administering to the subject (e.g., a mammalian subject), an amount or a therapeutic amount of an ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or pharmaceutical composition thereof, sufficient to treat the disease, delay the growth of, or treat the symptoms thereof (e.g., ALK-positive cancers (e.g., neuroblastoma)).
[0412] In some embodiments, the methods herein include administering to the subject (including a human subject identified as in need of such treatment) an effective amount of an ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or a pharmaceutical composition thereof, as described herein to produce such effect. The treatment methods are suitably administered to subjects, particularly humans, suffering from, are susceptible to, or at risk of having a disease, or symptoms thereof, namely, cancer (e.g., ALK-positive cancers (e.g., neuroblastoma)). Nonlimiting examples of ALK-positive cancers include non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, melanoma, or a combination thereof. In some embodiments, the ALK-positive cancer is neuroblastoma.
[0413] The ALK-positive cancer may be caused by an oncogenic ALK gene that either forms a fusion gene with other genes, gains additional gene copies, or is genetically mutated. In some embodiments, the ALK-positive cancer is caused by an ALK fusion gene encoding an ALK fusion protein. In some embodiments, the ALK-positive cancer is caused by a fusion between the ALK gene and the nucleophosmin (NPM) gene encoding a NPM-ALK fusion protein. In some embodiments, the ALK-positive cancer is caused by a fusion between the ALK gene and the echinoderm microtubule-associated protein-like 4 (EML4) gene encoding an ELM4-ALK fusion protein. In some embodiments, the ALK-positive cancer is caused by a point mutation. In some embodiments, the point mutation is F1174L (ALK.sup.F1174L).
[0414] Identifying a subject in need of such treatment can be based on the judgment of the subject or of a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). Briefly, the determination of those subjects who are in need of treatment or who are “at risk” or “susceptible” can be made by any objective or subjective determination by a diagnostic test (e.g., blood sample, biopsy, genetic test, enzyme or protein marker assay), marker analysis, family history, and the like, including an opinion of the subject or a health care provider. The ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or pharmaceutical compositions thereof, as described herein, may also be used in the treatment of any other disorders in which disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations may be implicated. A subject undergoing treatment can be a non-human mammal, such as a veterinary subject, or a human subject (also referred to as a “patient”).
[0415] In addition, prophylactic methods of preventing or protecting against a disease (e.g., ALK-positive cancers (e.g., neuroblastoma)), or symptoms thereof, are provided. Such methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR as described herein to a subject (e.g., a mammal, such as a human), in particular, prior to development or onset of a disease (e.g., ALK-positive cancers (e.g., neuroblastoma)).
[0416] In another embodiment, a method of monitoring the progress of a disease (e.g., ALK-positive cancers (e.g., neuroblastoma)), or monitoring treatment of the disease is provided. The method includes a diagnostic measurement (e.g., CT scan, screening assay or detection assay) in a subject suffering from or susceptible to disease or symptoms thereof (e.g., ALK-positive cancers (e.g., neuroblastoma)), in which the subject has been administered an amount (e.g., a therapeutic amount) of an ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or a pharmaceutical composition thereof, as described herein, sufficient to treat the disease or symptoms thereof. The diagnostic measurement in the method can be compared to samples from healthy, normal controls; in a pre-disease sample of the subject; or in other afflicted/diseased patients to establish the treated subject's disease status. For monitoring, a second diagnostic measurement may be obtained from the subject at a time point later than the determination of the first diagnostic measurement, and the two measurements can be compared to monitor the course of disease or the efficacy of the therapy/treatment. In certain embodiments, a pre-treatment measurement in the subject (e.g., in a sample or biopsy obtained from the subject or CT scan) is determined prior to beginning treatment as described; this measurement can then be compared to a measurement in the subject after the treatment commences and/or during the course of treatment to determine the efficacy of (monitor the efficacy of) the disease treatment.
[0417] The ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or pharmaceutical compositions thereof, can be administered to a subject by any of the routes normally used for introducing a recombinant protein or composition containing the recombinant protein into a subject. Routes and methods of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intrathecal, parenteral, such as intravenous (IV) or subcutaneous (SC), vaginal, rectal, intranasal, inhalation, intraocular, intracranial, or oral. Parenteral administration, such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection (immunization). Injectables can be prepared in conventional forms and formulations, either as liquid solutions or suspensions, solid forms (e.g., lyophilized forms) suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local.
[0418] The ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or pharmaceutical compositions thereof, can be administered in any suitable manner, such as with pharmaceutically acceptable carriers, diluents, or excipients as described supra. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, a pharmaceutical composition comprising the ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, can be prepared using a wide variety of suitable and physiologically and pharmaceutically acceptable formulations. In some embodiments, the disclosed methods include isolating T cells from a subject, transducing the T cells with an expression vector (e.g., a lentiviral vector) encoding the ALK CAR, and administering the ALK CAR-expressing T cells to the subject for treatment of a disease ((e.g., ALK-positive cancers (e.g., neuroblastoma)) in the subject.
[0419] Administration of the ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or pharmaceutical compositions thereof, can be accomplished by single or multiple doses. The dose administered to a subject should be sufficient to induce a beneficial therapeutic response in a subject over time, such as to inhibit, block, reduce, ameliorate, protect against, or prevent disease (e.g., ALK-positive cancers (e.g., neuroblastoma)). The dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, by the severity of the cancer being treated, by the particular composition being used and by the mode of administration. An appropriate dose can be determined by a person skilled in the art, such as a clinician or medical practitioner, using only routine experimentation. One of skill in the art is capable of determining therapeutically effective amounts of ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or pharmaceutical compositions, that provide a therapeutic effect or protection against disease (e.g., ALK-positive cancers (e.g., neuroblastoma)) suitable for administering to a subject in need of treatment or protection.
[0420] In some embodiments, an ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or a pharmaceutical composition thereof, is administered as a maximum-tolerated dose (MTD). In some embodiments, MTD is the dose with estimated probability of dose limiting toxicity (DLT) closest to the target toxicity rate of 20%. In some embodiments, an ALK CAR, a polynucleotide encoding an ALK CAR, or engineered immune cell comprising an ALK CAR, or a pharmaceutical composition thereof, is administered in a therapeutically effective dose for a mammal. In some embodiments, the mammal is a mouse. In some embodiments, a mouse is administered a dose of 0.5 million to 15 million ALK CAR-T cells. In some embodiments, the mammal is a human. In some embodiments, a human is administered a dose of at least about 0.25×10.sup.6 CAR.sup.+ cells/kg, at least about 0.5×10.sup.6 CAR.sup.+ cells/kg, at least about 1×10.sup.6 CAR.sup.+ cells/kg, or at least about 1.5×10.sup.6 CAR.sup.+ cells/kg.
