Anti-lamp5 antibodies, engineered immune cells expressing Anti-lamp5 antibodies, and uses thereof

Abstract

Provided herein are compositions, kits, and methods for manufacturing immunoglobulin- related compositions (e.g., antibodies or antigen binding fragments thereof) that can bind to the LAMP5 protein, and uses thereof. Additionally, provided herein are compositions, kits, and methods for manufacturing cells for adoptive cell therapy comprising engineered immune cells that express a LAMP5-specific receptor (e.g., a LAMP5-specific chimeric antigen receptor), and uses thereof. Additionally, provided herein are methods for identifying the risk of cancer in individuals of African American descent based on elevated LAMP5 levels in cancer cells.

Description

Atty. Dkt. No.: 642631-0168 ANTI-LAMP5 ANTIBODIES, ENGINEERED IMMUNE CELLS EXPRESSING ANTI- LAMP5 ANTIBODIES, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 601,418, filed November 21, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present technology relates generally to the preparation of immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) that specifically bind LAMP5 protein and uses of the same. In particular, the present technology relates to the preparation of LAMP5 binding antibodies and their use in detecting and treating cancer. The present technology also relates generally to compositions including engineered immune cells that express a LAMP5-specific receptor (e.g., a LAMP5-specific chimeric antigen receptor), and uses thereof. The present technology also generally relates to methods for identifying the risk of cancer to individuals of African American descent based on elevated LAMP5 levels in cancerous cells. BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] The blood-based cancers myeloma and leukemia represent a serious public health problem, afflicting hundreds of thousands of people in the United States alone. Myeloma is a cancer of the plasma cells, wherein the rapidly dividing plasma cells accumulate in the bone marrow and interfere with normal bodily processes, increasing susceptibility to diseases. Myeloma patients and those at risk of myeloma can be further broken down into clinical sub- type groups including monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, newly diagnosed multiple myeloma, or relapsed / refractory multiple myeloma. These subgroups represent varying degrees of risk and predicted disease progression. Leukemia, in contrast, is a disease of the white blood cells, and more specifically 1 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 lymphocytes or myeloid cells. As with Melanoma, there are multiple sub-types of leukemia, including plasma cell leukemia, mixed lineage leukemia, and acute myeloid leukemia.

[0005] Early diagnosis, risk stratification, and effective treatment are critical to myeloma and leukemia patient survival. However, there are currently limited options for diagnosing and treating patients, and an inaccurate risk stratification can prove disastrous for a patient’s overall prognosis. Accordingly, there is a need for new tools for the treatment and diagnosis of myeloma and leukemia, including for assigning an accurate risk value to individual patients. SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein: the VH comprises a VH-CDR1 sequence of GYTFTDFTI (SEQ ID NO: 5), a VH-CDR2 sequence of IIPNNGNA (SEQ ID NO: 6), and a VH-CDR3 sequence of ARARYYFDY (SEQ ID NO: 7); and the VL comprises a VL-CDR1 sequence of RSSSGAVTTSNSAN (SEQ ID NO: 12), a VL-CDR2 sequence of GTNNRPS (SEQ ID NO: 13), and a VL-CDR3 sequence of ALWNSNHWV (SEQ ID NO: 14)

[0007] In some embodiments, the antibody or antigen binding fragment VH comprises an amino acid sequence of SEQ ID NO: 4; and / or the antibody or antigen binding fragment VL comprises an amino acid sequence of SEQ ID NO: 11.

[0008] In some embodiments, the antibody or antigen binding fragment further comprises a Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

[0009] In some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

[0010] Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising a S228P mutation.

[0011] In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, F(ab’)2, Fab’, scFv, and Fv.

[0012] In some embodiments the antibody or antigen binding fragment binds to a LAMP5 polypeptide. 2 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0013] In certain embodiments, the antibody further comprises a heavy chain (HC) amino acid sequence comprising SEQ ID NO: 2, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO: 9.

[0014] In another aspect, the present disclosure provides an antibody comprising (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 11; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 4.

[0015] In yet another aspect the present disclosure provides an antibody comprising: (a) a LC sequence that is at least 95% identical to the LC sequence present in SEQ ID NO: 9; and / or (b) a HC sequence that is at least 95% identical to the HC sequence present in SEQ ID NO: 2.

[0016] In some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

[0017] In some embodiments, the antibody comprises an IgG4 constant region comprising a S228P mutation.

[0018] In any of the preceding embodiments, the antibody lacks α-1,6-fucose modifications.

[0019] In any of the preceding embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or multi-specific antibody.

[0020] In some embodiments, the multi-specific antibody or antigen binding fragment binds to T cells, B-cells, myeloid cells, plasma cells, or mast-cells.

[0021] In certain embodiments, the multi-specific antibody or antigen binding fragment binds to CD3, GPA33, HER2 / neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V intron V sequence (N- acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, LMP2, p53, lung resistance protein (LRP), Bcl-2, prostate specific antigen 3 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, PlGF, insulin-like growth factor (ILGF), tenascin, platelet- derived growth factor, IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis Y (Ley) antigen, E-cadherin, V- cadherin, GPC3, EpCAM, CD4, CD8, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, CD56, DLL3, PD-1, PD-L1, CD28, CD137, CD99, GloboH, CD24, STEAP1, B7H3, Polysialic Acid, OX40, OX40-ligand, or peptide MHC complexes (with peptides derived from TP53, KRAS, MYC, EBNA1-6, PRAME, MART, tyronsinase, MAGEA1-A6, pmel17, LMP2, or WT1).

[0022] In another aspect, the present disclosure provides a recombinant nucleic acid sequence encoding the antibody or antigen binding fragment of any of the preceding embodiments.

[0023] In one aspect, the present disclosure provides a recombinant nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1 and 8.

[0024] In a different aspect, the present disclosure provides a host cell or vector comprising the recombinant nucleic acid sequence of the preceding embodiments.

[0025] In another aspect, the present disclosure provides a composition comprising the antibody or antigen binding fragment of any of the preceding embodiments and a pharmaceutically-acceptable carrier, wherein the antibody or antigen binding fragment is optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof.

[0026] In a different aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding fragment or the composition of any of the preceding embodiments.

[0027] In certain embodiments, the cancer is selected from the group consisting of myeloma, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, multiple myeloma, relapsed / refractory multiple myeloma, leukemia, plasma cell 4 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 leukemia, mixed lineage leukemia, acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, and glioma and metastases thereof.

[0028] In some embodiments, the antibody or antigen binding fragment or the composition is administered to the subject separately, sequentially or simultaneously with an additional therapeutic agent.

[0029] In certain embodiments, the additional therapeutic agent is one or more of alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, and bisphosphonate therapy agents.

[0030] In one aspect, the present disclosure provides a method for detecting cancer in a subject in vivo comprising (a) administering to the subject an effective amount of the antibody or antigen binding fragment of any of the preceding embodiments, wherein the antibody or antigen binding fragment is configured to localize to a cancer cell expressing LAMP5 and is labeled with a radioisotope; and (b) detecting the presence of a cancer in the subject by detecting radioactive levels emitted by the antibody or antigen binding fragment that are higher than a reference value.

[0031] In certain embodiments, the subject is diagnosed with or is suspected of having cancer.

[0032] In some embodiments, the radioactive levels emitted by the antibody or antigen binding fragment are detected using positron emission tomography or single photon emission computed tomography.

[0033] In some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising the antibody or antigen binding fragment of any of the preceding embodiments conjugated to a radionuclide.

[0034] In certain embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger-emitter, or any combination thereof. 5 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0035] In certain embodiments, the beta particle-emitting isotope is selected from the group consisting of 86Y, 90Y, 89Sr, 165Dy, 186Re, 188Re, 177Lu, and 67Cu.

[0036] In another aspect, the present disclosure provides a kit comprising the antibody or antigen binding fragment of any of the preceding embodiments and instructions for use.

[0037] In certain embodiments, the antibody or antigen binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

[0038] In some embodiments, the kit comprises a secondary antibody that specifically binds to the antibody of any of the preceding embodiments.

[0039] In a different aspect, the present disclosure provides a method for detecting LAMP5 protein expression levels in a biological sample comprising contacting the biological sample with the antibody or antigen binding fragment of any of the preceding embodiments, and detecting binding to LAMP5 protein in the biological sample.

[0040] In some embodiments, the biological sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsied body tissue.

[0041] In some embodiments, the LAMP5 protein expression levels are detected via enzyme linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay, FACS, immunohistochemistry, FRET, or immunofluorescence.

[0042] In one aspect, the present disclosure provides a method for determining the prognosis of an individual of African American descent at risk for myeloma or leukemia comprising (a) detecting LAMP5 mRNA and / or protein expression levels in a sample obtained from the individual; and (b) determining that the prognosis of the individual is negative when the expression levels of LAMP5 mRNA and / or protein is elevated compared to a control sample obtained from a healthy individual of African American descent or a predetermined threshold.

[0043] In some embodiments, the LAMP5 protein expression levels are detected with the antibody or antigen binding fragment of any of the preceding embodiments. 6 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0044] In some embodiments, the method further comprises administering to the individual an effective amount of an anti-cancer therapy.

[0045] In certain embodiments, the anti-cancer therapy is selected from among radiation therapy, hormonal therapy, chemotherapy, immunotherapy or combinations thereof.

[0046] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antibody or antigen binding fragment of any of the preceding embodiments.

[0047] In some embodiments, the chimeric antigen receptor comprises (i) an extracellular antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain.

[0048] In certain embodiments, the extracellular antigen binding domain is an scFv, a Fab, or a F(ab)2.

[0049] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 60.

[0050] In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 60.

[0051] In some embodiments, the extracellular antigen binding domain comprises a signal peptide that is covalently joined to the N-terminus of the extracellular antigen binding domain.

[0052] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain.

[0053] In any of the preceding embodiments, the intracellular domain comprises one or more costimulatory domains.

[0054] In certain embodiments, the one or more costimulatory domains are selected from among a CD28 costimulatory domain, a 4-1BB costimulatory domain, an OX40 costimulatory domain, an ICOS costimulatory domain, a DAP-10 costimulatory domain, a PD-1 costimulatory domain, a CTLA-4 costimulatory domain, a LAG-3 costimulatory domain, a 2B4 costimulatory domain, a BTLA costimulatory domain, a CD3ζ-chain, or any combination thereof. 7 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0055] In one aspect, the present disclosure provides an engineered immune cell comprising the CAR of any of the preceding embodiments, wherein the engineered immune cell is a lymphocyte.

[0056] In some embodiments, the engineered immune cell is a B cell, a T cell, a tumor infiltrating lymphocyte, a CD4+ T cell, a CD8+ T cell or a natural killer (NK) cell.

[0057] In some embodiments, the engineered immune cell is derived from an autologous donor or an allogenic donor.

[0058] In one aspect, the present disclosure provides a nucleic acid encoding the CAR of any of the preceding embodiments.

[0059] In a different aspect, the present disclosure provides a vector comprising the nucleic acid of certain embodiments.

[0060] In one aspect, the present disclosure provides a host cell comprising the nucleic acid of some embodiments or the vector of some embodiments.

[0061] In one aspect, the present disclosure provides a kit comprising the engineered immune cell of any of the preceding embodiments, and instructions for use.

[0062] In another aspect, the present disclosure provides a method for preparing immune cells for cancer therapy comprising isolating immune cells from a donor subject; and transducing the immune cells with (a) the nucleic acid of certain embodiments or (b) the vector of certain embodiments.

[0063] In one aspect, the present disclosure provides a method of treatment comprising isolating immune cells from a donor subject; transducing the immune cells with (a) the nucleic acid of certain embodiments or (b) the vector of certain embodiments; and administering the transduced immune cells to a recipient subject.

[0064] In certain embodiments, the donor subject and the recipient subject are the same or different.

[0065] In some embodiments, the immune cells isolated from the donor subject comprise one or more lymphocytes. 8 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0066] In certain embodiments, the one or more lymphocytes is a T cell, a B cell, or a natural killer (NK) cell.

[0067] In certain embodiments, the T cell is a CD4+ T cell or a CD8+ T cell.

[0068] In some embodiments, the immune cells isolated from the donor subject comprise tumor infiltrating lymphocytes.

[0069] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the engineered immune cell of any the preceding embodiments.

[0070] In some embodiments, the method further comprises administering to the subject a cancer specific monoclonal antibody.

[0071] In one aspect, the present disclosure provides a method for treating of inhibiting tumor growth or metastasis in a subject with cancer comprising contacting a tumor cell with an effective amount of the engineered immune cell of any of the preceding embodiments.

[0072] In some embodiments, the engineered immune cell is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratumorally.

[0073] In some embodiments, the method further comprises administering an additional cancer therapy.

[0074] In some embodiments, the additional cancer therapy is selected from among chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti-cancer viruses or microorganisms, and any combinations thereof.

[0075] In some embodiments, the method further comprises administering a cytokine to the subject.

[0076] In certain embodiments, the cytokine is selected from the group consisting of interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23- 1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, 9 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

[0077] In some embodiments, the cancer or tumor is selected from among myeloma, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, multiple myeloma, relapsed / refractory multiple myeloma, leukemia, plasma cell leukemia, mixed lineage leukemia, acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, and glioma and metastases thereof.

[0078] In one aspect, the present disclosure provides a composition comprising the antibody or antigen binding fragment of any one of the preceding embodiments, conjugated to an agent selected from the group consisting of drugs, toxins, or any combination thereof.

[0079] In some embodiments, the drugs comprise one or more of Tesirine or MMAE.

[0080] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the composition of any one of the preceding embodiments and an additional cancer therapy.

[0081] In one aspect, the present disclosure provides a method for treating or inhibiting tumor growth or metastasis in a subject with cancer comprising administering to the subject an effective amount of the composition of any one of the preceding embodiments and an additional cancer therapy.

[0082] In some embodiments, the additional cancer therapy comprises one or more of Venetoclax or Idarubicin. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIGs.1A-1C show the impact of LAMP5 knockdown on the proliferation and apoptosis of myeloma (MM.1S) cells. MM.1S cells were lentivirally transduced with a doxycycline-inducible promoter fused to a LAMP5 shRNA construct and a GFP construct for selection. To measure proliferation (FIG.1A) transduced cells were mixed with wild-type unlabeled MM.1S cells and grown with or without doxycycline for 12 days. Population GFP positivity was measured at days 0, 6, and 12 post treatment. To measure apoptosis (FIG.1B) 10 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 transduced cells were grown with or without doxycycline for 0, 6, or 12 days, at which point cell death was measured using annexin V-pacific blue and Toppro-3 staining and flow cytometry. To measure LAMP5 specificity (FIG.1C), three cell lines (H929, JJN3, and MM.1S) were transduced with an RFP-Lc3 fusion expressing cassette and with either doxycycline inducible LAMP5 shRNA (sh1-2) or non-targeted shRNA (NT), and were grown with or without 1 ug / ml doxycycline for 72 hours, before RFP expression and cellular viability (annexin V-pacific blue and Toppro-3 staining) were measured using flow cytometry.

[0084] FIGs.2A-2D shows the generation of novel anti-LAMP5 antibodies via hybridoma screening. FIG.2A shows murine fibroblast L-cells were successfully transduced with the human LAMP5 ORF, as detected by flow cytometry. FIG.2B shows the results of ELISA binding assays for hybridoma clone supernatant against L-Lamp5 cells (undiluted supernatant, 1 / 15 diluted, and 1 / 225 diluted) and against parental L cells. FIG.2C is a Western Blot demonstrating the LAMP5-specific binding capabilities of monoclonal antibodies C13 and C17 against protein from L-cells, with and without exogenous LAMP5 expression, Ba / F3 cells, with and without exogenous LAMP5 expression, and MM.1S cells. FIG.2D shows the CDR sequences of C17.

[0085] FIGs.3A-3D show the binding affinity and epitope location for the C17 antibody. FIG.3A shows the results of an Octet binding analysis assay measuring the dissociation constant of C17 for LAMP5 (KD= 1.2 x 10-10). FIG.3B is a western blot showing C17 binding to the following LAMP5 epitope regions: full length, residues 1-100, residues 78-155, residues 128-194, and residues 175-238. FIG.3C is a western blot showing C17 binding to the following LAMP5 epitope regions: full length (GFP-FL), residues 1-100 (GFP-F1), residues 78-155 (GFP-F2), residues 1-55 and 78-100 (GFP-F1-del). FIG.3D is the results of an ELISA assay demonstrating the binding affinity of C17 to a series of overlapping peptides encoding regions of LAMP5.