Combination Therapies
[0421] The anaplastic lymphoma kinase chimeric antigen receptor (ALK CAR) or engineered immune cell comprising an ALK CAR as described herein can be administered alone or in combination with other therapeutic agents in a subject for the treatment of cancer (e.g., ALK-positive cancer (e.g., neuroblastoma)). For example, the ALK CAR or engineered immune cell comprising an ALK CAR can be administered with an adjuvant, such as alum, Freund's incomplete adjuvant, Freund's complete adjuvant, biological adjuvant, or immunostimulatory oligonucleotides (such as CpG oligonucleotides). The adjuvant may be conjugated to an amphiphile as previously described (H. Liu et al., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 5199522 (2014)). In some embodiments, the amphiphile conjugated to the adjuvant is N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE).
[0422] One or more cytokines, including but not limited to, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), the protein memory T-cell attractant “Regulated Activation, on Normal T Expressed and Secreted” (RANTES), granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), or interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α); one or more molecules such as the TNF ligand superfamily member 4 ligand (OX40L) or the type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL), or combinations of these molecules, can be used as biological adjuvants, if desired or warranted (see, e.g., Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl 1):561-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically (or locally) to a subject.
[0423] The ALK CAR or engineered immune cell comprising an ALK CAR can also be administered as a combination therapy with one or more other therapeutic agents, such as an ALK peptide or fusion protein, ALK peptide vaccine, ALK inhibitors, tyrosine kinase inhibitors (TKIs), and/or immune checkpoint inhibitors. Non-limiting examples of ALK inhibitors include lorlatinib (Lobrena®). Non-limiting examples of checkpoint inhibitors include programmed cell death protein 1 (PD-1) inhibitors, programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) inhibitors. Nonlimiting examples of PD-1 inhibitors include pembrolizumab (Keytruda®) and nivolumab)(Opdivo®). Nonlimiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy®). Non-limiting examples of TKI inhibitors include crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib. In some embodiments, one or more ALK peptides or fusion proteins, ALK peptide vaccines, ALK inhibitors, immune checkpoint inhibitors, and/or TKI inhibitors is/are administered simultaneously or sequentially with ALK CAR or engineered immune cell comprising an ALK CAR to a subject (e.g., human).
[0424] In some embodiments, the ALK CAR or engineered immune cell comprising an ALK CAR is administered simultaneously or sequentially with an ALK peptide vaccine. In particular embodiments, the ALK peptide vaccine contains antigenic determinants that serve to elicit an immune response in a subject (e.g., the production of activated T-cells) that can treat and/or protect a subject against disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers) and symptoms thereof. In some embodiments, the immune response includes producing T-lymphocytes. In some embodiments, the ALK peptide vaccine contains at least one ALK antigen or peptide or fragment thereof. In some embodiments, the ALK peptide vaccine contains two or more ALK peptides or antigens or fragments thereof. In some embodiments, the ALK peptides or antigens or fragments thereof are fragments of the cytoplasmic portion of an ALK protein, which bind a human leukocyte antigen (HLA). In some embodiments the ALK peptides or antigens or fragments thereof are modified with an amphiphilic conjugate to increase T-cell expansion and greatly enhance anti-tumor efficacy. In some embodiments, the amphiphile is N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE).
Kits
[0425] Also provided are kits containing the anaplastic lymphoma kinase chimeric antigen receptor (ALK CAR) or engineered immune cell comprising an ALK CAR as described, or a pharmaceutically acceptable composition containing the ALK CAR and a pharmaceutically acceptable carrier, diluent, or excipient, for administering to a subject, for example. In some embodiments, the kit is provided for treating cancer (e.g., ALK-positive cancer (e.g., neuroblastoma)) in a subject (e.g., human). In some embodiments, the kit is provided for making an ALK CAR as provided herein. In some embodiments, the kit will contain one or more of an ALK antibody or antigen binding fragment thereof, nucleic acid molecule encoding for an ALK peptide, ALK CAR or T cell expressing an ALK CAR as disclosed herein. The ALK CAR may be in the form of a polypeptide or a polynucleotide encoding an ALK CAR, as described herein. In some embodiments, the kit comprises a vector containing a nucleotide sequence encoding an ALK CAR as disclosed herein. As will be appreciated by the skilled practitioner in the art, such a kit may contain one or more containers, labels, carriers, diluents or excipients, as necessary, and instructions for use.
[0426] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Useful techniques for particular embodiments will be discussed in the sections that follow.
[0427] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES
[0428] The following examples are provided to illustrate certain particular features and/or embodiments. The examples should not be construed to limit the disclosure to the particular features or embodiments described.
Example 1: Generation of ALK CAR-T Constructs
[0429] More than 70% of neuroblastoma express the Anaplastic Lymphoma Kinase (ALK) receptor (Chiarle R, et al., The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer 2008 January; 8(1):11-23). About 10% of neuroblastoma cases have an activating point mutation in the ALK protein (e.g., ALK.sup.F1174L), correlating to advanced disease stage and poor prognosis (Passoni L, et al., Mutation-independent anaplastic lymphoma kinase overexpression in poor prognosis neuroblastoma patients. Cancer Res 2009 Sep. 15; 69(18):7338-46; Mosse Y P, et al., Identification of ALK as a major familial neuroblastoma predisposition gene. Nature 2008 Oct. 16; 455(7215):930-5). Promising antitumor effects have been obtained with ALK tyrosine kinase inhibitors (TKIs), but almost invariably disease progresses (Mosse Y P, Anaplastic Lymphoma Kinase as a Cancer Target in Pediatric Malignancies. Clin Cancer Res 2016 Feb. 1; 22(3):546-52). Thus, while ALK remains a promising target in neuroblastoma, it is clinically evident that alternative strategies to TKIs must be implemented to target ALK. In particular, ALK protein has several features suitable to be targeted by immunotherapy. For instance, ALK has almost no expression in normal tissues and is naturally immunogenic in humans (Blasco R B, et al., Comment on “ALK is a therapeutic target for lethal sepsis,” Sci Transl Med 2018 Dec. 12; 10(471)). Patients with ALK-rearranged lymphoma and lung cancer can indeed spontaneously develop immune responses against ALK (Awad M M, et al., Epitope mapping of spontaneous autoantibodies to anaplastic lymphoma kinase (ALK) in non-small cell lung cancer. Oncotarget 2017 Nov. 3; 8(54):92265-74; Ait-Tahar K, et al., Correlation of the autoantibody response to the ALK oncoantigen in pediatric anaplastic lymphoma kinase-positive anaplastic large cell lymphoma with tumor dissemination and relapse risk. Blood 2010 Apr. 22; 115(16):3314-9). Importantly, ALK is a potent driver oncogene required for tumor survival and growth, which minimizes the chances of escape of ALK-negative tumor cells (Chiarle R, et al., The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer 2008 January; 8(1):11-23; Voena C, et al., Efficacy of a Cancer Vaccine against ALK-Rearranged Lung Tumors. Cancer Immunol Res 2015 December; 3(12):1333-43). Specifically, in neuroblastoma, therapeutic effects are achieved by ALK knock-down (Di P D, et al., Neuroblastoma-targeted nanoparticles entrapping siRNA specifically knockdown ALK. Mol Ther 2011 June; 19(6):1131-40), inhibition (Infarinato N R, et al., The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma. Cancer Discov 2016 January; 6(1):96-107) or antibody-mediated drug delivery (Sano R, et al., An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma. Sci Transl Med 2019 Mar. 13; 11(483)). ALK-specific cancer immunotherapy based on CAR-Ts may represent an opportunity to increase clinical benefits. Accordingly, a series of ALK-specific CARs (ALK CARs) were developed from ALK antibodies that recognize both human and murine ALK and validated in preclinical models of neuroblastoma.
[0430] Seven (7) ALK-specific antibodies (ALK Antibodies #1-#7) directed against the extracellular domain of human ALK receptor were evaluated for use in CAR-based immunotherapy. These antibodies demonstrated specificity to the ALK extracellular domain (ECD) with various activities on ALK signaling (Table 5). ALK Antibodies #4 and #7 were agonistic of ALK signaling, ALK Antibodies #2, #3, #5, and #6 inhibited ALK signaling, and ALK Antibody #1 had no effect on ALK signaling. These antibodies also demonstrated different biology affinity and bound to various portions of human ALK receptor. For example, ALK antibodies #5, #6 and #7) recognize both the human and murine ALK, and can thus be utilized for toxicity studies in mice.
TABLE-US-00168 TABLE 5 Characterization of ALK Antibodies ALK ALK K.sub.D.sup.app ALK Mouse-ALK Antibody Phosphorylation [nM] Turnover Binding 1 No activity 0.35 no moderate 2 Inhibitor 0.2 no moderate 3 Weak inhibitor 0.5 no no 4 Strong agonist 0.5 strong no 5 Inhibitor 0.5 no strong 6 Inhibitor 0.4 strong strong 7 Weak agonist 0.5 strong strong
[0431] To develop a chimeric antigen receptor (CAR)-based immunotherapy for the treatment of neuroblastoma, CAR-T cells were constructed by fusing each of the seven ALK antibodies to T cell receptor intracellular domains for the activation of T cells. Specifically, the VH and VL regions were cloned from each of the seven antibodies to generate scFvs. The ALK scFvs were cloned into a murine CAR backbone, i.e., SFG-m1928z-GFP CAR-T retroviral construct. The SFG-m1928z-GFP CAR construct has been shown to be very efficacious in targeting CD19+ cells in mouse models (Dr. M Sadelain (MSKCC, NY)).
[0432] The cloning strategy is shown in
Example 2: Transduction of ALK CAR-T Constructs into Mouse T Cells
[0433] To generate CAR-T cells with the ALK CAR constructs, retrovirus vectors expressing CD19 (m1928Z-GFP) scFv or ALK CARs were transduced into T cell splenocytes from C57BL/6J mice. Mouse T cells were purified from spleen, activated with anti-CD3/CD28+IL2, and transduced with a CAR retroviral construct containing GFP as a reporter. Efficiency of transduction was evaluated 48 hours after viral infection by GFP reporter expression. Transduction efficiency was evaluated by measuring the percentage of GFP-positive T cells (
Example 3: ALK-Specific Cytolytic Activity and Cytokine Release of ALK CAR-T Cell Constructs In Vitro
[0434] Cytokine release by ALK-specific CAR constructs was evaluated. Specifically, IFNγ and GM-CSF production by ALK CAR-T cells was measured. Retrovirally transduced CAR-T cells were incubated at a 1:1 effector cell (GFP+CAR-T cells) to target cell ratio (E:T ratio). Target cells included NIH3T3, Eμ-myc, SH-SY5Y (expresses normal low levels of mutated ALK.sup.F1174L) and SK-N-BE (expresses high levels of amplified wild-type ALK).
[0435] IFNγ production was measured in NIH3T3 and Eμ-myc cells transduced with full-length human ALK retroviral vector or mock vector in the presence of CAR-T cells with ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7 (
[0436] The in vitro killing activity of ALK CAR-T cells was also evaluated. Eμ-myc cells overexpressing mock vector or full-length human ALK were stained with CFSE and co-incubated with effector ALK CAR-T cells at E:T ratios of 1:1, 5:1 or 10:1. The cell numbers of CAR-T cells were normalized based on the percentage of GFP positive cells transduced with the CAR construct ALK #1, ALK #2, ALK #3, ALK #4, ALK #5, ALK #6, or ALK #7. After 18 and 24 hours, the cytolytic activity was calculated by determining the fraction of alive target cells with the formula: cytolytic activity=100−% of CSFE+/CD19+ alive cells. CD19 CAR-T cells were used as golden standard control as they efficiently target CD19+Eμ-Myc cells (see Davila et al., PlosOne 2013). Eμ-Myc vector (ALK−) cells were used as control to determine the specificity of ALK-directed cytolytic activity. Strong cytolytic activity of CAR-T cells against Eμ-myc/ALK cells was found in in the CFSE assay (
Example 4: Validation of ALK CAR Constructs In Vivo
[0437] The CD19+/ALK+systemic leukemia model was used to validate and rank cytolytic activity of ALK CARs in vivo. Adoptive transfer of ALK CAR-T cells was conducted in mice with Eμ-myc/ALK systemic tumors. Mice were treated with cytophspamide (CTX, 100 mg/kg) alone (n=8), cytophosphamide plus CAR-CD19 (15×10.sup.6 based on GFP+) (n=8), or cytophosphamide plus CAR-ALK #5 (15×10.sup.6 based on GFP+) (n=8). Untreated mice (n=6) were used as a negative control.