[0086] FIGS.4A-4C establish that C17 is capable of detecting patient-derived myeloma cells. FIG.4A shows that the LAMP5 positive MM.1S cells were successfully bound by a C17-Alexa Fluor 647 conjugate (1890.0 signal intensity) compared to an isotype control (80.5 signal intensity). FIGs.4B-4C show a population of patient-derived myeloma cells treated 11 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 with either isotype FITC-A conjugate antibodies (FIG.4B) or C17 FITC-A antibodies (FIG. 4C).

[0087] FIGs.5A-5B show the conversion of the C17 antibody into an antibody drug conjugate to monomethyl auristatin E (MMAE) via maleimide. FIG.5A is a hydrophobic interaction chromatogram for C17-MMAE conjugate (upper, blue trace) and the C17 antibody (lower, black trace) including the drug to antibody ratio (DAR) percentage. FIG.5B shows the deconvoluted m / z mass spectrometry data comparing the C17 antibody light chain (LC; upper left) and heavy chain (HC; upper right) to the C17-MMAE antibody drug conjugate light chain (bottom left) and heavy chain (bottom right), with LC+1 and HC+1 representing successful 1:1 C17:MMAE conjugates.

[0088] FIGs.6A-6B demonstrate that the C17-MMAE antibody drug conjugate (ADC) is highly potent and specific. FIG.6A shows a cell viability assay wherein MM.1S cells or MM.1S cells expressing exogenous LAMP5 were treated with either C17 antibody, or C17- MMAE ADC at increasing concentrations while cell viability was measured. FIG.6B shows a cell viability assay wherein RPMI 8226 cells and RPMI 8226 cells transduced with a LAMP5 expression cassette were treated with increasing concentrations of C17-MMAE while cell viability was measured.

[0089] FIGs.7A-7B show the conversion of the C17 antibody into an antibody drug conjugate to Tesirine via maleimide. FIG.7A is a hydrophobic interaction chromatogram for C17-Tesirine conjugate (upper, blue trace) and the C17 antibody (lower, black trace) including the drug to antibody ratio (DAR) percentage. FIG.7B shows the deconvoluted m / z mass spectrometry data comparing the C17 antibody light chain (Lamp5 mAb LC; upper) and heavy chain (Lamp5 mAb HC; upper) to the C17-Tesirine antibody drug conjugate light chain (lower) and heavy chain (lower), with LC+1 and HC+1 representing successful 1:1 C17:Tesirine conjugates.

[0090] FIGs.8A-8C demonstrate that the C17-Tesirine antibody drug conjugate (ADC) is highly potent and specific. FIG.8A shows a cell viability assay wherein RPMI 8226 cells or RPMI 8226 cells expressing exogenous LAMP5 were treated with either C17 antibody, isotype antibody, C17-Tesirine ADC, or isotype-Tesirine ADC at increasing concentrations while cell viability was measured. FIG.8B shows a cell viability assay wherein MM.1S cells or R 12 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 MM.1S cells expressing exogenous LAMP5 were treated with either C17-Tesirine ADC or isotype-Tesirine ADC at increasing concentrations while cell viability was measured. FIG.8C shows a cell viability assay wherein H929 (LAMP5 positive) cells were treated with increasing concentrations of C17-Tesirine ADC or isotype-Tesirine ADC while cell viability was measured.

[0091] FIGs.9A-9B show that chimeric antigen receptor (CAR) T-cells expressing a C17 CAR were highly specific and potent. FIG.9A shows a cell viability assay for RPMI 8226 vector control cells and RPMI 8226-LAMP5 cells, when treated with T-cells transduced with a blank vector as a negative control (NC), CAR T-Cells targeted to the melanoma antigen BCMA, or C17 CAR T-cells generated with antibody plasmid clone 12 (C17-12) and 13 (C17- 13).. FIG.9B shows a cell viability assay using an H929-GFP / Luciferase (LUC) viability reporter target cell line treated with media as a negative control (NC), CAR T-Cells targeted to the melanoma antigen BCMA, or C17 CAR T-cells, at an effector to target (E:T) ratio of 20:1 and 10:1, for 16 hours, after which viability was measured.

[0092] FIG.10 shows a cell viability assay for the acute myelogenous leukemia (AML) cell lines Molm 13, OCI-AML3, MV411, U937, and HL-60 when treated with increasing concentrations of the ADC C17-MMAE. All of the cell lines except HL-60 are LAMP5 positive.

[0093] FIGs.11A-11C show the correlation of LAMP5 mRNA expression levels with survival probability in Black or African American patients and White patients with newly diagnosed myeloma, further sub-categorized as having “high” (FIG.11A), “medium” (FIG. 11B), or “low” (FIG.11C) LAMP5 expression, with p values representing the results a Wald test for significant differences in survival probability between the patient populations.

[0094] FIGs.12A-12B show the expression of LAMP5 in different cancer types as measured by RNA-seq (FIG.12A) and by Affymetrix-based microarray studies (FIG.12B).

[0095] FIGs.13A-13C show anti-LAMP5 DCs reduced the viability of LAMP5-positive myeloma cells and increased survival of a MM1.S-luc NGS xenograft mouse model. FIG. 13A: Bioluminescence-based tumor quantification in an MM1.S-based xenograft in immunodeficient mice treated with one single dose of IgG2A-Tesirine or C17-Tesirine (1 mg / kg) compared to a vehicle (PBS) control (n=10 per group). Statistical analyses for these 13 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 experiments were conducted using the Mann Whitney test. FIG.13B: Survival curve of mice treated with a single dose of PBS, IgG2A-Tesirine, or C17-Tesirine as detailed above and then monitored for up to 6 months, with statistical analyses performed using the Log-rank Mantel- Cox test. Four mice were still alive in the C17-Tesi group at 177 days after drug injection. FIG.13C: Fluorescence imaging of mice at 4 weeks post treatment.

[0096] FIGs.14A-14D show anti-LAMP5 DCs reduced the viability of LAMP5-positive myeloma cells and increased survival of a MV4-11-luc NOD scid xenograft mouse model. FIG.14A: total flux change fold post treatment in mice treated with Lp5-C17-MMAE or IgG2A-MMAE (Iso-MMAE) or PBS (vehicle) or naked IgG2a (Iso-Ab) or C17(Lp5-C17-Ab) antibody controls. FIG.14B: Bioluminescence-based tumor quantification in mice treated with Lp5-C17-MMAE or IgG2A-MMAE (Iso-MMAE) or PBS (vehicle) or naked IgG2a (Iso-Ab) or C17(Lp5-C17-Ab) antibody controls. FIG.14C: Fluorescence imaging of mice at 4 weeks post treatment. 14D: luciferase activity change fold and raw luciferase activity values for each treatment group throughout the experiment.

[0097] FIGs.15A-15C show combination treatment results. FIG.15A shows the combination index at different cell killing fractions for Molm13, OCI-AML3, and MV4-11 cells treated with Idarubicin(nM) and Lp5-C17-MMAE(ng / ml) at a constant ratio of 5:100, 10:200 and 5:100 respectively. FIG.15B shows the percent survival for OCI-AML3 treated with Venetoclax (Vene), Lp5-C17-MMAE (MMAE), or Venetoclax and Lp5-C17-MMAE (Ve-MMAE), at a constant ratio of 500:200. FIG.15C shows the combination index at different Fa for OCI-AML3 treated with Lp5-C17-MMAE and Venetoclax.

[0098] FIG.16 shows sequences of the present disclosure. DETAILED DESCRIPTION

[0099] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0100] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof), which can specifically bind to LAMP5 polypeptides. Accordingly, the various aspects of the present methods relate to the preparation, 14 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 characterization, and manipulation of anti-LAMP5 antibodies. The immunoglobulin-related compositions of the present technology are useful alone or in combination with additional therapeutic agents for treating and detecting cancer and the detection and purification of LAMP5. In some embodiments, the immunoglobulin-related composition is a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multi-specific antibody.

[0101] The present disclosure also generally provides compositions including engineered immune cells that express a LAMP5-specific receptor (e.g., a LAMP5-specific chimeric antigen receptor). Accordingly, the various aspects of the present methods relate to the preparation, characterization, and manipulation of such engineered immune cells. The engineered immune cell compositions of the present technology are useful alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the engineered immune cells express an anti-LAMP5 monoclonal antibody, humanized antibody, chimeric antibody, bispecific antibody, or multi-specific antibody.

[0102] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0103] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA 15 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)). Definitions

[0104] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.

[0105] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & 16 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0106] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0107] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 1.0 or 0.1, as appropriate or alternatively by a variation of + / − 20% or + / − 15%, or alternatively 10% or alternatively 5% or alternatively 2%. As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.

[0108] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like.

[0109] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to 17 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.

[0110] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally, topically, or other suitable routes herein described. Administration includes self-administration and the administration by another. “Administration” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer. 18 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0111] An “adjuvant” refers to one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens or antibodies. An adjuvant may be administered to a subject before, in combination with, or after administration of the vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponins), bacterial and fungal cell wall components (e.g., lipopolysaccarides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

[0112] As used herein “adoptive cell therapeutic composition” refers to any composition comprising cells suitable for adoptive cell transfer. In exemplary embodiments, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of a tumor infiltrating lymphocyte (TIL), TCR (i.e. heterologous T-cell receptor) modified lymphocytes (e.g., eTCR T cells and caTCR T cells) and CAR (i.e. chimeric antigen receptor) modified lymphocytes (e.g., CAR T cells). In another embodiment, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells and peripheral blood mononuclear cells. In another embodiment, TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition. In one embodiment, the adoptive cell therapeutic composition comprises T cells.

[0113] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In 19 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0114] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0115] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0116] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations and fragments thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. Antibodies and antibody fragments can be wholly or partially derived from mammals (e.g., humans, non- human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits and sheep) or non- mammalian antibody producing animals (e.g., chickens, ducks, geese, snakes, and urodele amphibians). The antibodies and antibody fragments can be produced in animals or produced outside of animals, such as from yeast or phage (e.g., as a single antibody or antibody fragment or as part of an antibody library). As used herein, “antibodies” (includes intact immunoglobulins) and “antigen binding fragments” specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M-1greater, at least 104M-1greater or at least 105M-1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York, 1997. 20 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 Antibodies may comprise whole native antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), single domain antibodies (e.g., nanobodies and single domain camelid antibodies), VNAR fragments, Bi- specific T-cell engager (BiTE) antibodies, minibodies, disulfide-linked Fvs (sdFv), and anti- idiotypic (anti-Id) antibodies, intrabodies, fusion polypeptides, unconventional antibodies and antigen binding fragments of any of the above.

[0117] More particularly, antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. The heavy chain constant (CH) region is comprised of three domains, CH1, CH2, and CH3. Together, the VHregion and the VLregion are responsible for binding the antigen recognized by the antibody. Typically, an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions (FR) of different light or heavy chains are relatively conserved within a species. Each VHand VLis composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, largely adopt a β-sheet 21 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 conformation and the CDRs form loops which connect, and in some cases form part of, the β- sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

[0118] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 and are also typically identified by the chain in which the particular CDR is located. Thus, a VHCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VLCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds LAMP5 protein will have a specific VHregion and VLregion sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs.

[0119] Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). “Immunoglobulin-related compositions” as used herein, refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multi-specific antibodies, bispecific antibodies, etc.,) as well as antibody fragments. An antibody or antigen binding fragment thereof specifically binds to an antigen.

[0120] As used herein, the term “antibody-related polypeptide” means antigen-binding antibody fragments, including single-chain antibodies, that can comprise the variable region(s) alone, or in combination, with all or part of the following polypeptide elements: hinge region, CH1, CH2, and CH3domains of an antibody molecule. Also included in the technology are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3domains. Antibody- related molecules useful in the present methods, e.g., but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VLor VHdomain. Examples include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1domains; (ii) a F(ab′)2fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1domains; (iv) a Fv fragment consisting 22 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 of the VLand VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of a VHdomain; and (vi) an isolated complementarity determining region (CDR). As such “antibody fragments” or “antigen binding fragments” can comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments or antigen binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single- chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

[0121] "Bispecific antibody" or “BsAb”, as used herein, refers to an antibody that can bind simultaneously to two targets that have a distinct structure, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen binding moiety in a bispecific antibody includes VHand / or VLregions; in some such embodiments, the VHand / or VLregions are those found in a particular monoclonal antibody. In some embodiments, the bispecific antibody contains two antigen binding moieties, each including VHand / or VLregions from different monoclonal antibodies. In some embodiments, the bispecific antibody contains two antigen binding moieties, wherein one of the two antigen binding moieties includes an immunoglobulin molecule having VHand / or VLregions that contain CDRs from a first monoclonal antibody, and the other antigen binding moiety includes an antibody fragment (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.) having VHand / or VLregions that contain CDRs from a second monoclonal antibody.

[0122] As used herein, the term “conjugated” refers to the association of two molecules by any method known to those in the art. Suitable types of associations include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for instance, hydrogen bonds, dipolar interactions, van der Waal forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.

[0123] As used herein, the term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VHVL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two 23 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 antigen binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). As used herein, the terms “single-chain antibodies” or “single-chain Fv (scFv)” refer to an antibody fusion molecule of the two domains of the Fv fragment, VLand VH. Single-chain antibody molecules may comprise a polymer with a number of individual molecules, for example, dimer, trimer or other polymers. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker (e.g., about 10, 15, 20, 25 amino acids), which connects the N-terminus of the VHwith the C-terminus of the VL, or the C-terminus of the VHwith the N-terminus of the VL. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. In certain embodiments, the linker comprises amino acids having GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18). In certain embodiments, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 19 is ggcggcggcggatctggaggtggtggctcaggtggcggaggctccggtggcggaggctcc (SEQ ID NO: 19). In one embodiment, the linker sequence is a (G4S)n (SEQ ID NO: 20), wherein n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15. Such single-chain antibodies can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies. Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988)). See, also, U.S. Patent Nos.5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al., J Imunol 183(4):2277-85 (2009); Giomarelli et al., Thromb Haemost 97(6):955-63 (2007); Fife eta., J Clin Invst 116(8):2252-61 (2006); Brocks et al., Immunotechnology 3(3): 173-84 (1997); Moosmayer et al., Ther Immunol 2(10):31- 40 (1995). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 25278(38):36740-7 (2003); Xie et al., Nat Biotech 15(8):768-71 24 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 (1997); Ledbetter et al., Crit Rev Immunol 17(5-6):427-55 (1997); Ho et al., Bio Chim Biophys Acta 1638(3):257-66 (2003)).

[0124] Any of the above-noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0125] As used herein, an “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a polypeptide (e.g., a LAMP5 polypeptide). An antigen may also be administered to an animal to generate an immune response in the animal.

[0126] The term “antigen binding fragment” refers to a fragment of the whole immunoglobulin structure which possesses a part of a polypeptide responsible for binding to antigen. Examples of the antigen binding fragment useful in the present technology include scFv, (scFv)2, scFvFc, Fab, Fab′ and F(ab′)2, but are not limited thereto.

[0127] By “binding affinity” is meant the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by standard methods known in the art, including those described herein. Without wishing to be bound by theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes (e.g., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, comprising use of binding experiments to calculate affinity. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay). A low-affinity complex contains an antibody 25 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration.

[0128] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0129] As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and in some aspects, the term may be used interchangeably with the term “tumor.” The term “cancer or tumor antigen” refers to an antigen known to be associated and expressed in a cancer cell or tumor cell (such as on the cell surface) or tissue, and the term “cancer or tumor targeting antibody” refers to an antibody that targets such an antigen. In some embodiments, the cancer or tumor antigen is not expressed in a non-cancer cell or tissue. In some embodiments, the cancer or tumor antigen is expressed in a non-cancer cell or tissue at a level significantly lower compared to a cancer cell or tissue. In some embodiments, the cancer is myeloma, including monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, newly diagnosed multiple myeloma, or relapsed / refractory multiple myeloma, and / or the cancer is leukemia, including plasma cell leukemia, mixed lineage leukemia, or acute myeloid leukemia. In some embodiments, the cancer is neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, 26 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, and glioma, among others shown to express LAMP5 (FIGs.12A-12B). In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer reaches a remission, but can relapse. In some embodiments, the cancer is unresectable.

[0130] As used herein, the term “CDR-grafted antibody” means an antibody in which at least one CDR of an “acceptor” antibody is replaced by a CDR “graft” from a “donor” antibody possessing a desirable antigen specificity.

[0131] As used herein, the term “cell population” refers to a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1×106cells, at least about 1×107cells, at least about 1×108cells, at least about 1×109cells, at least about 1×1010cells, at least about 1×1011cells, at least about 1×1012cells, or more cells expressing similar or different phenotypes.

[0132] As used herein, the term “chimeric co-stimulatory receptor” or “CCR” refers to a chimeric receptor that binds to an antigen and provides co-stimulatory signals, but does not provide a T-cell activation signal.

[0133] As used herein, the term “chimeric antibody” means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced, using recombinant DNA techniques, with an Fc constant region from an antibody of another species (e.g., a human Fc constant region). See generally, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No.4,816,567; Cabilly et al., European Patent Application 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; 27 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst.80: 1553-1559, 1988.

[0134] As used herein, the term “consensus FR” means a framework (FR) antibody region in a consensus immunoglobulin sequence. The FR regions of an antibody do not contact the antigen.

[0135] As used herein, a “cleavable peptide”, which is also referred to as a “cleavable linker,” means a peptide that can be cleaved, for example, by an enzyme. One translated polypeptide comprising such cleavable peptide can produce two final products, therefore, allowing expressing more than one polypeptides from one open reading frame. One example of cleavable peptides is a self-cleaving peptide, such as a 2A self-cleaving peptide. 2A self- cleaving peptides, is a class of 18-22 aa-long peptides, which can induce the cleaving of the recombinant protein in a cell. In some embodiments, the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A and BmCPV2A. See, for example, Wang Y, et al. Sci Rep. 2015;5:16273. Published 2015 Nov 5. As used herein, the terms “T2A” and “2A peptide” are used interchangeably to refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide comprising the requisite amino acids in a relatively short peptide sequence (on the order of 20 amino acids long depending on the virus of origin) containing the consensus polypeptide motif D-V / I-E-X-N-P-G-P (SEQ ID NO: 21), wherein X refers to any amino acid generally thought to be self-cleaving.

[0136] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 28 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0137] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a nanoparticle, detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include carriers, such as pharmaceutically acceptable carriers. In some embodiments, the carrier (such as the pharmaceutically acceptable carrier) comprises, or consists essentially of, or yet further consists of a nanoparticle, such as an polymeric nanoparticle carrier or an lipid nanoparticle that can be used alone or in combination with another carrier, such as an adjuvant or solvent. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, 29 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 lactitol, xylitol sorbitol (glucitol) and myoinositol. A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0138] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0139] As used herein, the term, “co-stimulatory signaling domain,” or “co-stimulatory domain”, refers to the portion of the CAR comprising the intracellular domain of a co- stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD3ζ, CD7, LIGHT, NKD2C, B7-H2 and a ligand that specifically binds CD83. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from CD28, 4-1BB, and CD3ζ, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more co-stimulatory signaling domains can enhance the efficacy and expansion of T cells expressing CAR receptors. The intracellular signaling and co-stimulatory signaling domains can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0140] As used herein, the phrase “derived” means isolated, purified, mutated, or engineered, or any combination thereof. For example, an immune cell derived from a donor refers to the immune cell isolated from a biological sample of the donor and optionally engineered.

[0141] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms 30 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the effective concentration of the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0142] As used herein, the term “effector cell” means an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and carry out specific immune functions. An effector cell can induce antibody-dependent cell- mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes which express FcαR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens.

[0143] As used herein, the term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In some embodiments, an “epitope” of the LAMP5 protein is a region of the protein to which the anti-LAMP5 antibodies of the present technology 31 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 specifically bind. In some embodiments, the epitope is a conformational epitope or a non- conformational epitope. To screen for anti-LAMP5 antibodies which bind to an epitope, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if an anti-LAMP5 antibody binds the same site or epitope as an anti- LAMP5 antibody of the present technology. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. In a different method, peptides corresponding to different regions of LAMP5 protein can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

[0144] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long- term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0145] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of the compositions disclosed herein. The term “expression” also refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription) within a cell; (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation) within a cell; (3) translation of an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presentation of a polypeptide or protein on the cell surface; and (6) 32 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 secretion or presentation or release of a polypeptide or protein from a cell. The level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.

[0146] As used herein, an "expression vector" includes vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0147] As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0148] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments. 33 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0149] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0150] As used herein, the term “heterologous nucleic acid molecule or polypeptide” refers to a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is either not normally expressed or is expressed at an aberrant level in a cell or sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0151] As used herein, a "host cell" is a cell that is used to receive, maintain, reproduce and amplify an expression vector. A host cell also can be used to express the polypeptide encoded by the expression vector. The nucleic acid contained in the expression vector is replicated when the host cell divides, thereby amplifying the nucleic acids.

[0152] “Homology” or “identity” or “percent identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by ═HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. 34 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 The percent homology between two amino acid sequences can be also determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0153] As used herein, “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab′, F(ab′)2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 35 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). See e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0154] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR”. These CDR regions have been described by Kabat et al., J. Biol. Chem.252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol.196:901-917 (1987); and MacCallum et al., J. Mol. Biol.262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. Table A CDR Definitions Kabat1Chothia2MacCallum3IMGT4AHo5VHCDR1 31-35 26-32 30-35 27-38 25-40 VHCDR2 50-65 53-55 47-58 56-65 58-77 VHCDR3 95-102 96-101 93-101 105-117 109-137 VLCDR1 24-34 26-32 30-36 27-38 25-40 VLCDR2 50-56 50-52 46-55 56-65 58-77 VLCDR3 89-97 91-96 89-96 105-117 109-137

[0155] 1Residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991). 36 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0156] 2Residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997).

[0157] 3Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008).

[0158] 4Residue numbering follows the nomenclature of Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001).

[0159] 5Residue numbering follows the nomenclature of Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001).

[0160] As used herein, the term “intact antibody” or “intact immunoglobulin” means an antibody that has at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0161] As used herein, the term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term “engineered immune cell” refers to an immune cell that is genetically 37 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 modified. As used herein, the term “native immune cell” refers to an immune cell that naturally occurs in the immune system.

[0162] As used herein, the term “increase” or “enhance” means to alter positively by at least about 5%, including, but not limited to, alter positively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

[0163] As used herein, the term “isolated,” “purified,” or “biologically pure” refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or polypeptide of the presently disclosed subject matter is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0164] As used herein, the term “isolated cell” refers to a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0165] As used herein, the term “ligand” refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and / or interaction.

[0166] The term “linker” refers to synthetic sequences (e.g., amino acid sequences) that connect or link two sequences, e.g., that link two polypeptide domains. In some embodiments, the linker comprises from a total of 1 to 200 amino acid residues; or about 1 to 10 amino acid residues, or alternatively 8 amino acids, or alternatively 6 amino acids, or alternatively 5 amino acids that may be repeated from 1 to 10, or alternatively to about 8, or alternatively to about 6, 38 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 or alternatively to about 5, or alternatively, to about 4, or alternatively to about 3, or alternatively to about 2 times. For example, the linker may comprise up to 15 amino acid residues consisting of a pentapeptide repeated three times. In one embodiment, the linker sequence is a (G4S)n (SEQ ID NO: 20), wherein n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15.

[0167] The term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from lymphoid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-1 cells, B-2 cells, and anergic AN1 / T3 cell populations.

[0168] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, e.g., but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (See, e.g., U.S. Patent No.4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described 39 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991), for example.

[0169] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0170] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0171] As used herein, the term “pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle, such as an polymeric nanoparticle carrier or an lipid nanoparticle (LNP). Additionally or alternatively, pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- 40 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They can be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0172] As used herein, the term “polyclonal antibody” means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0173] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof, in modified or unmodified form. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics 41 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 applications such as functional genomics and homology searching. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0174] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non-naturally occurring amino acid, e.g., an amino acid analog. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

[0175] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0176] As used herein, the term “reduce” means to alter negatively by at least about 5%, including, but not limited to, alter negatively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%. 42 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0177] As used herein, “regulatory sequence” of a nucleic acid molecule means a cis-acting nucleotide sequence that influences expression, positively or negatively, of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0178] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Polymerase II and III are examples of promoters. A polymerase II or “pol II” promoter catalyzes the transcription of DNA to synthesize precursors of mRNA, and most shRNA and microRNA. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase (“PGK”) promoter; EF1-alpha; CMV (minimal cytomegalovirus promoter); and LTRs from retroviral and lentiviral vectors. In some embodiments, the promoter is a constitutive promoter. As used herein, the term “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control in all or most tissues of a subject at all or most developing stages. Non-limiting examples of the constitutive promoters include a CMV promoter, a simian virus 40 (SV40) promoter, a polyubiquitin C (UBC) promoter, an EF1-alpha promoter, a PGK promoter and a CAG promoter. In some embodiments, the promoter is a conditional promoter, which allows for continual transcription of the coding sequence or gene under certain conditions. In further embodiments, the conditional promoter is an immune cell specific promoter, which allows for continual transcription of the coding sequence or gene in an immune cell. Non-limiting examples of the immune cell specific promoters include a promoter of a B29 gene promoter, a CD14 gene promoter, a CD43 gene promoter, a CD45 43 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 gene promoter, a CD68 gene promoter, a IFN-β gene promoter, a WASP gene promoter, a T- cell receptor β-chain gene promoter, a V9 γ (TRGV9) gene promoter, a V2 δ (TRDV2) gene promoter, and the like.

[0179] Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0180] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0181] As used herein, the term “sample” refers to clinical samples obtained from a subject. In certain embodiments, a sample is obtained from a biological source (i.e., a "biological sample"), such as tissue, bodily fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), whole blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0182] As used herein, the term “secreted” in reference to a polypeptide means a polypeptide that is released from a cell via the secretory pathway through the endoplasmic reticulum, Golgi apparatus, and as a vesicle that transiently fuses at the cell plasma membrane, releasing the proteins outside of the cell. Small molecules, such as drugs, can also be secreted by diffusion through the membrane to the outside of cell.

[0183] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes. 44 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0184] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0185] As used herein, “specifically binds” refers to a molecule (e.g., an antibody or antigen binding fragment thereof) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a KDfor the molecule to which it binds to of about 10−4M, 10−5M, 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, or 10−12M. The term “specifically binds” may also refer to binding where a molecule (e.g., an antibody or antigen binding fragment thereof) binds to a particular polypeptide (e.g., a LAMP5 polypeptide), or an epitope on a particular polypeptide, without substantially binding to any other polypeptide, or polypeptide epitope.

[0186] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0187] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, or a mammal and may include humans, non-human primates, rodents, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested). In certain embodiments, the individual, patient or subject is a human.

[0188] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0189] The terms “substantially homologous” or “substantially identical” mean a polypeptide or nucleic acid molecule that exhibits at least 50% or greater homology or identity 45 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). For example, such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% homologous or identical at the amino acid level or nucleic acid to the sequence used for comparison (e.g., a wild-type, or native, sequence). In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more amino acid substitutions, insertions, or deletions relative to the sequence used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more non-natural amino acids or amino acid analogs, including, D-amino acids and retroinverso amino, to replace homologous sequences.

[0190] Nucleic acid molecules useful in the presently disclosed subject matter include any nucleic acid molecule that encodes a polypeptide or a fragment thereof. In certain embodiments, nucleic acid molecules useful in the presently disclosed subject matter include nucleic acid molecules that encode an antibody or an antigen binding portion thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial homology” or “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger, Methods Enzymol.152:399 (1987); Kimmel, A. R. Methods Enzymol.152:507 (1987)). For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% w / v formamide, or at least about 50% w / v formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, 46 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In certain embodiments, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% w / v SDS. In certain embodiments, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% w / v SDS, 35% w / v formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In certain embodiments, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% w / v SDS, 50% w / v formamide, and 200 µg ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0191] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25°C, at least about 42°C, or at least about 68°C. In certain embodiments, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% w / v SDS. In certain embodiments, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% w / v SDS. In certain embodiments, wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% w / v SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180 (1977)); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA 72:3961 (1975)); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0192] As used herein, "synthetic," with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods. As used herein, production by recombinant means by using recombinant DNA methods means the use of the well-known methods of molecular biology for expressing 47 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 proteins encoded by cloned DNA.

[0193] As used herein, the term “T-cell” includes naïve T cells, CD4+T cells, CD8+T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEMcells and TEMRA cells), activated T cells, anergic T cells, tolerant T cells, chimeric B cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and γδ T cells, and antigen-specific T cells.

[0194] As used herein, “T cell receptor” or “TCR”, is a protein complex found on the surface of T cells, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex molecules. TCR is composed of two disulfide-linked protein chains. Cells expressing a TCR containing the highly variable alpha (α) and beta (β) chains are referred to as αβ T cells. Cells expressing an alternate TCR, formed by variable gamma (γ) and delta (δ) chains, are referred to as γδ T cells. When the TCR engages with antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors. In some embodiments, the TCR is a native T cell receptor that is endogenous to the immune cells. In some embodiments, the TCR is an artificial receptor that mimics native TCR function, i.e., recognizing peptide antigens of key intracellular proteins in the context of MHC on the cell surface.

[0195] As used herein “tumor-infiltrating immune cells” or “TILs” refer to immune cells that have left the bloodstream and migrated into a tumor.

[0196] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0197] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, 48 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By “treating a cancer” is meant that the symptoms associated with the cancer are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0198] It is also to be appreciated that the various modes of treatment of diseases as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0199] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0200] Amino acid sequence modification(s) of the anti-LAMP5 antibodies, antigen binding fragments, or CAR compositions described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the compositions. Amino acid sequence variants of the anti-LAMP5 antibodies, antigen binding fragments, or CAR are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, 49 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to obtain the compositions of interest, as long as the obtained composition possesses the desired properties. The modification also includes the change of the pattern of glycosylation of the protein. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. As used herein, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the presently disclosed antibodies, antigen binding fragments or CAR (e.g., the extracellular antigen binding domain of the CAR) comprising the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of the presently disclosed antibodies, antigen binding fragments or CAR by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (1) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence or a CDR region are altered. “Conservative substitutions” of amino acids are shown in Table B below. Table B. Amino Acid Substitutions OriginalExemplConservative Residueary SubstitutionsSubstitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) Ala ala His (H) asn; gln; lys; arg arg 50 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 Table B. Amino Acid Substitutions Original eExConservative Residuemplary SubstitutionsSubstitutions Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) Ala ala Ser (S) Thr thr Thr (T) Ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

[0201] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with similar or superior properties in one or more relevant assays may be selected for further development. 51 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 Immunoglobulin-related Compositions of the Present Technology

[0202] The present technology in part describes methods and compositions for the generation and use of anti-LAMP5 immunoglobulin-related compositions (e.g., anti-LAMP5 antibodies or antigen binding fragments thereof). The anti-LAMP5 immunoglobulin-related compositions of the present disclosure may be useful in the diagnosis, or treatment of cancer. Anti-LAMP5 immunoglobulin-related compositions within the scope of the present technology include, e.g., but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies and diabodies that specifically bind the target polypeptide, a homolog, derivative or a fragment thereof. The present disclosure also provides antigen binding fragments of any of the anti- LAMP5 antibodies disclosed herein, wherein the antigen binding fragment is selected from the group consisting of Fab, F(ab)'2, Fab’, scFv, and Fv. Disclosed herein are anti-LAMP5 antibodies or antigen binding fragments thereof comprising the VHCDR1-3 sequences of SEQ ID NO: 4; and the VLCDR1-3 sequences of SEQ ID NO: 11 as defined in accordance with Kabat, Chothia, IMGT, MacCallum or Aho numbering scheme. The CDR of the VHand VLof humanized LAMP5 antibody based on the Kabat annotation system are summarized below: RegionDefinitionSequence Fragment Residues LengthCDR-H1KabatGYTFTDFTI (SEQ ID NO: 5) 26-34 9CDR-H2 Kabat IIPNNGNA (SEQ ID NO: 6) 51-58 8 CDR-H3Kabat ARARYYFDY (SEQ ID NO: 7) 97-1059CDR-L1Kabat RSSSGAVTTSNSAN (SEQ ID NO: 12) 23-3614CDR-L2Kabat GTNNRPS (SEQ ID NO: 13 52-587CDR-L3Kabat ALWNSNHWV (SEQ ID NO: 14) 91-999

[0203] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein (a) the VHcomprises a VH-CDR1 sequence of GYTFTDFTI (SEQ ID NO: 5), a VH-CDR2 sequence of IIPNNGNA (SEQ ID NO: 6), and a VH-CDR3 sequence of ARARYYFDY (SEQ ID NO: 7); and / or; (b) the VLcomprises a VL-CDR1 sequence of RSSSGAVTTSNSAN (SEQ ID NO: 12), a VL-CDR2 sequence of GTNNRPS (SEQ ID NO: 13), and a VL-CDR3 sequence of ALWNSNHWV (SEQ ID NO: 14). 52 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0204] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein: (a) the VHcomprises SEQ ID NO: 4; and / or (b) the VLcomprises SEQ ID NO: 11.