[0438] Using FACS analysis, CD19+/ALK+ cells were found in one mouse treated with CTX alone. Circulating CD19+/ALK+ tumor cells were found in peripheral blood (
Example 5: Treatment of Transgenic Mice with ALK CAR-T Constructs
[0439] The generated ALK CARs were used for studies in both in immunocompetent and immunodeficient mice. ALK CAR-T constructs were investigated in two transplantable neuroblastoma mouse models: i) ALK.sup.F1174L/MYCN (Brentjens R J, et al., CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 2013 Mar. 20; 5(177):177ra38) transgenic mice; and ii) NSG immunodeficient mice with orthotopic grafts of human neuroblastoma cells.
[0440] Neuroblastoma in ALK.sup.F1174L/MYCN transgenic mice is driven by overexpression of human mutant ALK.sup.F1174L. ALK.sup.F1174L/MYCN transgenic mice express ALK at levels comparable to the human neuroblastoma cell line SH-SY5Y, a line expressing low ALK levels (Heczey A, et al., CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol Ther 2017 Sep. 6; 25(9):2214-24).
[0441] NSG immunodeficient mice with orthotopic grafts of human neuroblastoma cells were implanted s.c. with 1×10.sup.6SH-SY5Y cells in the kidney capsule to model orthotopic human neuroblastoma. The mice were then injected with 10×10.sup.6 ALK #5 CAR-T cells or 10×10.sup.6 CD19 CAR-T cells as a positive control. Neuroblastoma growth delay induced by ALK CAR-Ts with tumor volume was measured daily (two-tailed p value <0.0001, unpaired t test).
Example 6: Comparison of Anti-Tumor Activity of ALK CAR-T Cells to Lorlatinib in an Immunocompetent Model of Metastatic Neuroblastoma
[0442] The antitumor effect of ALK CAR-T cells was evaluated in a fully syngeneic neuroblastoma model. ALK.sup.F1174L/MYCN neuroblastoma was transplanted s.c. into BALB/c mice. ALK #5 CAR-T cells or CD19 CAR-T cells were generated from BALB/c purified T cells and injected i.v. weekly for three weeks. Lorlatinib was administered by oral gavage (4 mg/kg/day) for three weeks. Tumor volumes were measured at day 23. As shown in
[0443] The potential toxicity of ALK #5 CAR-T cells was also evaluated. In normal cells low ALK expression is confined to few neurons in the brain and in the testicle (Kabir T F, et al., Immune Checkpoint Inhibitors in Pediatric Solid Tumors: Status in 2018. Ochsner J 2018; 18(4):370-6). No signs of toxicity induced by ALK #5 CAR-T cells were detected as measured by weight loss, temperature changes and IL-6 release following injection of ALK #5 CAR-T cells. Histology examination showed no evidence of brain inflammation and mice did not show any obvious neurological symptoms.
Example 7: In Vitro Validation of Human ALK CAR-T Cells
[0444] In order to validate hALK CAR-T cells in vitro, fully human ALK #5 CAR (hALK #5 CAR) based on a humanized version of the ALK #5 scFv was generated. Human T cells were transduced with the hALK #5 CAR and neuroblastoma tumor cells were targeted in vitro. ALK CAR expression was measured in human T cells at day 4 after transduction evaluated by flow cytometry (
Example 8: Validation of ALK CARs Against Human Neuroblastoma Lines with Different ALK Expression Levels In Vitro and In Vivo
[0445] The level of expression of the target molecule on cancer cells is a critical determinant of CAR-T cell anti-tumor activity. ALK is expressed at variable levels on the surface of neuroblastoma cells, ranging from cases with low expression of the wild-type ALK receptor, to cases with moderate or high expression of a wild-type or mutated ALK receptor, including in some cases cells with ALK gene amplification (Heczey A, et al., CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol Ther 2017 Sep. 6; 25(9):2214-24). Several neuroblastoma cell lines are representative of the various genetic mutations and varying ALK expression: cell lines with high ALK expression, i.e. NB-1 (ALK WT amplified) and Felix (mutated ALK.sup.F1245C); cell lines with moderate ALK expression, i.e. IMR-32 (ALK WT), NBL-S(ALK WT) and COG-N-453 (mutated ALK.sup.F1174L); cell lines with low ALK expression, i.e. SH-SY5Y (mutated ALK.sup.F1174L) and COG-N-424x (ALK WT). All these lines grow well in vitro and engraft in NSG mice (Heczey A, et al., CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol Ther 2017 Sep. 6; 25(9):2214-24).
[0446] The antitumor activity of human T cells expressing the hALK #5 CAR was tested in vitro by measuring cytotoxic activity, cytokine release and T cell proliferation as previously described (Chen Y, et al., Eradication of Neuroblastoma by T Cells Redirected with an Optimized GD2-Specific Chimeric Antigen Receptor and Interleukin-15. Clin Cancer Res 2019 Jan. 7).
[0447] The anti-tumor activity of hALK #5 CAR-Ts in vivo in models of neuroblastoma in NSG mice was validated by implanting four patient-derived xenografts (PDXs) (SJNBL013762, SJNBL013761, SJNBL046148 and SJNBL046) (St. Jude Children's Research Hospital), which express the luciferase reporter (FFLuc), into NSG mice and injecting the mice with the hALK #5 CAR-Ts.
Example 9: Testing NK Cells as a Cell Platform for CAR Expression
[0448] Genetic modification of NK cells remains challenging using either γ-retrovirus or lentivirus vectors. The use of α-retroviruses has a novel gene delivery system in NK cells. A split packaging design was developed for α-retrovirus based vectors in which viral coding sequences (gag/pol, env) were integrated into virus packaging cells without packaging sequences and without sequence overlaps (Awad M M, et al., Epitope mapping of spontaneous autoantibodies to anaplastic lymphoma kinase (ALK) in non-small cell lung cancer. Oncotarget 2017 Nov. 3; 8(54):92265-74). To improve viral titers, codon optimized α-virus packaging sequences are available with enhanced titers of several orders of magnitude due to enhanced gag/pol expression. Resulting α-virus vectors pseudotyped for infection of murine cells transduce hematopoietic stem cells murine (HSCs) with high efficiency (Ait-Tahar K, et al., Correlation of the autoantibody response to the ALK oncoantigen in pediatric anaplastic lymphoma kinase-positive anaplastic large cell lymphoma with tumor dissemination and relapse risk. Blood 2010 Apr. 22; 115(16):3314-9). Transduction of NK cells with an α-retroviral vector containing a CD19 CAR expression cassette selectively enhanced NK cell cytotoxicity towards CD19-expressing leukemia cells (Voena C, et al., Efficacy of a Cancer Vaccine against ALK-Rearranged Lung Tumors. Cancer Immunol Res 2015 December; 3(12):1333-43).