[0205] In any of the above embodiments, the antibody further comprises a Fc domain of any isotype, e.g., but are not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1and IgA2), IgD, IgE, or IgM, and IgY. Non-limiting examples of constant region sequences include:

[0206] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 22) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRR DSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQ AEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTP AVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQH SRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAAS WLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQ PATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0207] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO: 23) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0208] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 24) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSRE 53 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 EMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0209] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 25) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPK SCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIF SCSVMHEALHNRFTQKSLSLSPGK

[0210] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 26) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVL RGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFV PPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTT YKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASI FLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICED DWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCL VTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGET YTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0211] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 27) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0212] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO: 28) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDAS GDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSP 54 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 SCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDL CGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSE ELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSI LRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0213] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 29) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDA SGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHR PALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLP GCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLT CLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWK KGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0214] Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 30) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0215] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or is 100% identical to SEQ ID NOS: 22-29. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or is 100% identical to SEQ ID NO: 30.

[0216] In some embodiments, the immunoglobulin-related compositions of the present technology bind to the extracellular domain of a LAMP5 polypeptide. In certain embodiments, the epitope is a conformational epitope or non-conformational epitope. In some embodiments, the LAMP5 polypeptide has the amino acid sequence of SEQ ID NO: 15.

[0217] NCBI Ref: NP_036393.1 Homo sapiens Lysosome-associated membrane glycoprotein 5 (LAMP5) (SEQ ID NO: 15): MDLQGRGVPSIDRLRVLLMLFHTMAQIMAEQEVENLSGLSTNPEKDIFVVRENGTTCL MAEFAAKFIVPYDVWASNYVDLITEQADIALTRGAEVKGRCGHSQSELQVFWVDRAY ALKMLFVKESHNMSKGPEATWRLSKVQFVYDSSEKTHFKDAVSAGKHTANSHHLSA 55 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 LVTPAGKSYECQAQQTISLASSDPQKTVTMILSAVHIQPFDIISDFVFSEEHKCPVDERE QLEETLPLILGLILGLVIMVTLAIYHVHHKMTANQVQIPRDRSQYKHMG

[0218] Additionally or alternatively, in some embodiments, the antibody or antigen binding fragment binds to the extracellular domain of a LAMP5 polypeptide. In certain embodiments, the extracellular domain comprises SEQ ID NO: 16.

[0219] LAMP5 predicted extracellular domain (SEQ ID NO: 16): EQEVENLSGLSTNPEKDIFVVRENGTTCLMAEFAAKFIVPYDVWASNYVDLITEQADIA LTRGAEVKGRCGHSQSELQVFWVDRAYALKMLFVKESHNMSKGPEATWRLSKVQFV YDSSEKTHFKDAVSAGKHTANSHHLSALVTPAGKSYECQAQQTISLASSDPQKTVTMI LSAVHIQPFDIISDFVFSEEHKCPVDEREQLEE

[0220] LAMP5 DNA sequence NM_012261 (SEQ ID NO: 17): GGATTGCTTTCAGCACTCGCAGCCGTGGACCGCCGTGCGGTCCTTTCCTCCGCAGTGAG CCGATTTGCTCTGCCAGCAGCTGTCGGTGCCGCGCTCGACACCGAGTCCTAGCTAGGCG CTCACAGAATACGCGCTCCCTCCCTCCCCCTTCTCTGTCCCCCGCCTCTCGCTCACCCC GGCCCACTCCAGCGGCGACTTTGAGGGATTCCCTCTCTGGCGGCCTCTGCAGCAGCACA GCCGGCCTCATTCGGGGCACTGCGAGTATGGATCTCCAAGGAAGAGGGGTCCCCAGCA TCGACAGACTTCGAGTTCTCCTGATGTTGTTCCATACAATGGCTCAAATCATGGCAGAACA AGAAGTGGAAAATCTCTCAGGCCTTTCCACTAACCCTGAAAAAGATATATTTGTGGTGCG GGAAAATGGGACGACGTGTCTCATGGCAGAGTTTGCAGCCAAATTTATTGTACCTTATGA TGTGTGGGCCAGCAACTACGTAGATCTGATCACAGAACAGGCCGATATCGCATTGACCC GGGGAGCTGAGGTGAAGGGCCGCTGTGGCCACAGCCAGTCGGAGCTGCAAGTGTTCTG GGTGGATCGCGCATATGCACTCAAAATGCTCTTTGTAAAGGAAAGCCACAACATGTCCAA GGGACCTGAGGCGACTTGGAGGCTGAGCAAAGTGCAGTTTGTCTACGACTCCTCGGAGA AAACCCACTTCAAAGACGCAGTCAGTGCTGGGAAGCACACAGCCAACTCGCACCACCTC TCTGCCTTGGTCACCCCCGCTGGGAAGTCCTATGAGTGTCAAGCTCAACAAACCATTTCA CTGGCCTCTAGTGATCCGCAGAAGACGGTCACCATGATCCTGTCTGCGGTCCACATCCA ACCTTTTGACATTATCTCAGATTTTGTCTTCAGTGAAGAGCATAAATGCCCAGTGGATGAG CGGGAGCAACTGGAAGAAACCTTGCCCCTGATTTTGGGGCTCATCTTGGGCCTCGTCAT CATGGTAACACTCGCGATTTACCACGTCCACCACAAAATGACTGCCAACCAGGTGCAGAT CCCTCGGGACAGATCCCAGTATAAGCACATGGGCTAGAGGCCGTTAGGCAGGCACCCC CTATTCCTGCTCCCCCAACTGGATCAGGTAGAACAACAAAAGCACTTTTCCATCTTGTACA CGAGATACACCAACATAGCTACAATCAAACAGGCCTGGGTATCTGAGGCTTGCTTGGCTT 56 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 GTGTCCATGCTTAAACCCACGGAAGGGGGAGACTCTTTCGGATTTGTAGGGTGAAATGG CAATTATTCTCTCCATGCTGGGGAGGAGGGGAGGAGGGTCTCAGACAGCTTTCGTGCTC ATGGTGGCTTGGCTTTGACTCTCCAAAGAGCAATAAATGCCACTTGGAGCTGTATCTGGC CCCAAAGTTTAGGGATTGAAAACATGCTTCTTTGAGGAGGAAACCCCTTTAGGTTCAGAA GAATATGGGGTGCTTTGCTCCCTTGGACACAGCTGGCTTATCCTATACAGTTGTCAATGC ACACAGAATACAACCTCATGCTCCCTGCAGCAAGACCCCTGAAAGTGATTCATGCTTCTG GCTGGCATTCTGCATGTTTAGTGATTGTCTTGGGAATGTTTCACTGCTACCCGCATCCAG CGACTGCAGCACCAGAAAACGACTAATGTAACTATGCAGAGTTGTTTGGACTTCTTCCTG TGCCAGGTCCAAGTCGGGGGACCTGAAGAATCAATCTGTGTGAGTCTGTTTTTCAAAATG AAATAAAACACACTATTCTCTGGC (SEQ ID NO: 17)

[0221] In another aspect, the present disclosure provides an isolated immunoglobulin- related composition (e.g., an antibody or antigen binding fragment thereof) comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO: 2, or a variant thereof having one or more conservative amino acid substitutions. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO: 9, or a variant thereof having one or more conservative amino acid substitutions. In some embodiments, the immunoglobulin-related compositions of the present technology comprise a HC amino acid sequence and a LC amino acid sequence comprising SEQ ID NO: 2 and SEQ ID NO: 9.

[0222] In any of the above embodiments of the immunoglobulin-related compositions, the HC and LC immunoglobulin variable domain sequences form an antigen binding site that binds to the extracellular domain of a LAMP5 polypeptide. In certain embodiments, the extracellular domain comprises SEQ ID NO: 16. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope.

[0223] In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0224] In some embodiments, the immunoglobulin-related compositions of the present technology bind specifically to at least one LAMP5 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology bind at least one LAMP5 57 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 polypeptide with a dissociation constant (KD) of about 10−3M, 10−4M, 10−5M, 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, or 10−12M. In certain embodiments, the immunoglobulin- related compositions are monoclonal antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multi-specific antibodies. In some embodiments, the antibodies comprise a human antibody framework region.

[0225] In certain embodiments, the immunoglobulin-related composition includes one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 11; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 4. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a “conservative substitution” as defined herein.

[0226] In another aspect, the present disclosure provides an antibody comprising (a) a LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the LC sequence present in SEQ ID NO: 9; and / or (b) a HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the HC sequence present in SEQ ID NO: 2.

[0227] Additionally or alternatively, in some embodiments, the multi-specific antibodies of the present disclosure bind to LAMP5, CD3, GPA33, HER2 / neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N-acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC- 17, tyrosinase, Pmel 17 (gp100), GnT-V intron V sequence (N- acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, LMP2, p53, lung resistance protein (LRP), Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), HLA- DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, PlGF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth factor, IL-6, CD20, CD19, PSMA, CD33, CD123, 58 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis Y (Ley) antigen, E-cadherin, V-cadherin, GPC3, EpCAM, CD4, CD8, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, CD56, DLL3, PD-1, PD-L1, CD28, CD137, CD99, GloboH, CD24, STEAP1, B7H3, Polysialic Acid, OX40, OX40-ligand, or peptide MHC complexes (with peptides derived from TP53, KRAS, MYC, EBNA1-6, PRAME, MART, tyronsinase, MAGEA1-A6, pmel17, LMP2, or WT1).

[0228] In some aspects, the anti-LAMP5 immunoglobulin-related compositions described herein contain structural modifications to facilitate rapid binding and cell uptake and / or slow release. In some aspects, the anti-LAMP5 immunoglobulin-related composition of the present technology (e.g., an antibody) may contain a deletion in the CH2 constant heavy chain region to facilitate rapid binding and cell uptake and / or slow release. In some aspects, a Fab fragment is used to facilitate rapid binding and cell uptake and / or slow release. In some aspects, a F(ab)'2fragment is used to facilitate rapid binding and cell uptake and / or slow release.

[0229] In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1 and 8.

[0230] In another aspect, the present technology provides a host cell expressing any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0231] The immunoglobulin-related compositions of the present technology (e.g., an anti- LAMP5 antibody) can be monospecific, bispecific, trispecific or of greater multi-specificity. Multi-specific antibodies can be specific for different epitopes of one or more LAMP5 polypeptides or can be specific for both the LAMP5 polypeptide(s) as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g., WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol.147: 60- 69 (1991); U.S. Pat. Nos.5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol.148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related compositions are chimeric. In certain embodiments, the immunoglobulin-related compositions are humanized. 59 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0232] The immunoglobulin-related compositions of the present technology can further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Pat. No.5,314,995; and EP 0396387.

[0233] In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen binding fragment may be optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof. For a chemical bond or physical bond, a functional group on the immunoglobulin- related composition typically associates with a functional group on the agent. Alternatively, a functional group on the agent associates with a functional group on the immunoglobulin-related composition. In some embodiments, the immunoglobulin-related compositions of the present technology may be conjugated to at least one chemotherapeutic agent, optionally selected from the group consisting of cytarabine (cytosine arabinoside or ara-C), anthracycline drugs (such as daunorubicin (daunomycin) or idarubicin), cladribine (2-CdA), fludarabine, mitoxantrone, etoposide (VP-16), 6-thioguanine (6-TG), hydroxyurea, corticosteroid drugs (e.g., prednisone or dexamethasone), methotrexate (MTX), 6-mercaptopurine (6-MP), azacytidine, decitabine, Monomethyl auristatin E (MMAE) and Tesirine. In some embodiments the at least one chemotherapeutic agent is MMAE or Tesirine.

[0234] The functional groups on the agent and immunoglobulin-related composition can associate directly. For example, a functional group (e.g., a sulfhydryl group) on an agent can associate with a functional group (e.g., sulfhydryl group) on an immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can associate through a cross-linking agent (i.e., linker). Some examples of cross-linking agents are described below. The cross-linker can be attached to either the agent or the immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions in a conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of 60 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 agents associated with a conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with an agent depends on the number of functional groups present on the agent.

[0235] In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated to one agent. In one embodiment, a conjugate comprises at least one agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bonded to an immunoglobulin-related composition by any method known to those in the art. For example, a functional group on the agent may be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate and hydroxyl.

[0236] The agent may also be chemically bonded to the immunoglobulin-related composition by means of cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can, for example, be obtained from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. web-site can provide assistance. Additional cross-linking agents include the platinum cross-linking agents described in U.S. Pat. Nos.5,580,990; 5,985,566; and 6,133,038 of Kreatech Biotechnology, B.V., Amsterdam, The Netherlands.

[0237] Alternatively, the functional group on the agent and immunoglobulin-related composition can be the same. Homobifunctional cross-linkers are typically used to cross-link identical functional groups. Examples of homobifunctional cross-linkers include EGS (i.e., ethylene glycol bis[succinimidylsuccinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidylpropionate])), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis- maleimidohexane). Such homobifunctional cross-linkers are also available from Pierce Biotechnology, Inc.

[0238] In other instances, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The web-site of Pierce Biotechnology, Inc. described above can also provide assistance to one skilled in the art in choosing suitable cross-linkers 61 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 which can be cleaved by, for example, enzymes in the cell. Thus the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), Sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), Sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2- aminoethyl)dithio]propionic acid HCl).

[0239] In another embodiment, a conjugate comprises at least one agent physically bonded with at least one immunoglobulin-related composition. Any method known to those in the art can be employed to physically bond the agents with the immunoglobulin-related compositions. For example, the immunoglobulin-related compositions and agents can be mixed together by any method known to those in the art. The order of mixing is not important. For instance, agents can be physically mixed with immunoglobulin-related compositions by any method known to those in the art. For example, the immunoglobulin-related compositions and agents can be placed in a container and agitated, by for example, shaking the container, to mix the immunoglobulin-related compositions and agents.

[0240] The immunoglobulin-related compositions can be modified by any method known to those in the art. For instance, the immunoglobulin-related composition may be modified by means of cross-linking agents or functional groups, as described above. A. Methods of Preparing Anti-LAMP5 Antibodies of the Present Technology

[0241] General Overview. Initially, a target polypeptide is chosen to which an antibody of the present technology can be raised. For example, an antibody may be raised against the full- length LAMP5 protein, or to a portion of the extracellular domain of the LAMP5 protein. Techniques for generating antibodies directed to such target polypeptides are well known to those skilled in the art. Examples of such techniques include, for example, but are not limited to, those involving display libraries, xeno or human mice, hybridomas, and the like. Target polypeptides within the scope of the present technology include any polypeptide derived from LAMP5 protein containing the extracellular domain which is capable of eliciting an immune 62 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 response. In certain embodiments, the extracellular domain comprises SEQ ID NO: 16, or a portion thereof.

[0242] It should be understood that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, which are directed to LAMP5 protein and fragments thereof are suitable for use in accordance with the present disclosure.

[0243] Anti-LAMP5 antibodies that can be subjected to the techniques set forth herein include monoclonal and polyclonal antibodies, and antibody fragments such as Fab, Fab′, F(ab′)2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains and / or antibody fragments. Methods useful for the high yield production of antibody Fv-containing polypeptides, e.g., Fab′ and F(ab′)2antibody fragments have been described. See U.S. Pat. No. 5,648,237.

[0244] Generally, an antibody is obtained from an originating species. More particularly, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain or both, of an originating species antibody having specificity for a target polypeptide antigen is obtained. An originating species is any species which was useful to generate the antibody of the present technology or library of antibodies, e.g., rat, mouse, rabbit, chicken, monkey, human, and the like.

[0245] Phage or phagemid display technologies are useful techniques to derive the antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology, can be carried out in E. coli.

[0246] Due to the degeneracy of nucleic acid coding sequences, other sequences which encode substantially the same amino acid sequences as those of the naturally occurring proteins may be used in the practice of the present technology These include, but are not limited to, nucleic acid sequences including all or portions of the nucleic acid sequences encoding the above polypeptides, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. It is appreciated that the nucleotide sequence of an immunoglobulin according to the present technology tolerates sequence homology variations of up to 25% as calculated by standard methods (“Current Methods in Sequence Comparison and Analysis,” Macromolecule 63 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 Sequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1998, Alan R. Liss, Inc.) so long as such a variant forms an operative antibody which recognizes LAMP5 proteins. For example, one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligands, etc. Additionally, an immunoglobulin encoding nucleic acid sequence can be mutated in vitro or in vivo to create and / or destroy translation, initiation, and / or termination sequences or to create variations in coding regions and / or form new restriction endonuclease sites or destroy pre-existing ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to in vitro site directed mutagenesis, J. Biol. Chem.253:6551, use of Tab linkers (Pharmacia), and the like.

[0247] Preparation of Polyclonal Antisera and Immunogens. Methods of generating antibodies or antibody fragments of the present technology typically include immunizing a subject (generally a non-human subject such as a mouse or rabbit) with a purified LAMP5 protein or fragment thereof or with a cell expressing the LAMP5 protein or fragment thereof. An appropriate immunogenic preparation can contain, e.g., a recombinantly-expressed LAMP5 protein or a chemically-synthesized LAMP5 peptide. The extracellular domain of the LAMP5 protein, or a portion or fragment thereof, can be used as an immunogen to generate an anti- LAMP5 antibody that binds to the LAMP5 protein, or a portion or fragment thereof using standard techniques for polyclonal and monoclonal antibody preparation. In certain embodiments, the extracellular domain comprises a SEQ ID NO: 16 or a portion thereof. In some embodiments, the antigenic LAMP5 peptide comprises at least 10, at least 20, at least 30, 64 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 amino acid residues, at least 120 amino acid residues, at least 140 amino acid residues, at least 160 amino acid residues, at least 180 amino acid residues, or at least 200 amino acid residues. Longer antigenic peptides are sometimes desirable over shorter antigenic peptides, depending on use and according to methods well known to those skilled in the art. Multimers of a given epitope are sometimes more effective than a monomer.

[0248] If needed, the immunogenicity of the LAMP5 protein (or fragment thereof) can be increased by fusion or conjugation to a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. One can also combine the LAMP5 protein with a conventional adjuvant such as Freund’s complete or incomplete adjuvant to increase the subject’s immune reaction to the polypeptide. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0249] In describing the present technology, immune responses may be described as either “primary” or “secondary” immune responses. A primary immune response, which is also described as a “protective” immune response, refers to an immune response produced in an individual as a result of some initial exposure (e.g., the initial “immunization”) to a particular antigen, e.g., LAMP5 protein. In some embodiments, the immunization can occur as a result of vaccinating the individual with a vaccine containing the antigen. For example, the vaccine can be a LAMP5 vaccine comprising one or more LAMP5 protein-derived antigens. A primary immune response can become weakened or attenuated over time and can even disappear or at least become so attenuated that it cannot be detected. Accordingly, the present technology also relates to a “secondary” immune response, which is also described here as a “memory immune response.” The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has already been produced.

[0250] Thus, a secondary immune response can be elicited, e.g., to enhance an existing immune response that has become weakened or attenuated, or to recreate a previous immune 65 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 response that has either disappeared or can no longer be detected. The secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells that were generated at the first presentation of the antigen. Delayed type hypersensitivity (DTH) reactions are a type of cellular secondary or memory immune response that are mediated by CD4+T cells. A first exposure to an antigen primes the immune system and additional exposure(s) results in a DTH.

[0251] Following appropriate immunization, the anti-LAMP5 antibody can be prepared from the subject’s serum. If desired, the antibody molecules directed against the LAMP5 protein can be isolated from the mammal (e.g., from the blood) and further purified by well- known techniques, such as polypeptide A chromatography to obtain the IgG fraction.

[0252] Monoclonal Antibody. In one embodiment of the present technology, the antibody is an anti-LAMP5 monoclonal antibody. For example, in some embodiments, the anti-LAMP5 monoclonal antibody may be a human or a mouse anti-LAMP5 monoclonal antibody. For preparation of monoclonal antibodies directed towards the LAMP5 protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (See, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (See, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be utilized in the practice of the present technology and can be produced by using human hybridomas (See, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). For example, a population of nucleic acids that encode regions of antibodies can be isolated. PCR utilizing primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding portions of antibodies from the population and then DNAs encoding antibodies or fragments thereof, such as variable domains, are reconstructed from the amplified sequences. Such amplified sequences also can be fused to DNAs encoding other 66 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 proteins − e.g., a bacteriophage coat, or a bacterial cell surface protein − for expression and display of the fusion polypeptides on phage or bacteria. Amplified sequences can then be expressed and further selected or isolated based, e.g., on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on the LAMP5 protein. Alternatively, hybridomas expressing anti-LAMP5 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject’s spleen using routine methods. See, e.g., Milstein et al., (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods will produce monoclonal antibodies of varying specificity (i.e., for different epitopes) and affinity. A selected monoclonal antibody with the desired properties, e.g., LAMP5 binding, can be used as expressed by the hybridoma, it can be bound to a molecule such as polyethylene glycol (PEG) to alter its properties, or a cDNA encoding it can be isolated, sequenced and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains, e.g., lysine, to enhance the immunogenic properties of LAMP5 protein. Also, CPG-dinucleotide techniques can be used to enhance the immunogenic properties of the LAMP5 protein. Other manipulations include substituting or deleting particular amino acyl residues that contribute to instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve affinity of the antibody of the LAMP5 protein.

[0253] Hybridoma Technique. In some embodiments, the antibody of the present technology is an anti-LAMP5 monoclonal antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma techniques include those known in the art and taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art.

[0254] Phage Display Technique. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology. For example, anti-LAMP5 antibodies, can be prepared using various phage display methods 67 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. Phages with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with an antigen, typically an antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either the phage gene III or gene VIII protein. In addition, methods can be adapted for the construction of Fab expression libraries (See, e.g., Huse, et al., Science 246: 1275-1281, 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a LAMP5 polypeptide, e.g., a polypeptide or derivatives, fragments, analogs or homologs thereof. Other examples of phage display methods that can be used to make the antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol.24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994; PCT / GB91 / 01134; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et al.); WO 97 / 08320 (Morphosys); WO 92 / 01047 (CAT / MRC); WO 91 / 17271 (Affymax); and U.S. Pat. Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. Methods useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides via disulfide bonds have been described by Lohning, U.S. Pat. No. 6,753,136. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2fragments can also be employed using methods known in the art such as those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988. 68 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0255] Generally, hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and / or screening.

[0256] Single-Chain Antibodies. In one embodiment, the anti-LAMP5 antibody of the present technology is a single-chain anti-LAMP5 antibody. According to the present technology, techniques can be adapted for the production of single-chain antibodies specific to a LAMP5 protein (See, e.g., U.S. Pat. No.4,946,778). Examples of techniques which can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. Nos.4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0257] Chimeric and Humanized Antibodies. In one embodiment, the anti-LAMP5 antibody of the present technology is a chimeric anti-LAMP5 antibody. In one embodiment, the anti-LAMP5 antibody of the present technology is a humanized anti-LAMP5 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody.

[0258] Recombinant anti-LAMP5 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques, and are within the scope of the present technology. For some uses, including in vivo use of the anti-LAMP5 antibody of the present technology in humans as well as use of these agents in in vitro detection assays, it is possible to use chimeric or humanized anti-LAMP5 antibodies. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, 69 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 e.g., but are not limited to, methods described in International Application No. PCT / US86 / 02269; U.S. Pat. No.5,225,539; European Patent No.184187; European Patent No. 171496; European Patent No.173494; PCT International Publication No. WO 86 / 01533; U.S. Pat. Nos.4,816,567; 5,225,539; European Patent No.125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol.139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res.47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst.80: 1553-1559; Morrison (1985) Science 229: 1202- 1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988. Science 239: 1534; Morrison, Science 229: 1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Pat. No.5,807,715; and Beidler, et al., 1988. J. Immunol.141: 4053-4060. For example, antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0239400; WO 91 / 09967; U.S. Pat. No.5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0592106; EP 0519596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain shuffling (U.S. Pat. No.5,565,332). In one embodiment, a cDNA encoding a murine anti-LAMP5 monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted (See Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No.4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521- 3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80: 1553-1559; U.S. Pat. No.6,180,370; U.S. Pat. Nos.6,300,064; 6,696,248; 6,706,484; 6,828,422.

[0259] In one embodiment, the present technology provides the construction of humanized anti-LAMP5 antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter 70 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 referred to as “HAMA”) response, while still having an effective antibody effector function. As used herein, the terms “human” and “humanized”, in relation to antibodies, relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides for a humanized anti- LAMP5 antibodies, heavy and light chain immunoglobulins.

[0260] CDR Antibodies. In some embodiments, the anti-LAMP5 antibody of the present technology is an anti-LAMP5 CDR antibody. Generally the donor and acceptor antibodies used to generate the anti-LAMP5 CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody. The graft may be of a single CDR (or even a portion of a single CDR) within a single VHor VLof the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the VHand VL. Frequently, all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, though one needs to replace only as many as necessary to permit adequate binding of the resulting CDR-grafted antibody to LAMP5 protein. Methods for generating CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Pat. No.5,585,089; U.S. Pat. No.5,693,761; U.S. Pat. No.5,693,762; and Winter U.S.5,225,539; and EP 0682040. Methods useful to prepare VHand VLpolypeptides are taught by Winter et al., U.S. Pat. Nos.4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP0451216; and EP0120694.

[0261] After selecting suitable framework region candidates from the same family and / or the same family member, either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs from the originating species) can be produced by oligonucleotide synthesis and / or PCR. The nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with 71 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 suitable ligation enzymes. Alternatively, the framework regions of the variable chains of the originating species antibody can be changed by site-directed mutagenesis.

[0262] Since the hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0263] This process typically does not alter the acceptor antibody’s FRs flanking the grafted CDRs. However, one skilled in the art can sometimes improve antigen binding affinity of the resulting anti-LAMP5 CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (See, e.g., US 5,585,089, especially columns 12-16). Or one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or a human consensus FR. Techniques for making these modifications are known in the art. Particularly if the resulting FR fits a human consensus FR for that position, or is at least 90% or more identical to such a consensus FR, doing so may not increase the antigenicity of the resulting modified anti-LAMP5 CDR-grafted antibody significantly compared to the same antibody with a fully human FR.

[0264] Bispecific Antibodies (BsAbs). A bispecific antibody is an antibody that can bind simultaneously to two targets that have a distinct structure, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or epitope on a target antigen. BsAbs can be made, for example, by combining heavy chains and / or light chains that recognize different epitopes of the same or different antigen. In some embodiments, by molecular function, a bispecific binding agent binds one antigen (or epitope) on one of its two binding arms (one VH / VL pair), and binds a different antigen (or epitope) on its second arm (a different VH / VL pair). By this definition, a bispecific binding agent has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds. 72 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0265] Multi-specific antibodies, such as bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) have at least one arm that specifically binds to, for example, LAMP5 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B-cell, a T-cell, a myeloid cell, a plasma cell, or a mast-cell. Additionally or alternatively, in certain embodiments, the second target antigen is selected from the group consisting of CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46 and KIR. In certain embodiments, the BsAbs are capable of binding to cancer cells that express LAMP5 antigen on the cell surface. In some embodiments, the BsAbs have been engineered to facilitate killing of cancer cells by directing (or recruiting) cytotoxic T cells to a cancer site.

[0266] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed that either utilize the full immunoglobulin framework (e.g., IgG), single chain variable fragment (scFv), or combinations thereof. In some embodiments, the bispecific fusion protein is divalent, comprising, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is divalent, comprising, for example, an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs composed of two scFv units in tandem have been shown to be a clinically successful bispecific antibody format. In some embodiments, BsAbs comprise two single chain variable fragments (scFvs) in tandem have been designed such that an scFv that binds a cancer antigen (e.g., LAMP5) is linked with an scFv that engages T cells (e.g., by binding CD3). In this way, T cells are recruited to a cancer site such that they can mediate cytotoxic killing of the cancer cells. See e.g., Dreier et al., J. Immunol.170:4397-4402 (2003); Bargou et al., Science 321 :974- 977 (2008)).

[0267] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins linking 73 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g., Coloma et al., Nature Biotech.15:159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0268] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some certain embodiments, a BsAb according to the present technology comprises an immunoglobulin, which immunoglobulin comprises a heavy chain and a light chain, and an scFv. In some certain embodiments, the scFv is linked to the C- terminal end of the heavy chain of any LAMP5 immunoglobulin disclosed herein. In some certain embodiments, scFvs are linked to the C-terminal end of the light chain of any LAMP5 immunoglobulin disclosed herein. In various embodiments, scFvs are linked to heavy or light chains via a linker sequence. Appropriate linker sequences necessary for the in-frame connection of the heavy chain Fd to the scFv are introduced into the VLand Vkappadomains through PCR reactions. The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and ligated into a vector containing a DNA sequence encoding the VHregion of a LAMP5 antibody. The resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell for the expression of the bispecific fusion protein.

[0269] In some embodiments, a linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., first and / or second antigen binding sites). In some embodiments, a linker is employed in a BsAb described herein based on specific properties imparted to the BsAb such as, for example, an increase in stability. In some embodiments, a BsAb of the present technology comprises a G4S linker. In some certain embodiments, a BsAb of the present technology comprises a (G4S)nlinker (SEQ ID NO: 20), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0270] Fc Modifications. In some embodiments, the anti-LAMP5 antibodies of the present technology comprise a variant Fc region, wherein said variant Fc region comprises at least one 74 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 amino acid modification relative to a wild-type Fc region (or the parental Fc region), such that said molecule has an altered affinity for an Fc receptor (e.g., an FcγR), provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make a direct contact with an Fc receptor such as an FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G) loop.

[0271] In some embodiments, an anti-LAMP5 antibody of the present technology has an altered affinity for activating and / or inhibitory receptors, having a variant Fc region with one or more amino acid modifications, wherein said one or more amino acid modification is a N297 substitution with alanine, a S228 substitution with proline, or a K322 substitution with alanine.

[0272] Glycosylation Modifications. In some embodiments, anti-LAMP5 antibodies of the present technology have an Fc region with variant glycosylation as compared to a parent Fc region. In some embodiments, variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation results from expression in GnT1-deficient CHO cells.

[0273] In some embodiments, the antibodies of the present technology, may have a modified glycosylation site relative to an appropriate reference antibody that binds to an antigen of interest (e.g., LAMP5), without altering the functionality of the antibody, e.g., binding activity to the antigen. As used herein, "glycosylation sites" include any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach.

[0274] Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine. For example, an Fc-glycoform (hLAMP5-IgGln) that lacks certain oligosaccharides including fucose and terminal N- acetylglucosamine may be produced in special CHO cells and exhibit enhanced ADCC effector function. 75 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0275] In some embodiments, the carbohydrate content of an immunoglobulin-related composition disclosed herein is modified by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, e.g., U.S. Patent No.6,218,149; EP 0359096B1; U.S. Patent Publication No. US 2002 / 0028486; International Patent Application Publication WO 03 / 035835; U.S. Patent Publication No.2003 / 0115614; U.S. Patent No.6,218,149; U.S. Patent No.6,472,511; all of which are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some certain embodiments, the present technology includes deleting the glycosylation site of the Fc region of an antibody, by modifying position 297 from asparagine to alanine.

[0276] Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al., 1999, Nat. Biotechnol.17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem.277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem.278:3466-3473; U.S. Patent No.6,602,684; U.S. Patent Application Serial No. 10 / 277,370; U.S. Patent Application Serial No.10 / 113,929; International Patent Application Publications WO 00 / 61739A1 ; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication WO 00 / 061739; U.S. Patent Application Publication No.2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49. 76 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0277] Fusion Proteins. In one embodiment, the anti-LAMP5 antibody of the present technology is a fusion protein. The anti-LAMP5 antibodies of the present technology, when fused to a second protein, can be used as an antigenic tag. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but can occur through linker sequences. Moreover, fusion proteins of the present technology can also be engineered to improve characteristics of the anti-LAMP5 antibodies. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the anti-LAMP5 antibody to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties can be added to an anti- LAMP5 antibody to facilitate purification. Such regions can be removed prior to final preparation of the anti-LAMP5 antibody. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art. The anti-LAMP5 antibody of the present technology can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In select embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0278] Thus, any of these above fusion proteins can be engineered using the polynucleotides or the polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein show an increased half-life in vivo.

[0279] Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can be more efficient in binding and neutralizing other molecules compared to the monomeric secreted protein or protein fragment alone. Fountoulakis et al., J. Biochem.270: 3958-3964, 1995.