[0449] The use of α-retrovirus system was used to facilitate the manufacturing of ALK CAR expressing NK cells for targeting ALK-positive cells. An avian α-retroviral vector backbone was used to more effectively mediate CAR delivery to NK cells. As shown in
[0450] For all these 6 vectors, stable producer clones were generated and NK-92 cells were transduced using the RD114/TR-pseudotyped α-retroviral particles. About 50 clones were screened in order to isolate a producer line with titers >1×10.sup.6/ml of infectious particles (tittered on HT1080, a standard human cell line used for such tittering).
[0451] Quantification of in vitro killing activity of NK-92 cells transduced with an hALK CAR construct was conducted after 24 hrs incubation with HT1080 cells expressing the human ALK receptor. NK-92 cells transduced with an MPSV.ALK5.CAR construct efficiently and specifically killed target cells expressing the ALK receptor (
Example 10: Combination of ALK CAR-T Cells and ALK Blockade
[0452] First generation ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, have limited therapeutic efficacy in neuroblastoma, while the third generation ALK TKI, lorlatinib, is effective against mutated neuroblastoma (Infarinato N R, et al., The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma. Cancer Discov 2016 January; 6(1):96-107). The effects of lorlatinib on ALK viability and expression in neuroblastoma cells was evaluated. Several neuroblastoma cell lines with various ALK genetic alterations, including NB-1 (ALK WT amplified), IMR-32 (ALK WT), NBL-S(ALK WT), SH-SY5Y (mutated ALK.sup.F1174L), Kelly (mutated ALK.sup.F1174L), were treated with increasing doses of lorlatinib (
[0453] Expression of surface ALK was measured on Kelly and IMR-32 cells by flow cytometry on Kelly and IMR-32 cells treated with 10 nM lorlatinib for 24 hours (
[0454] To examine the synergic killing effect of hALK CAR-T cells in combination with lorlatinib, hALK CAR-T cells alone or in combination with lorlatinib were administered against neuroblastoma cell lines. First, hALK CAR-T cells alone or in combination with lorlatinib at 10 nM and 100 nM were administered against two ALK.sup.F1174L mutated neuroblastoma cell lines (Kelly and SH-SY5Y), which express relatively low levels of ALK (
[0455] Lorlatinib was evaluated for potentiating the activity of ALK CAR-T cells, not only by affecting the viability of tumor cells, but also by increasing ALK expression. The mechanisms by which lorlatinib enhances expression of ALK on the surface of neuroblastoma cells and increases the targeting by ALK CAR-T cells is shown in
[0456] Western blot analysis was further conducted to evaluate the expression of ALK in LAN-5 (R1275Q), SH-SY5Y (F1174L), SK-N-SH (F1174L), NGP (D1529E), NBL-S(WT), IMR-32 (WT), SK-N-FI (WT), Kelly (WT) neuroblastoma cells when used in combination with 10 nM and 100 nM of lorlatinib (
[0457] To test whether the increased expression of surface ALK during treatment with lorlatinib enhances the killing activity of ALK CAR-Ts, IMR-32 and Kelly cell lines, that upregulate ALK expression without significant effects on cell viability at 10 nM lorlatinib, were incubated with increasing amounts of ALK #5 CAR-T cells or control CD19 CAR-T cells (10:1, 1:1, 1:5, 1:10 tumor:T cell ratios) for 3 days. The neuroblastoma cell viability was then measured by flow cytometry.
[0458] To test whether the anti-proliferative effect, combined with the increased expression of surface ALK induced by lorlatinib, enhanced the killing activity of hALK CAR-Ts in vitro and in vivo, neuroblastoma cell lines were incubated with 10 nM or 100 nM lorlatinib, plus increasing amounts of hALK5 CAR-T cells or control CD19 CAR-T cells (10:1, 1:1, 1:5, 1:10 tumor:T cell ratios) for 5 days, and then NB cell viability and ALK surface expression of the residual tumor cells was measured by flow cytometry.
[0459] For in vivo therapeutic experiments, immunocompetent BALB/c mice were injected i.v. with 1×10.sup.6 ALK.sup.F1174L/MYCN syngeneic neuroblastoma cells and immunodeficient NSG mice were injected i.v. with NB-1 (ALK WT amplified), IMR-32 (ALK WT), or Kelly or SH-SY5Y (mutated ALK.sup.F1174L) cells. One week after tumor injection lorlatinib was administered by oral gavage (4 mg/kg/day-10 mg/kg/day) for three weeks. hALK CAR-T cells or control CD19 CAR-T cells were injected one week after the first lorlatinib treatment. Tumor growth was measured by luciferase activity and survival was compared in mice treated with CAR-T cells alone or in combination with lorlatinib.