[0280] Similarly, EP-A-O 464533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or a fragment thereof. In many cases, the Fc part in a fusion 77 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties. See EP-A 0232262. Alternatively, deleting or modifying the Fc part after the fusion protein has been expressed, detected, and purified, may be desired. For example, the Fc portion can hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, e.g., human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0281] Labeled Anti-LAMP5 antibodies. In one embodiment, the anti-LAMP5 antibody of the present technology is coupled with a label moiety, i.e., detectable group. The particular label or detectable group conjugated to the anti-LAMP5 antibody is not a critical aspect of the technology, so long as it does not significantly interfere with the specific binding of the anti- LAMP5 antibody of the present technology to the LAMP5 protein. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,3H,14C,35S,125I,121I,131I,112In,99mTc), other imaging agents such as microbubbles (for ultrasound imaging),18F,11C,15O,89Zr (for Positron emission tomography),99mTC,111In (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads. Patents that describe the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6thEd., Molecular Probes, Inc., Eugene OR.).

[0282] The label can be coupled directly or indirectly to the desired component of an assay according to methods well known in the art. As indicated above, a wide variety of labels can 78 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 be used, with the choice of label depending on factors such as required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[0283] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti- ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally-occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-LAMP5 antibody.

[0284] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties, include, but are not limited to, e.g., fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties, include, but are not limited to, e.g., luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal-producing systems which can be used, see U.S. Pat. No.4,391,904.

[0285] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be detected simply by observing the color associated with the 79 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0286] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies, e.g., the anti- LAMP5 antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection. B. Identifying and Characterizing the Anti-LAMP5 Antibodies of the Present Technology

[0287] Methods for identifying and / or screening the anti-LAMP5 antibodies of the present technology. Methods useful to identify and screen antibodies against LAMP5 polypeptides for those that possess the desired specificity to LAMP5 protein (e.g., those that bind to the extracellular domain of LAMP5 protein, include any immunologically-mediated techniques known within the art. Components of an immune response can be detected in vitro by various methods that are well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and the lysis of these target cells detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0288] Similarly, products of an immune response in either a model organism (e.g., mouse) or a human subject can also be detected by various methods that are well known to those of ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., an ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over 80 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 scratch site (Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reaction can be measured using3H-thymidine; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells together with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of the PBMCs expressing these markers.

[0289] In one embodiment, anti-LAMP5 antibodies of the present technology are selected using display of LAMP5 peptides on the surface of replicable genetic packages. See, e.g., U.S. Pat. Nos.5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585287; EP 605522; EP 616640; EP 1024191; EP 589877; EP 774511; EP 844 306. Methods useful for producing / selecting a filamentous bacteriophage particle containing a phagemid genome encoding for a binding molecule with a desired specificity has been described. See, e.g., EP 774511; US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0290] In some embodiments, anti-LAMP5 antibodies of the present technology are selected using display of LAMP5 peptides on the surface of a yeast host cell. Methods useful for the isolation of scFv polypeptides by yeast surface display have been described by Kieke et al., Protein Eng.1997 Nov; 10(11): 1303-10.

[0291] In some embodiments, anti-LAMP5 antibodies of the present technology are selected using ribosome display. Methods useful for identifying ligands in peptide libraries using ribosome display have been described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

[0292] In certain embodiments, anti-LAMP5 antibodies of the present technology are selected using tRNA display of LAMP5 peptides. Methods useful for in vitro selection of ligands using tRNA display have been described by Merryman et al., Chem. Biol., 9: 741-46, 2002.

[0293] In one embodiment, anti-LAMP5 antibodies of the present technology are selected using RNA display. Methods useful for selecting peptides and proteins using RNA display libraries have been described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 81 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods useful for selecting peptides and proteins using unnatural RNA display libraries have been described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0294] In some embodiments, anti-LAMP5 antibodies of the present technology are expressed in the periplasm of gram negative bacteria and mixed with labeled LAMP5 protein. See WO 02 / 34886. In clones expressing recombinant polypeptides with affinity for LAMP5 protein, the concentration of the labeled LAMP5 protein bound to the anti-LAMP5 antibodies is increased and allows the cells to be isolated from the rest of the library as described in Harvey et al., Proc. Natl. Acad. Sci.22: 9193-982004 and U.S. Pat. Publication No. 2004 / 0058403.

[0295] After selection of the desired anti-LAMP5 antibodies, it is contemplated that said antibodies can be produced in large volume by any technique known to those skilled in the art, e.g., prokaryotic or eukaryotic cell expression and the like. The anti-LAMP5 antibodies which are, e.g., but not limited to, anti-LAMP5 hybrid antibodies or fragments can be produced by using conventional techniques to construct an expression vector that encodes an antibody heavy chain in which the CDRs and, if necessary, a minimal portion of the variable region framework, that are required to retain original species antibody binding specificity (as engineered according to the techniques described herein) are derived from the originating species antibody and the remainder of the antibody is derived from a target species immunoglobulin which can be manipulated as described herein, thereby producing a vector for the expression of a hybrid antibody heavy chain.

[0296] Measurement of LAMP5 Binding. In some embodiments, a LAMP5 binding assay refers to an assay format wherein LAMP5 protein and an anti-LAMP5 antibody are mixed under conditions suitable for binding between the LAMP5 protein and the anti-LAMP5 antibody and assessing the amount of binding between the LAMP5 protein and the anti- LAMP5 antibody. The amount of binding is compared with a suitable control, which can be the amount of binding in the absence of the LAMP5 protein, the amount of the binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assay methods include, e.g., ELISA, radioimmunoassays, scintillation proximity assays, fluorescence energy transfer assays, liquid 82 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 chromatography, membrane filtration assays, and the like. Biophysical assays for the direct measurement of LAMP5 protein binding to anti-LAMP5 antibody are, e.g., nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chips) and the like. Specific binding is determined by standard assays known in the art, e.g., radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy and the like. If the specific binding of a candidate anti-LAMP5 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-LAMP5 antibody, the candidate anti-LAMP5 antibody is useful as an anti-LAMP5 antibody of the present technology. Adoptive Cell Therapy (ACT)

[0297] CAR T cell therapy has gained momentum after several promising clinical trials for the treatment of B-cell neoplasms and the FDA approval of a CD19 targeted CAR T cell for treatment of B cell acute lymphoid leukemia (Sadelain et al., Nature 545:423-431 (2017); Yu et al., J Hematol Oncol.10:78 (2017); Kakarla and Gottschalk, Cancer J.20:151-155 (2014); Wang et al., J Hematol Oncol.10:53 (2017)). CAR T cell therapy involves isolating a patient’s own T cells, engineering them to express a CAR, and reinfusing the engineered T cells back into the patient. The CAR contains an extracellular single-chain variable fragment (scFv), a transmembrane domain, and an intracellular signaling domain. Surface expression of a tumor- targeted scFv on the T cell results in cancer antigen-directed T cell activation and specific cancer killing via its signaling domain. However, many patients with hematologic cancers treated with CAR T cell therapy relapse with antigen loss variants as a result of cancer editing (Wang et al., J Hematol Oncol.10:53 (2017)). Furthermore, translation of CAR T cell therapy to solid tumors has been difficult due to the immunosuppressive tumor environment (TME) (Yu et al., J Hematol Oncol.10:78 (2017); Kakarla and Gottschalk, Cancer J.20:151-155 (2014)).

[0298] In some embodiments, the engineered immune cells provided herein express a receptor or other cell-surface ligand that binds to a LAMP5 antigen. In some embodiments, the receptor is a wild-type or native receptor. In some embodiments, the receptor is an engineered receptor or a non-native receptor. In some embodiments, the engineered receptor is an engineered TCR (eTCR). In some embodiments, the engineered receptor is a chimeric 83 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 antibody TCR (caTCR). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR).

[0299] In exemplary embodiments, the engineered immune cells provided herein express a native receptor, a non-native receptor, or an engineered receptor (e.g., a CAR, caTCR, or eTCR) or other cell-surface ligand that binds to a LAMP5 antigen. A. Chimeric Antigen Receptors

[0300] In some embodiments, the engineered immune cells provided herein express at least one chimeric antigen receptor (CAR). CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell. For example, CARs can be used to graft the specificity of a monoclonal antibody onto an immune cell, such as a T cell. In some embodiments, transfer of the coding sequence of the CAR is facilitated by nucleic acid vector, such as a retroviral vector.

[0301] There are currently three generations of CARs. In some embodiments, the engineered immune cells provided herein express a “first generation” CAR. “First generation” CARs are typically composed of an extracellular antigen binding domain (e.g., a single-chain variable fragment (scFv)) fused to a transmembrane domain fused to cytoplasmic / intracellular domain of the T cell receptor (TCR) chain. “First generation” CARs typically have the intracellular domain from the CD3ζ chain, which is the primary transmitter of signals from endogenous TCRs. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0302] In some embodiments, the engineered immune cells provided herein express a “second generation” CAR. “Second generation” CARs add intracellular domains from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. “Second generation” CARs comprise those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (e.g., CD3ζ). Preclinical studies have indicated that “Second Generation” CARs can improve the antitumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was 84 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL).

[0303] In some embodiments, the engineered immune cells provided herein express a “third generation” CAR. “Third generation” CARs comprise those that provide multiple co- stimulation (e.g., CD28 and 4-1BB) and activation (e.g., CD3ζ).

[0304] In accordance with the presently disclosed subject matter, the CARs of the engineered immune cells provided herein comprise an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain. Further, the activity of the engineered immune cells can be adjusted by selection of co-stimulatory molecules included in the chimeric antigen receptor.

[0305] Extracellular Antigen-Binding Domain of a CAR. In certain embodiments, the extracellular antigen-binding domain of a CAR specifically binds a LAMP5 antigen. In certain embodiments, the extracellular antigen-binding domain is derived from a monoclonal antibody (mAb) that binds to a LAMP5 antigen. In some embodiments, the extracellular antigen- binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises a Fab, which is optionally crosslinked. In some embodiments, the extracellular binding domain comprises a F(ab)2.In some embodiments, any of the foregoing molecules are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen- binding domain comprises a human scFv that binds specifically to a LAMP5 antigen. In certain embodiments, the scFv is identified by screening scFv phage library with a LAMP5 antigen-Fc fusion protein.

[0306] In certain embodiments, the extracellular antigen-binding domain of a presently disclosed CAR has a high binding specificity and high binding affinity to a LAMP5 antigen. For example, in some embodiments, the extracellular antigen-binding domain of the CAR (embodied, for example, in a human scFv or an analog thereof) binds to a particular LAMP5 antigen with a dissociation constant (Kd) of about 1 × 10-5M or less. In certain embodiments, the Kdis about 5 × 10-6M or less, about 1 × 10-6M or less, about 5 × 10-7M or less, about 1 × 10-7M or less, about 5 × 10-8M or less, about 1 × 10-8M or less, about 5 × 10-9or less, about 4 85 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 × 10-9or less, about 3 × 10-9or less, about 2 × 10-9or less, or about 1 × 10-9M or less. In certain non-limiting embodiments, the Kdis from about 3 × 10-9M or less. In certain non- limiting embodiments, the Kdis from about 3 × 10-9to about 2 × 10-7.

[0307] Binding of the extracellular antigen-binding domain (embodiment, for example, in an scFv or an analog thereof) of a presently disclosed LAMP5-specific CAR can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the LAMP5-specific CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the scFv of a presently disclosed LAMP5-specific CAR is labeled with GFP.

[0308] In some embodiments, the extracellular antigen-binding domain of the expressed CAR binds to a LAMP5 antigen that is expressed by a cancer cell. In some embodiments, the extracellular antigen-binding domain of the expressed CAR binds to a LAMP5 antigen that is expressed on the surface of a cancer cell.

[0309] In certain embodiments, the extracellular antigen-binding domain (e.g., human scFv) comprises a heavy chain variable (VH) region and a light chain variable (VL) region, optionally linked with a linker sequence, for example a linker peptide (e.g., SEQ ID NO: 18), between the heavy chain variable (VH) region and the light chain variable (VL) region.

[0310] In certain non-limiting embodiments, an extracellular antigen-binding domain of the 86 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 presently disclosed CAR can comprise a linker connecting the heavy chain variable (VH) region and light chain variable (VL) region of the extracellular antigen-binding domain. As used herein, the term “linker” refers to a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a “peptide linker” refers to one or more amino acids used to couple two proteins together (e.g., to couple VHand VLdomains). In certain embodiments, the linker comprises amino acids having the sequence set forth in SEQ ID NO: 18. In certain embodiments, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 is set forth in SEQ ID NO: 19.

[0311] Additionally or alternatively, in some embodiments, the extracellular antigen- binding domain can comprise a leader or a signal peptide sequence that directs the nascent protein into the endoplasmic reticulum. The signal peptide or leader can be essential if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader sequence can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of the newly synthesized proteins that direct their entry to the secretory pathway.

[0312] In certain embodiments, the signal peptide is covalently joined to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a human CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 31 as provided below: MALPVTALLLPLALLLHAARP (SEQ ID NO: 31).

[0313] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 31 is set forth in SEQ ID NO: 32, which is provided below: ATGGCCCTGCCAGTAACGGCTCTGCTGCTGCCACTTGCTCTGCTCCTCCATGCAGCC AGGCCT (SEQ ID NO: 32).

[0314] In certain embodiments, the signal peptide comprises a human CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 33 as provided below: MALPVTALLLPLALLLHA (SEQ ID NO: 33).

[0315] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33 is set 87 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 forth in SEQ ID NO: 34, which is provided below: ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCA (SEQ ID NO: 34).

[0316] In certain embodiments, the signal peptide comprises a mouse CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 35 as provided below: MASPLTRFLSLNLLLLGESII (SEQ ID NO: 35).

[0317] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35 is set forth in SEQ ID NO: 36, which is provided below: ATGGCCAGCCCCCTGACCAGGTTCCTGAGCCTGAACCTGCTGCTGCTGGGCGAGAG CATCATC (SEQ ID NO: 36).

[0318] In certain embodiments, the signal peptide comprises a mouse CD8 signal polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 37 as provided below: MASPLTRFLSLNLLLLGE (SEQ ID NO: 37).

[0319] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37 is set forth in SEQ ID NO: 38, which is provided below: ATGGCCAGCCCCCTGACCAGGTTCCTGAGCCTGAACCTGCTGCTGCTGGGCGAG (SEQ ID NO: 38).

[0320] Transmembrane Domain of a CAR. In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In accordance with the presently disclosed subject matter, the transmembrane domain of the CAR can comprise a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (e.g., a transmembrane peptide not based on a protein associated with the immune response), or a combination thereof.

[0321] In certain embodiments, the transmembrane domain of a presently disclosed CAR 88 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 comprises a CD28 polypeptide. The CD28 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a UniProtKB Reference No: P10747 or NCBI Reference No: NP006130 (SEQ ID NO: 39), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 39 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Additionally or alternatively, in non- limiting various embodiments, the CD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 39. In certain embodiments, the CAR of the present disclosure comprises a transmembrane domain comprising a CD28 polypeptide, and optionally an intracellular domain comprising a co- stimulatory signaling region that comprises a CD28 polypeptide. In certain embodiments, the CD28 polypeptide comprised in the transmembrane domain and the intracellular domain has an amino acid sequence of amino acids 114 to 220 of SEQ ID NO: 39. In certain embodiments, the CD28 polypeptide comprised in the transmembrane domain has an amino acid sequence of amino acids 153 to 179 of SEQ ID NO: 39.

[0322] SEQ ID NO: 39 is provided below: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNALSCKYSYNLFSREFRASLHKGLDS AVEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYQTDIYFCKIEVMYPPP YLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 39)

[0323] In accordance with the presently disclosed subject matter, a “CD28 nucleic acid molecule” refers to a polynucleotide encoding a CD28 polypeptide. In certain embodiments, the CD28 nucleic acid molecule encoding the CD28 polypeptide comprised in the transmembrane domain (and optionally the intracellular domain (e.g., the co-stimulatory signaling region)) of the presently disclosed CAR (e.g., amino acids 114 to 220 of SEQ ID NO: 39 or amino acids 153 to 179 of SEQ ID NO: 39) comprises at least a portion of the sequence set forth in SEQ ID NO: 40 as provided below. attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtccc 89 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 ctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattatt ttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcatt accagccctatgccccaccacgcgacttcgcagcctatcgctcc (SEQ ID NO: 40)

[0324] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. The CD8 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%) homologous to SEQ ID NO: 41 (homology herein may be determined using standard software such as BLAST or FASTA) as provided below, or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 41 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Additionally or alternatively, in various embodiments, the CD8 polypeptide has an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 235 of SEQ ID NO: 41.

[0325] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPT SGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRREN EGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGDKPS LSARYV (SEQ ID NO: 41)

[0326] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide comprising amino acids having the sequence set forth in SEQ ID NO: 42 as provided below:

[0327] PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCN (SEQ ID NO: 42)

[0328] In accordance with the presently disclosed subject matter, a “CD8 nucleic acid molecule” refers to a polynucleotide encoding a CD8 polypeptide. In certain embodiments, the CD8 nucleic acid molecule encoding the CD8 polypeptide comprised in the transmembrane domain of the presently disclosed CAR (SEQ ID NO: 42) comprises nucleic acids having the sequence set forth in SEQ ID NO: 43 as provided below. 90 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0329] CCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGATCG CGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGC AGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGG CCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAC (SEQ ID NO: 43)

[0330] In certain non-limiting embodiments, a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition while preserving the activating activity of the CAR. In certain non-limiting embodiments, the spacer region can be the hinge region from IgGl, the CH2CH3region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide (e.g., SEQ ID NO: 39), a portion of a CD8 polypeptide (e.g., SEQ ID NO: 41), a variation of any of the foregoing which is at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous thereto, or a synthetic spacer sequence. In certain non-limiting embodiments, the spacer region may have a length between about 1-50 (e.g., 5-25, 10-30, or 30-50) amino acids.

[0331] Intracellular Domain of a CAR. In certain non-limiting embodiments, an intracellular domain of the CAR can comprise a CD3ζ polypeptide, which can activate or stimulate a cell (e.g., a cell of the lymphoid lineage, e.g., a T cell). CD3ζ comprises 3 ITAMs, and transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., a T cell) after antigen is bound. The CD3ζ polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No: NP_932170 (SEQ ID NO: 44), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0332] In certain embodiments, the CD3ζ polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 44 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Additionally or alternatively, in various embodiments, the CD3ζ polypeptide has an amino acid sequence of amino acids 1 to 164, 1 to 91 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 50, 50 to 100, 100 to 150, or 150 to 164 of SEQ ID NO: 44. In certain embodiments, the CD3ζ polypeptide has an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 44.

[0333] SEQ ID NO: 44 is provided below: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 44)

[0334] In certain embodiments, the CD3ζ polypeptide has the amino acid sequence set forth in SEQ ID NO: 45, which is provided below: RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 45)

[0335] In certain embodiments, the CD3ζ polypeptide has the amino acid sequence set forth in SEQ ID NO: 46, which is provided below: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46)

[0336] In accordance with the presently disclosed subject matter, a “CD3ζ nucleic acid molecule” refers to a polynucleotide encoding a CD3ζ polypeptide. In certain embodiments, the CD3ζ nucleic acid molecule encoding the CD3ζ polypeptide (SEQ ID NO: 45) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 47 as provided below. AGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAA GAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCA GGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCC CCTCGCG (SEQ ID NO: 47) 92 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0337] In certain embodiments, the CD3ζ nucleic acid molecule encoding the CD3ζ polypeptide (SEQ ID NO: 46) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 48 as provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAA GAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCA GGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCC CCTCGCTAA (SEQ ID NO: 48)

[0338] In certain non-limiting embodiments, an intracellular domain of the CAR further comprises at least one signaling region. The at least one signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP- 10 polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with the immune response), or a combination thereof.

[0339] In certain embodiments, the signaling region is a co-stimulatory signaling region.

[0340] In certain embodiments, the co-stimulatory signaling region comprises at least one co-stimulatory molecule, which can provide optimal lymphocyte activation. As used herein, “co-stimulatory molecules” refer to cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigen. The at least one co-stimulatory signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. The co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on cell surface that upon binding to its receptor produces a co-stimulatory response, i.e., an intracellular response that effects the stimulation provided when an antigen binds to its CAR molecule. Co-stimulatory ligands, include, but are not limited to CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD- Ll. As one example, a 4-1BB ligand (i.e., 4-1BBL) may bind to 4-1BB (also known as “CD 137”) for providing an intracellular signal that in 93 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 combination with a CAR signal induces an effector cell function of the CAR+T cell. CARs comprising an intracellular domain that comprises a co-stimulatory signaling region comprising 4-1BB, ICOS or DAP-10 are disclosed in U.S.7,446,190, which is herein incorporated by reference in its entirety. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that comprises two co-stimulatory molecules: CD28 and 4-1BB or CD28 and OX40.

[0341] 4-1BB can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. The 4-1BB polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a UniProtKB Reference No: P41273 or NCBI Reference No: NP_001552 (SEQ ID NO: 49) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0342] SEQ ID NO: 49 is provided below: MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQ RTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKG CKDCCFGTFNDQKRGICRPWTNCSLDGKSVLGTKERDWCGPSPADLSPGASSVTPPAP AREPGHSPQIISFFLALTSTALLFLLFFLTLRFSWKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCEL (SEQ ID NO: 49)

[0343] In certain embodiments, the 4-1BB co-stimulatory domain has the amino acid sequence set forth in SEQ ID NO: 50, which is provided below: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 50)

[0344] In accordance with the presently disclosed subject matter, a “4-1BB nucleic acid molecule” refers to a polynucleotide encoding a 4-1BB polypeptide. In certain embodiments, the 4-1BB nucleic acid molecule encoding the 4-1BB polypeptide (SEQ ID NO: 50) comprised in the intracellular domain of the presently disclosed CAR comprises a nucleotide sequence as set forth in SEQ ID NO: 51 as provided below. AAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAG TACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGA 94 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 AGGAGGATGTGAACTG (SEQ ID NO: 51)

[0345] An OX40 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a UniProtKB Reference No: P43489 or NCBI Reference No: NP_003318 (SEQ ID NO: 52), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0346] SEQ ID NO: 52 is provided below: MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCS RSQNTVCRPCGPGFYNDWSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQP LDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPA TQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAI LLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 52)

[0347] In accordance with the presently disclosed subject matter, an “OX40 nucleic acid molecule” refers to a polynucleotide encoding an OX40 polypeptide.

[0348] An ICOS polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: NP_036224 (SEQ ID NO: 53) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0349] SEQ ID NO: 53 is provided below: MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKG GQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPP FKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVWCILGCILICWLTKKKYSSSVHDPNG EYMFMRATAKKSRLTDVTL (SEQ ID NO: 53)

[0350] In accordance with the presently disclosed subject matter, an “ICOS nucleic acid molecule” refers to a polynucleotide encoding an ICOS polypeptide.

[0351] CTLA-4 is an inhibitory receptor expressed by activated T cells, which when 95 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 engaged by its corresponding ligands (CD80 and CD86; B7-1 and B7-2, respectively), mediates activated T cell inhibition or anergy. In both preclinical and clinical studies, CTLA-4 blockade by systemic antibody infusion, enhanced the endogenous anti-tumor response albeit, in the clinical setting, with significant unforeseen toxicities.

[0352] CTLA-4 contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. Alternate splice variants, encoding different isoforms, have been characterized. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. The intracellular domain is similar to that of CD28, in that it has no intrinsic catalytic activity and contains one YVKM motif (SEQ ID NO: 54) able to bind PI3K, PP2A and SHP-2 and one proline-rich motif able to bind SH3 containing proteins. One role of CTLA-4 in inhibiting T cell responses seem to be directly via SHP-2 and PP2A dephosphorylation of TCR-proximal signaling proteins such as CD3 and LAT. CTLA-4 can also affect signaling indirectly via competing with CD28 for CD80 / 86 binding. CTLA-4 has also been shown to bind and / or interact with PI3K, CD80, AP2M1, and PPP2R5A.

[0353] In accordance with the presently disclosed subject matter, a CTLA-4 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss-Prot Ref. No.: P16410.3 (SEQ ID NO: 55) (homology herein may be determined using standard software such as BLAST or FASTA) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0354] SEQ ID NO: 55 is provided below: MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAWLASSRGIASFVCE YASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQLTIQGL RAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSF LLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 55)

[0355] In accordance with the presently disclosed subject matter, a “CTLA-4 nucleic acid molecule” refers to a polynucleotide encoding a CTLA-4 polypeptide. 96 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0356] PD-1 is a negative immune regulator of activated T cells upon engagement with its corresponding ligands PD-L1 and PD-L2 expressed on endogenous macrophages and dendritic cells. PD-1 is a type I membrane protein of 268 amino acids. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. The protein's structure comprises an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine- based inhibitory motif and an immunoreceptor tyrosine- based switch motif, that PD-1 negatively regulates TCR signals. SHP- I and SHP-2 phosphatases bind to the cytoplasmic tail of PD-1 upon ligand binding. Upregulation of PD-L1 is one mechanism tumor cells may evade the host immune system. In pre-clinical and clinical trials, PD-1 blockade by antagonistic antibodies induced anti -tumor responses mediated through the host endogenous immune system. In accordance with the presently disclosed subject matter, a PD-1 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to NCBI Reference No: NP_005009.2 (SEQ ID NO: 56) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0357] SEQ ID NO: 56 is provided below: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARR NDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGWG GLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREK TPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 56)

[0358] In accordance with the presently disclosed subject matter, a “PD-1 nucleic acid molecule” refers to a polynucleotide encoding a PD-1 polypeptide.

[0359] Lymphocyte-activation protein 3 (LAG-3) is a negative immune regulator of immune cells. LAG-3 belongs to the immunoglobulin (Ig) superfamily and contains 4 extracellular Ig-like domains. The LAG3 gene contains 8 exons. The sequence data, exon / intron organization, and chromosomal localization all indicate a close relationship of 97 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 LAG3 to CD4. LAG3 has also been designated CD223 (cluster of differentiation 223).

[0360] In accordance with the presently disclosed subject matter, a LAG-3 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss-Prot Ref. No.: P18627.5 (SEQ ID NO: 57) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0361] SEQ ID NO: 57 is provided below: MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPWWAQEGAPAQLPCSPTIPLQDLSLLRR AGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPL QPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMT ASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQV SPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVG TRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVT LAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLS QPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLV TGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL (SEQ ID NO: 57)

[0362] In accordance with the presently disclosed subject matter, a “LAG-3 nucleic acid molecule” refers to a polynucleotide encoding a LAG-3 polypeptide.

[0363] Natural Killer Cell Receptor 2B4 (2B4) mediates non-MHC restricted cell killing on NK cells and subsets of T cells. To date, the function of 2B4 is still under investigation, with the 2B4-S isoform believed to be an activating receptor, and the 2B4-L isoform believed to be a negative immune regulator of immune cells.2B4 becomes engaged upon binding its high- affinity ligand, CD48. 2B4 contains a tyrosine-based switch motif, a molecular switch that allows the protein to associate with various phosphatases. 2B4 has also been designated CD244 (cluster of differentiation 244).

[0364] In accordance with the presently disclosed subject matter, a 2B4 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, 98 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss-Prot Ref. No.: Q9BZW8.2 (SEQ ID NO: 58) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0365] SEQ ID NO: 58 is provided below: MLGQWTLILLLLLKVYQGKGCQGSADHWSISGVPLQLQPNSIQTKVDSIAWKKLLPSQ NGFHHILKWENGSLPSNTSNDRFSFIVKNLSLLIKAAQQQDSGLYCLEVTSISGKVQTA TFQVFVFESLLPDKVEKPRLQGQGKILDRGRCQVALSCLVSRDGNVSYAWYRGSKLIQ TAGNLTYLDEEVDINGTHTYTCNVSNPVSWESHTLNLTQDCQNAHQEFRFWPFLVIIVI LSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGST IYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPA RLSRKELENFDVYS (SEQ ID NO: 58)

[0366] In accordance with the presently disclosed subject matter, a “2B4 nucleic acid molecule” refers to a polynucleotide encoding a 2B4 polypeptide.

[0367] B- and T-lymphocyte attenuator (BTLA) expression is induced during activation of T cells, and BTLA remains expressed on Thl cells but not Th2 cells. Like PD1 and CTLA4, BTLA interacts with a B7 homolog, B7H4. However, unlike PD-1 and CTLA-4, BTLA displays T-Cell inhibition via interaction with tumor necrosis family receptors (TNF-R), not just the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immune responses. BTLA activation has been shown to inhibit the function of human CD8+cancer-specific T cells. BTLA has also been designated as CD272 (cluster of differentiation 272).

[0368] In accordance with the presently disclosed subject matter, a BTLA polypeptide can have an amino acid sequence that is at least about 85%>, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to UniProtKB / Swiss-Prot Ref. No.: Q7Z6A9.3 (SEQ ID NO: 59) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0369] SEQ ID NO: 59 is provided below: 99 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELECP VKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSA NFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCLFCC LRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFR MQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 59)

[0370] In accordance with the presently disclosed subject matter, a “BTLA nucleic acid molecule” refers to a polynucleotide encoding a BTLA polypeptide. B. Engineered Immune Cells

[0371] As described herein, immune cells can be engineered to constitutively or conditionally express a receptor including an anti-LAMP5 antigen binding fragment that binds to a LAMP5 antigen present on the cell surface of the cancer cells, such as myeloma, including monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, newly diagnosed multiple myeloma, or relapsed / refractory multiple myeloma, leukemia, including plasma cell leukemia, mixed lineage leukemia, or acute myeloid leukemia, neuroblastoma, B- cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, glioma, or other LAMP5 expressing cancers. The engineered immune cells of the present technology express a receptor comprising an anti- LAMP5 antigen binding fragment (e.g., scFv) that permits delivery of the immune cell to the target cancer cells. In some embodiments, the engineered immune cells provided herein express a T-cell receptor (TCR) (e.g., caTCR, or eTCR) or other cell-surface ligand that binds to a LAMP5 antigen. In some embodiments, the receptor is a chimeric antigen receptor (CAR). In exemplary embodiments provided herein, the engineered immune cells provided herein express a receptor (e.g., a CAR, caTCR, or eTCR) or other cell-surface ligand that binds to a LAMP5 antigen.

[0372] Provided herein are engineered immune cells (e.g., T cells) that express a LAMP5- specific antigen receptor (e.g., CAR, caTCR, or eTCR) that effectively target cancer cells. The engineered immune cells (e.g., T cells) provided herein that express a LAMP5-specific antigen receptor (e.g., CAR, caTCR, or eTCR) are useful in methods for eliminating cancer cells, 100 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 and / or treating cancer, such as myeloma, including monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, newly diagnosed multiple myeloma, or relapsed / refractory multiple myeloma, leukemia, including plasma cell leukemia, mixed lineage leukemia, or acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B- cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, glioma, or other LAMP5 expressing cancers in a subject in need thereof.

[0373] In certain embodiments, the engineered immune cells will proliferate extensively (e.g., 100 times or more) when it encounters a LAMP5 antigen at a tissue site, thus significantly increasing production of the receptor comprising the anti-LAMP5 antigen binding fragment. The engineered immune cells (e.g., T cells) can be generated by in vitro transduction of immune cells with a nucleic acid encoding the receptor comprising the anti-LAMP5 antigen binding fragment (e.g., CAR, caTCR, or eTCR). Further, the activity of the engineered immune cells (e.g., T cells) can be adjusted by selection of co-stimulatory molecules included in the receptor (e.g., CAR, caTCR, or eTCR).

[0374] In some embodiments, the receptor (e.g., a CAR, caTCR, or eTCR) comprises a LAMP5 antigen binding fragment (e.g., scFv) comprising a VHcomprising a VH-CDR1 sequence of GYTFTDFTI (SEQ ID NO: 5), a VH-CDR2 sequence of IIPNNGNA (SEQ ID NO: 6), and a VH-CDR3 sequence of ARARYYFDY (SEQ ID NO: 7). Additionally or alternatively, in some embodiments, the LAMP5 antigen binding fragment (e.g., scFv) comprises a VLcomprising a VL-CDR1 sequence of RSSSGAVTTSNSAN (SEQ ID NO: 12), a VL-CDR2 sequence of GTNNRPS (SEQ ID NO: 13), and a VL-CDR3 sequence of ALWNSNHWV (SEQ ID NO: 14).

[0375] Additionally or alternatively, in some embodiments, the amino acid sequence of the VHof the anti-LAMP5 antigen binding fragment (e.g., scFv) is:

[0376] EVQLQQSGPELVKPGASVKISCKTSGYTFTDFTIHWMRQSHGKSLEWIGGIIP NNGNAIYNQKFKGKATLTVDKSSSTAHMELRSLTSEHSAVYYCARARYYFDYWGQG TTLTVSS (SEQ ID NO: 4).

[0377] Additionally or alternatively, in some embodiments, the amino acid sequence of the 101 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 VLof the anti-LAMP5 antigen binding fragment (e.g., scFv) is:

[0378] QAVVTQESALTTSPGETVTLTCRSSSGAVTTSNSANWVQEKPDHLFTGLIGG TNNRPSGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWNSNHWVFGGGTKLTVL (SEQ ID NO: 11).