Example 11: Combination Therapy Using ALK CAR-T Cells with an ALK Vaccine
[0460] ALK.sup.F1174L/MYCN transgenic mice were used to evaluate combination therapy with CAR-T cells and an ALK vaccine. Immunocompetent BALB/c mice were injected s.c. with 1×10.sup.6 ALK.sup.F1174L/MYCN syngeneic neuroblastoma cells. After tumor injection, ALK CAR-T cells were injected in combination with an ALK vaccine as shown in the administration schedule of
[0461] ALK.sup.F1174L/MYCN transgenic mice were used to evaluate combination therapy with ALK CAR-T cells, ALK vaccine and lorlatinib. BALB/c mice were injected s.c. with 1×10.sup.6 cells of syngeneic ALK.sup.F1174L/MYCN neuroblastoma cells. Mice were vaccinated with an ALK vaccine and injected with 15×10.sup.6 ALK CAR-T cells at the indicated times as shown in
Example 12: Validation of hALK5 CAR-T Cells Against Human Neuroblastoma Cell Lines Showing Different Levels of ALK Expression
[0462] The level of expression of the target molecule on cancer cells is a critical determinant of CAR-T cell anti-tumor activity. ALK is expressed at variable levels on the surface of neuroblastoma cells, and it is thus critical to assess the antitumor effects of hALK CAR-Ts in neuroblastoma cells expressing different levels of ALK. Several neuroblastoma cell lines representative of the various genetic mutations and ALK expression levels are shown in Table 6. ALK expression in human neuroblastoma cell lines, LAN-1, SK-N-FI, NGP, SK-N-SH, SH-SY5Y, Kelly, LAN-5, NBL-S, Felix, IMR-32, and NB-1, is shown in
TABLE-US-00169 TABLE 6 List of Human Neuroblastoma (NB) Cells Lines NB Cell Line ALK MYCN LAN-1 F1174L Amplified SK-N-FI R1275Q Not amplified* NGP Wild-type Amplified SK-N-BE(2)C Wild-type Amplified SK-N-SH F1174L Not amplified SH-SY5Y F1174L Not amplified Kelly F1174L Amplified LAN-5 R1275Q Amplified NBL-S Wild-type Not amplified Felix F1245C Not amplified IMR-32 Wild-type Amplified NB-1 Amplified Amplified
[0463] To measure the in vitro killing activity of hALK CAR-T cells, residual tumor cells from two independent donors were measured after administration of hALK CAR-T cells against NBL-S, SK-N-FI, IMR-32, NGP, NB-1, LAN-5, SK-N-SH, Kelly, SH-SY5Y neuroblastoma cell lines (
[0464] The killing activity of human ALK CAR-T cells against several cell lines (NBL-S, SK-N-FI, IMR-32, NGP, NB-1, LANS, SK-N-SH, Kelly, SH-SY5Y, Raji) of human neuroblastoma was also examined at a 1:1 tumor:CAR-T ratio (
[0465] Antitumor activity of hALK5 CAR-Ts in vivo in NSG mice was examined by injecting by i.v. 10.sup.6 luciferase-transduced neuroblastoma cell lines with different ALK expression levels (NB-1, IMR-32, Kelly and SH-SY5Y). hALK CAR-T cells and CD19 CAR-T cells (5×10.sup.6 cells/mouse) were injected 2 weeks after neuroblastoma injection. Tumor growth was assessed by luciferase monitoring via IVIS instrumentation. Four patient-derived xenografts (PDXs) (SJNBL013762, SJNBL013761, SJNBL046148 and SJNBL046) were obtained from the PDX bank of the St. Jude Children's Research Hospital (stjude.org/research/resources-data/childhood-solid-tumor-network/available-resources.html#xenografts). PDXs also express FFLuc and can be implanted in NSG mice. Neuroblastoma PDXs can also be injected orthotopically in the kidney capsule.
Example 13: Testing of ALK CAR-T Cell Toxicity
[0466] To examine the toxicity of ALK CAR-T cells, changes in body weight, body temperature, interferon gamma (IFNγ) production, interleukin 6 (IL-6) production, and serum amyloid A 3 (mSAA3) production was measured in mice with and without tumors injected with ALK5 CAR T cells alone and in combination with lorlatinib (
Example 14: In Vivo Anti-Tumor Activity of Human ALK CAR-T Cells
[0467] The anti-tumor activity of hALK CAR-T cells was evaluated in vivo. NSG mice were injected with NB-1 cells, which express high levels of ALK, and then treated with one single injection of ALK CAR-T cells (
[0468] An experimental procedure was further developed for combining ALK CAR-T cells with three cycles of lorlatinib in vivo in NSG mice (
Example 15: Dose-Escalation with Autologous hALK CAR-T Cells in Patients with Relapsed/Refractory Neuroblastoma
[0469] Autologous T cells expressing hALK CAR can be evaluated without any additional gene modification such as IL-15 delivery. Presence of surface ALK expression by immunohistochemistry (IHC) can be used as an eligibility criterion. About >80% of neuroblastoma patients are expected to express detectable ALK levels by IHC. Using a hALK CAR transgene as shown in
Patient Eligibility
[0470] Eligible subjects will have: 1) written HIPAA authorization signed by legal guardian; 2) age greater than 18 months and less than 18 years at the time of consent; 3) adequate performance status as defined by Lansky or Karnofsky performance status of >60 (Lansky for <16 years of age); 4) life expectancy 12 weeks; 5) histological confirmation of neuroblastoma or ganglioneuroblastoma at initial diagnosis. Bone marrow samples are acceptable as confirmation of neuroblastoma. 6) high risk neuroblastoma with persistent or relapsed disease, defined as: first or greater relapse of neuroblastoma following completion of aggressive multi-drug frontline therapy; first episode of progressive NB during aggressive multi-drug frontline therapy; persistent/refractory neuroblastoma as defined by less than a complete response (by the revised INRC) at the conclusion of at least 4 cycles of aggressive multidrug induction chemotherapy on or according to a high-risk NB protocol (such as A3973 or ANBL0532); 7) measurable or evaluable disease per Revised International Neuroblastoma Response Criteria; 8) adequate central nervous system function (no known CNS disease, no seizure disorder requiring antiepileptic drug therapy); 9) adequate cardiac function (shortening fraction of >27% by echocardiogram); and 10) adequate pulmonary function (no chronic oxygen requirement and room air pulse oximetry >94%).
Treatment Plan
[0471] All patients will receive lymphodepleting chemotherapy before CAR-T cell infusion (Heczey A, et al., CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol. Ther. 2017; 25:2214-2224). Lymphodepletion will consist of cyclophosphamide 500 mg/m.sup.2/day IV on days 1-2 and fludarabine 30 mg/m.sup.2/day IV on days 1-4. The continual reassessment method (CRM) will be used to estimate the maximum-tolerated dose (MTD) of cells that can be administered in dose escalation cohorts comprised of 2-6 subjects. The final MTD will be the dose with estimated probability of dose limiting toxicity (DLT) closest to the target toxicity rate of 20%. Three cell doses will be evaluated: D1: 0.5×10.sup.6 CAR.sup.+ cells/kg; D2: 1×10.sup.6 CAR.sup.+ cells/kg; D3: 1.5×10.sup.6 CAR.sup.+ cells/kg. Cohort enrollment will be staggered and each subject must complete at least 2 weeks of cell treatment without incident of DLT before another subject can be enrolled at that dose level. A minimum of two subjects must complete the 4-week post-infusion DLT safety assessment period before cohort enrollment of subjects at the next higher dose level will be considered. If dose level 1 is determined to be above a tolerable dose, de-escalation would occur to dose level −1 where subjects would receive 0.25×10.sup.6 CAR.sup.+ cells/kg. Rimiducid (aka AP1903) (0.4 mg/kg), a dimerizing agent that is designed to engage and activate the iC9 to trigger T cell death, will be used to alleviate Grade 3 or 4 neurotoxicity or grade 3 pain symptoms unresponsive to standard of care (Di Stasi A., et al., Inducible apoptosis as a safety switch for adoptive cell therapy. N. Engl. J. Med. 2011; 365:1673-1683). In the dose expansion portion of the study, subjects may receive a second cell infusion (with prior lymphodepletion). Risk assessment will be evaluated per SOPs. Dose-limiting toxicity (DLT) will be evaluated per NCI CTCAE criteria v 5.0 or CRS and ICANS grading criteria if it occurs within the DLT reporting period (i.e., 4 weeks following CAR-T cell infusions).
Clinical Monitoring of Patients
[0472] Patient follow-up is directed by SOPs including a history and physical examination and routine laboratory investigations performed preinfusion and at 4 hours and 1, 2, 3, 4, 6 weeks, and months 3, 6, 9, and 12 post T-cell infusion, then every 6 months for 4 years. Patients are monitored for tumor progression or recurrence using standard criteria. Patients are evaluated at week 6 post-CAR-T cell infusion. Additional imaging obtained as part of standard clinical care will also be evaluated. Clinical response will be assessed using the revised International Neuroblastoma Response Criteria (INRC). Progression free survival (PFS) and overall survival (OS) will be estimated using the Kaplan-Meier method. Imaging will be obtained before and 6 weeks after CAR-T cell infusion. Imaging will then be performed at months 3, 6, 9, and 12 for study purposes. Patients will have bilateral bone marrow aspirates and biopsies obtained before and 6 weeks after CAR-T cell infusion. Repeat bone marrows will then be performed at months 3, 6, 9, and 12 for study purposes. If other tissue is obtained for clinical indications during the first year, a portion will be used to assess for presence of transduced T cells. If the patient dies, an autopsy will be requested and tissues assessed for the presence of CAR-T cells.
Example 16: Materials and Methods
Cell Lines and Cell Culture
[0473] Human neuroblastoma (NB) tumor cell lines IMR-32, NBL-S, NGP, LAN-5, LAN-1, SK-N-SH, SK-N-FI, SH-SY5Y and Felix, and human Burkitt's lymphoma cell line Raji were purchased from American Type Culture Collection (ATCC). NBL-S, NGP, LAN-5, LAN-1, SK-N-SH, SK-N-FI, SH-SY5Y, NB-1 and Raji were maintained in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (FBS)(Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Corning), and 2 mM of L-glutamine (Corning). Felix were maintained in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (FBS)(Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Corning), 2 mM of L-glutamine (Corning), and 1% Insulin/Transferrin/Selenium (ITS)(Corning). Phoenix-ECO and 293T packaging cells were obtained from DSMZ and cultured in Dulbecco's modified Eagle's medium (DMEM) (Corning) supplemented with 10% FBS (Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Corning), and 2 mM of L-glutamine (Corning).
[0474] Cells were maintained at 37° C. in humidified atmosphere with 5% CO.sub.2. NB cell lines were transduced with a retroviral vector encoding the GFP-Firefly-Luciferase (GFP-FFluc) gene, kindly provided by Prof. Giampietro Dotti (Vera et al., 2006). All cell lines were mycoplasma free and validated by flow cytometry for surface markers and functional readouts as needed. Lorlatinib was obtained from Pfizer.
CAR-T Plasmid Construction
[0475] The variable regions of the heavy and light chains of the ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7 mAbs were cloned from mouse hybridoma and then cloned as an scFv fragment into previously validated CAR formats that include the murine CD8α hinge and transmembrane domain, CD28 intracellular costimulatory domain, and CD3ζ intracellular signaling domain. The ALK CAR cassettes were cloned into the retroviral vector SFG. For the human version of the ALK5 CAR, murine CD8α, CD28 and CD3ζ were replaced by human CD8α, CD28 and CD3ζ, and the ALK5 scFv was modified to generate a humanized version (hALK5 CAR). The scFv specific for CD19 and GD2 were previously reported (Kochenderfer et al., Blood, 116(20): 4099-4102 (2010); Du H, et al., Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells. Cancer Cell 2019; 35:221-237).
Retrovirus Production
[0476] Retroviral supernatants used for the transduction of murine T cells were generated by cotransfecting Phoenix-ECO packaging cells. Phoenix-ECO cells were plated in a 10 cm dish. The following day, cells were transfected with the retroviral vectors and the pCL-Eco plasmid using the Xfect Transfection Reagent (Takara) according to the manufacturer's instruction. The media was changed 6 hours post-transfection. The viral supernatant was collected 48 hours after transfection, and filtered with 0.45 μm filters.
[0477] For the preparation of retroviral supernatants used for the transduction of human T cells, 2×10.sup.6 293 T cells were seeded in a 10 cm cell culture dish and transfected with the plasmid mixture of the retroviral vector, the Peg-Pam-e plasmid encoding MoMLV gag-pol, and the RDF plasmid encoding the RD114 envelope, using the GeneJuice transfection reagent (Merck Millipore) according to the manufacturer's instructions. Supernatants containing the retrovirus were collected 48 and 72 hours after transfection, and filtered with 0.45 μm filters.
Generation of Murine CAR-T Cells
[0478] Murine T cells were isolated using EasySep Mouse T Cell Isolation Kit (Stemcell) from splenocytes obtained from C57BL/6J mice and stimulated with 100 U/mL IL-2 and Dynabeads Mouse T-Activator CD3/CD28 (Gibco), according to the manufacturer's instructions, for 24 hours. Activated murine T lymphocytes were transduced with retroviral supernatants plus 6 μg/mL polybrene via spinfection at 2,000 rpm for 80 minutes, and expanded in complete medium (RPMI-1640 (Corning), 15% FBS (Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Corning), 2 mM of L-glutamine (Corning), 55 μM β-mercaptoethanol (Gibco), 1 mM Sodium Pyruvate (Corning), 10 mM Hepes (Corning), 1×MEM Nonessential Amino Acids (Corning)) with rhIL-2 (100 U/mL; R&D systems) changing medium every 2 days. On days 4-6, T cells were collected and used for functional assays in vitro and in vivo.
Co-Culture Experiments with Murine CAR-T Cells
[0479] Eμ-myc cells labeled with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) were seeded in 24-well plates at a concentration of 1×10.sup.5 cells/well, T cells were added to the culture at different ratios (E:T of 1:1, 2.5:1; 5:1; 10:1, or 20:1) without the addition of exogenous cytokines. Cells were analyzed 18 hours later to measure residual tumor cells by FACS. Target cells were identified by the expression of murine CD19-APC (130-102-546, Miltenyi Biotec) and their viability by the expression of CFSE.
Transduction and Expansion of Human T Cells
[0480] Apheresis leukoreduction collars from healthy donors were obtained from the Boston Children's Hospital Blood Donor Center, Boston, Mass. On day 0, lymphocytes were isolated with Ficoll-Paque Plus density separation (GE Healthcare), T cells were isolated with EasySep Human T Cell Isolation Kit (Stemcell) and activated with Dynabeads Human T-Activator CD3/CD28 (Gibco) according to the manufacturer's instructions. The day after, plates for transduction were prepared: non-tissue culture treated 24-well plates were coated overnight with 7 μg/mL retronectin (500 μL/well) (Takara Bio Inc., Shiga, Japan) in the cold room. On day 2, T cells were transduced. Briefly, non-tissue culture treated 24-well plates coated overnight with 7 mg/mL retronectin in the cold room were washed once with 1 mL medium, coated with 1 mL of the retroviral supernatant per well and centrifuged at 2000 g for 90 min. After removal of the supernatant, 5×10.sup.5 activated T cells were plated, and centrifuged at 1000 g for 10 min. Three days later, T cells were collected and expanded in complete medium (45% RPMI-1640 and 45% Click's medium (Irvine Scientific), 10% FBS (Gibco), 2 mM GlutaMAX (Gibco), 100 unit/mL of penicillin and 100 μg/mL of streptomycin (Corning) with rhIL-7 (10 ng/mL; PeproTech) and rhIL-15 (5 ng/mL; PeproTech), changing medium every 2-3 days. On day 12-14, cells were collected for in vitro and in vivo experiments. T cells were cultured in rhIL-7/IL-15 depleted medium for two days prior to being used in in vitro functional assays.
Co-culture Experiments with hCAR-T Cells
[0481] Tumor cells were seeded in 24-well plates at a concentration of 5×10.sup.5 cells/well 24 hours before co-cloture. T cells were added to the culture at different ratios (E:T of 1:1; 1:5, or 1:10) without the addition of exogenous cytokines. Cells were analyzed at day 5 to measure residual tumor cells and T cells by FACS. Dead cells were gated out by Zombie Aqua Dye (Biolegend) staining while T cells were identified by the expression of CD3 and tumor cells by the expression of GFP (NB cell lines) or CD19 (Raji cell line).
Flow Cytometry
[0482] Flow cytometry was performed using the following antibodies: human CD3 PerCP-cy5.5 (340948, BD Biosciences), human CD3 FITC (IM1281U, Beckman Coulter), human CD19 APC (IM2470U, Beckman Coulter), murine CD19 APC (130-102-546, Miltenyi Biotec). Expression of ALK in tumor cell lines was assessed with the ALK5 mAb conjugated with Alexa Fluor 647 using the Alexa Fluor Antibody Labeling Kit (Life technologies) according to manufacturer's instructions. Expression of the ALK CAR-T Cells was detected using F(ab′)2-Goat anti-Mouse IgG (H+L) Alexa Fluor 647 (Invitrogen). Samples were acquired with BD FACSCelesta flow cytometer using the BD Diva software (BD Biosciences). For each sample, a minimum of 10,000 events were acquired and data was analyzed using FlowJo 10.
ELISA
[0483] T cells (5×10.sup.4, 1×10.sup.5 or 5×10.sup.5) were co-cultured with tumor cells (5×10.sup.5) in 24-well plates without the addition of exogenous cytokines. After 24 hours, supernatant was collected and IFNγ and GM-CSF cytokines were measured in duplicate using specific ELISA kits (BioLegend or R&D system) following manufacturer's instructions.
T Cells Proliferation Assay
[0484] T cells were labeled with 1.5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) and plated with tumor cells at an E:T ratio of 1:1. CFSE dilution was measured on gated T cells on day 5 using flow cytometry.
Western Blot
[0485] Whole cell extracts were obtained using GST-FISH buffer (10 mM MgCl.sub.2, 150 mM NaCl, 1% NP-40, 2% Glycerol, 1 mM EDTA, 25 mM HEPES pH 7.5) supplemented with Protease Inhibitor Cocktail (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na.sub.3VO.sub.4. Extracts were cleared by centrifugation at 15,000 rpm for 20 min. The supernatants were collected and assayed for protein concentration using BCA protein assay method (Sigma). Equal amounts of protein lysates were resolved by Mini-PROTEAN TGX gels (BIO-RAD), transferred on nitrocellulose membrane (GE Healthcare), and probed with the following primary antibodies: ALK, rabbit ALK (D5F3) XP (Cell Signaling Technology, #3633), rabbit GFP (Cell Signaling Technology, #2555), rabbit polyclonal anti-β-actin (Sigma, #A5316), rabbit α-actinin (D6F6) XP (Cell Signaling Technology, #6487). Membranes were developed with ECL solution (GE Healthcare).
NB Cell Proliferation and Apoptosis Assays after Lorlatinib Treatment
[0486] In white 96-well plates, 3×10.sup.4 cells/mL were grown in triplicates. The treatment with lorlatinib was done after 24 hour. Cell growth was analyzed 5 days after treatment using Cell Titer-GloMax assay (Promega, Fitchburg, Wis., USA), according to the manufacturer's instructions. In 24-well plates, 5×10.sup.4 cells/mL were grown in triplicates. The treatment with lorlatinib was done after 24 hours. Apoptosis was measured 48 hours after treatment by flow cytometry after staining with the FITC Annexin V and propidium iodide (PI) Staining Solution Apoptosis Detection Kit I (BD Pharmingen) according to the manufacturer's instructions.
Quantification and Statistical Analysis
[0487] The unpaired and nonparametric Mann Whitney test with two tailed p value calculation was used to measure differences between two groups. For multiple group comparisons, one-way ANOVA or two-way ANOVA was used to determine statistically significant differences between samples. Holm-Sidak test adjusted p value <0.05 indicates a significant difference. Measurements were summarized as mean±SD. Difference between the survival curves were analyzed by the Chi-square test using GraphPad Prism v5. Graph generation and statistical analyses were performed using the GraphPad Prism software (GraphPad, La Jolla, Calif.).
Other Embodiments
[0488] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[0489] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of some embodiments herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
[0490] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. Particularly, WO 2017/035430, WO 2017/147383, U.S. Ser. No. 62/902,096, and Davila et al., CD19 CAR-Targeted T Cells Induce Long-Term Remission and B Cell Aplasia in an Immunocompetent Mouse Model of B Cell Acute Lymphoblastic Leukemia, PLoS ONE (2013), are incorporated herein by reference.