[0379] In some embodiments, the VHcomprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 4 and / or the VLcomprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 11. In some embodiments, the antigen binding fragment specifically binds to LAMP5. In certain embodiments, the antigen binding fragment is selected from the group consisting of Fab, F(ab’)2, Fab’, scFv, and Fv. The antigen binding fragment may be monoclonal, chimeric, humanized, or bispecific.

[0380] Additionally or alternatively, in some embodiments, the anti-LAMP5 antigen binding fragment (e.g., scFv) comprises an amino acid sequence selected from the group consisting of:

[0381] EVQLQQSGPELVKPGASVKISCKTSGYTFTDFTIHWMRQSHGKSLEWIGGIIP NNGNAIYNQKFKGKATLTVDKSSSTAHMELRSLTSEHSAVYYCARARYYFDYWGQG TTLTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSSGAVTT SNSANWVQEKPDHLFTGLIGGTNNRPSGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWNSNHWVFGGGTKLTVL (SEQ ID NO: 60).

[0382] Additionally or alternatively, in some embodiments, the anti-LAMP5 antigen binding fragment (e.g., scFv) comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 60. In some embodiments, the anti-LAMP5 antigen binding fragment (e.g., scFv) comprises an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 60. In some embodiments, the anti-LAMP5 antigen binding fragment is an scFv, a Fab, or a (Fab)2.

[0383] Additionally or alternatively, in some embodiments, the anti-LAMP5 antigen binding fragment (e.g., scFv) is encoded by a nucleic acid sequence which encodes the 102 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 polypeptide of SEQ ID NO: 60.

[0384] Additionally or alternatively, in some embodiments, the anti-LAMP5 antigen binding fragment (e.g., scFv) is encoded by a nucleic acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 61. In some embodiments, the anti-LAMP5 antigen binding fragment (e.g., scFv) is encoded by a nucleic acid that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61. GAGGTGCAACTGCAGCAGTCCGGACCCGAGCTGGTGAAGCCCGGCGCTTCGGTGA AGATCTCATGCAAGACCTCCGGCTACACGTTCACCGACTTCACCATTCACTGGATG CGCCAGAGTCACGGCAAGAGCCTAGAGTGGATCGGGGGCATCATCCCGAACAACG GCAACGCTATTTACAACCAGAAGTTCAAGGGAAAGGCCACTCTGACGGTAGACAA ATCGTCTAGCACAGCGCACATGGAATTGCGTTCCCTGACCTCGGAGCACTCCGCGG TGTATTACTGCGCCCGGGCTCGCTACTACTTCGACTATTGGGGACAGGGCACTACC CTGACCGTGTCCTCCGGCGGCGGGGGTTCCGGGGGTGGCGGGTCTGGCGGCGGCG GTTCAGGAGGCGGTGGGAGCCAGGCTGTGGTCACACAGGAGAGCGCGTTGACTAC GAGCCCTGGGGAGACTGTTACTCTTACCTGTAGATCCTCCAGCGGCGCTGTCACCA CCTCTAATAGCGCCAATTGGGTGCAGGAGAAGCCTGATCACCTGTTCACCGGCCTG ATCGGCGGCACCAACAACCGCCCCTCCGGCGTGCCTGCGCGCTTTTCTGGATCTCT CATCGGTGATAAGGCCGCCCTCACAATCACCGGTGCTCAGACCGAGGACGAGGCC ATCTACTTTTGTGCTCTTTGGAACTCTAACCACTGGGTGTTCGGGGGCGGCACCAA ACTGACGGTCCTG (SEQ ID NO: 61)

[0385] Additionally or alternatively, in certain embodiments, the LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR) of the present technology and a reporter or selection marker (e.g., GFP, LNGFR) are expressed as a single polypeptide linked by a self-cleaving linker, such as a P2A linker. In certain embodiments, the receptor (e.g., a CAR, caTCR, or eTCR) and a reporter or selection marker (e.g., GFP, LNGFR) are expressed as two separate polypeptides.

[0386] In any and all of the preceding embodiments, the CAR comprises an extracellular binding fragment (e.g., anti-LAMP5 scFv) that specifically binds to a LAMP5 antigen or polypeptide, a transmembrane domain comprising a CD28 polypeptide and / or a CD8 103 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 polypeptide, and an intracellular domain comprising a CD3ζ polypeptide and optionally a co- stimulatory signaling region disclosed herein. The CAR may also comprise a signal peptide or a leader sequence covalently joined to the N-terminus of the extracellular LAMP5 binding fragment. The signal peptide comprises amino acids having the sequence set forth in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37.

[0387] Additionally or alternatively, in some embodiments, the nucleic acid encoding the receptor (e.g., a CAR, caTCR, or eTCR) of the present technology is operably linked to an inducible promoter. In some embodiments, the nucleic acid encoding the receptor (e.g., a CAR, caTCR, or eTCR) of the present technology is operably linked to a constitutive promoter.

[0388] In some embodiments, the inducible promoter is a synthetic Notch promoter that is activatable in an immune cell including the receptor (e.g., CAR, caTCR, or eTCR), where the intracellular domain of the receptor contains a transcriptional regulator that is released from the membrane when engagement of the receptor (e.g., CAR, caTCR, or eTCR) with the LAMP5 antigen / polypeptide induces intramembrane proteolysis (see, e.g., Morsut et al., Cell 164(4): 780–791 (2016). Accordingly, further transcription of the LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR) is induced upon binding of the engineered immune cell with the LAMP5 antigen / polypeptide.

[0389] The presently disclosed subject matter also provides isolated nucleic acid molecules encoding the receptor (e.g., a CAR, caTCR, or eTCR) constructs described herein or a functional portion thereof.

[0390] In certain embodiments, the isolated nucleic acid molecule encodes an anti-LAMP5- targeted CAR comprising (a) an LAMP5 binding fragment (e.g., an anti-LAMP5 scFv) that specifically binds to a LAMP5 antigen, (b) a transmembrane domain comprising a CD8 polypeptide or CD28 polypeptide, and (c) an intracellular domain comprising a CD28 polypeptide and / or a CD3ζ polypeptide, and optionally one or more of (i) a co-stimulatory signaling region disclosed herein, (ii) a P2A self-cleaving peptide, and (iii) a reporter or selection marker (e.g., GFP, LNGFR) provided herein. The at least one co-stimulatory signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP- 10 polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a 104 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with the immune response), or a combination thereof. In certain embodiments, the isolated nucleic acid molecule encodes a LAMP5-targeted CAR comprising a LAMP5 binding fragment (e.g., an anti-LAMP5 scFv) that specifically binds to a LAMP5 antigen / polypeptide, fused to a synthetic Notch transmembrane domain and an intracellular cleavable transcription factor. In certain embodiments, the present disclosure provides an isolated nucleic acid molecule encoding a LAMP5-specific CAR that is inducible by release of the transcription factor of a synthetic Notch system.

[0391] In certain embodiments, the isolated nucleic acid molecule encodes a functional portion of a presently disclosed receptor (e.g., a CAR, caTCR, or eTCR) constructs. As used herein, the term “functional portion” refers to any portion, part or fragment of a receptor (e.g., a CAR, caTCR, or eTCR), which portion, part or fragment retains the biological activity of the parent receptor (e.g., a CAR, caTCR, or eTCR). For example, functional portions encompass the portions, parts or fragments of a LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR) that retains the ability to recognize a target cancer cell, to treat cancer to a similar, same, or even a higher extent as the parent receptor (e.g., a CAR, caTCR, or eTCR). In certain embodiments, an isolated nucleic acid molecule encoding a functional portion of a LAMP5- specific receptor (e.g., a CAR, caTCR, or eTCR) can encode a protein comprising, e.g., about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%, or more of the parent receptor (e.g., a CAR, caTCR, or eTCR).

[0392] The presently disclosed subject matter provides engineered immune cells expressing a LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR) or other ligand that comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, where the extracellular antigen-binding domain specifically binds a LAMP5 antigen / polypeptide. In certain embodiments immune cells can be transduced with a presently disclosed receptor (e.g., a CAR, caTCR, or eTCR) constructs such that the cells express the receptor (e.g., a CAR, caTCR, or eTCR). The presently disclosed subject matter also provides methods of using such cells for the treatment of cancer such as myeloma, including monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, newly diagnosed 105 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 multiple myeloma, or relapsed / refractory multiple myeloma, leukemia, including plasma cell leukemia, mixed lineage leukemia, or acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, glioma, or other LAMP5 expressing cancers.

[0393] The presently disclosed subject matter also provides methods of using such cells for the treatment of a tumor. The engineered immune cells of the presently disclosed subject matter can be cells of the lymphoid lineage or myeloid lineage. Examples of myeloid cells include but are not limited to, mast cells, monocytes, macrophages, dendritic cells, eosinophils, neutrophils, basophils. The lymphoid lineage, comprising B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells can be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells)), and two types of effector memory T cells: e.g., TEMcells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.

[0394] Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[0395] The engineered immune cells of the presently disclosed subject matter can express an extracellular LAMP5 binding domain (e.g., an anti-LAMP5 scFv, an anti-LAMP5 Fab that is optionally crosslinked, an anti-LAMP5 F(ab)2) that specifically binds to a LAMP5 antigen, for the treatment of cancer such as myeloma, including monoclonal gammopathy of 106 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 undetermined significance, smoldering multiple myeloma, newly diagnosed multiple myeloma, or relapsed / refractory multiple myeloma, leukemia, including plasma cell leukemia, mixed lineage leukemia, or acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, glioma, or other LAMP5 expressing cancers. Such engineered immune cells can be administered to a subject (e.g., a human subject) in need thereof for the treatment of cancer such as myeloma, including monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, newly diagnosed multiple myeloma, or relapsed / refractory multiple myeloma, leukemia, including plasma cell leukemia, mixed lineage leukemia, or acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, glioma, or other LAMP5 expressing cancers. In some embodiments, the immune cell is a lymphocyte, such as a T cell, a B cell or a natural killer (NK) cell. In certain embodiments, the engineered immune cell is a T cell. The T cell can be a CD4+T cell or a CD8+T cell. In certain embodiments, the T cell is a CD4+T cell. In certain embodiments, the T cell is a CD8+T cell.

[0396] The engineered immune cells of the present disclosure can further include at least one recombinant or exogenous co-stimulatory ligand. For example, the engineered immune cells of the present disclosure can be further transduced with at least one co-stimulatory ligand, such that the engineered immune cells co-expresses or is induced to co-express the LAMP5- specific receptor (e.g., a CAR, caTCR, or eTCR) and the at least one co-stimulatory ligand. The interaction between the LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR) and the at least one co-stimulatory ligand provides a non-antigen-specific signal important for full activation of an immune cell (e.g., T cell). Co-stimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Members of TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. TNF superfamily members include, without limitation, nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4- 1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, 107 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LT-α), lymphotoxin-beta (LΤ-β), CD257 / B cell-activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF Receptor ligand (GITRL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins — they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, or PD-L1 / (B7-H1) that are ligands for PD-1. In certain embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4- 1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the engineered immune cell comprises one recombinant co-stimulatory ligand (e.g., 4-1BBL). In certain embodiments, the engineered immune cell comprises two recombinant co-stimulatory ligands (e.g., 4-1BBL and CD80). CARs comprising at least one co-stimulatory ligand are described in U.S. Patent No.8,389,282, which is incorporated by reference in its entirety.

[0397] Furthermore, the engineered immune cells of the present disclosure can further comprise at least one exogenous cytokine. For example, a presently disclosed engineered immune cell can be further transduced with at least one cytokine, such that the engineered immune cell secretes the at least one cytokine as well as expresses the LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR). In certain embodiments, the at least one cytokine is selected from the group consisting of IL-2, IL- 3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, and IL-21.

[0398] The engineered immune cells can be generated from peripheral donor lymphocytes, e.g., those disclosed in Sadelain, M., et al., Nat Rev Cancer 3 :35-45 (2003) (disclosing peripheral donor lymphocytes genetically modified to express CARs), in Morgan, R.A. et al., Science 314: 126-129 (2006) (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex comprising the α and β heterodimer), in Panelli et al., J Immunol 164:495-504 (2000); Panelli et al., J Immunol 164:4382-4392 (2000) (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and in Dupont et al., Cancer Res 65:5417-5427 108 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 (2005); Papanicolaou et al., Blood 102:2498-2505 (2003) (disclosing selectively inv / Yro- expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells). The engineered immune cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0399] In certain embodiments, the engineered immune cells of the present disclosure (e.g., T cells) express from about 1 to about 5, from about 1 to about 4, from about 2 to about 5, from about 2 to about 4, from about 3 to about 5, from about 3 to about 4, from about 4 to about 5, from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, or from about 4 to about 5 vector copy numbers per cell of a presently disclosed LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR).

[0400] For example, the higher the receptor (e.g., a CAR, caTCR, or eTCR) expression level in an engineered immune cell, the greater cytotoxicity and cytokine production the engineered immune cell exhibits. An engineered immune cell (e.g., T cell) having a high LAMP5-specific receptor (e.g., a CAR, caTCR, or eTCR) expression level can induce antigen- specific cytokine production or secretion and / or exhibit cytotoxicity to a tissue or a cell having a low expression level of LAMP5, e.g., about 2,000 or less, about 1,000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, about 100 or less of LAMP5 antigen binding sites / cell. Additionally or alternatively, the cytotoxicity and cytokine production of a presently disclosed engineered immune cell (e.g., T cell) are proportional to the expression level of LAMP5 antigen in a target tissue or a target cell. For example, the higher the expression level of LAMP5 antigen in the target, the greater cytotoxicity and cytokine production the engineered immune cell exhibits.

[0401] The unpurified source of immune cells may be any source known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-immune cells initially. Monoclonal antibodies are particularly useful for identifying markers associated with particular 109 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 cell lineages and / or stages of differentiation for both positive and negative selections.

[0402] A large proportion of terminally differentiated cells can be initially removed by a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. Suitably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed prior to cell isolation.

[0403] Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g., plate, chip, elutriation or any other convenient technique.

[0404] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.

[0405] The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). Usually, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable (e.g., sterile), isotonic medium.

[0406] In some embodiments, the engineered immune cells comprise one or more additional modifications. For example, in some embodiments, the engineered immune cells comprise and express (are transduced to express) an antigen recognizing receptor that binds to a second antigen that is different than the first LAMP5 antigen. The inclusion of an antigen recognizing receptor in addition to a presently disclosed receptor (e.g., a CAR, caTCR, or eTCR) on the engineered immune cell can increase the avidity of the receptor (e.g., a CAR, caTCR, or eTCR) (or the engineered immune cell comprising the same) on a target cell, especially, the receptor (e.g., a CAR, caTCR, or eTCR) is one that has a low binding affinity to a particular LAMP5 antigen, e.g., a Kdof about 2 × 10-8M or more, about 5 × 10-8M or more, about 8 × 10-8M or more, about 9 × 10-8M or more, about 1 × 10-7M or more, about 2 × 10-7110 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 M or more, or about 5 × 10-7M or more.

[0407] In certain embodiments, the antigen recognizing receptor is a chimeric co- stimulatory receptor (CCR). CCR is described in Krause, et al., J. Exp. Med.188(4):619- 626(1998), and US20020018783, the contents of which are incorporated by reference in their entireties. CCRs mimic co-stimulatory signals, but unlike, CARs, do not provide a T-cell activation signal, e.g., CCRs lack a CD3ζ polypeptide. CCRs provide co-stimulation, e.g., a CD28-like signal, in the absence of the natural co-stimulatory ligand on the antigen-presenting cell. A combinatorial antigen recognition, i.e., use of a CCR in combination with a CAR, can augment T-cell reactivity against the dual-antigen expressing cells, thereby improving selective targeting. Kloss et al., describe a strategy that integrates combinatorial antigen recognition, split signaling, and, critically, balanced strength of T-cell activation and costimulation to generate T cells that eliminate target cells that express a combination of antigens while sparing cells that express each antigen individually (Kloss et al., Nature Biotechnology 31(l):71-75 (2013)). With this approach, T-cell activation requires CAR-mediated recognition of one antigen, whereas costimulation is independently mediated by a CCR specific for a second antigen. To achieve tumor selectivity, the combinatorial antigen recognition approach diminishes the efficiency of T-cell activation to a level where it is ineffective without rescue provided by simultaneous CCR recognition of the second antigen. In certain embodiments, the CCR comprises (a) an extracellular antigen-binding domain that binds to an antigen different than the first LAMP5 antigen, (b) a transmembrane domain, and (c) a co-stimulatory signaling region that comprises at least one co-stimulatory molecule, including, but not limited to, CD28, 4-1BB, OX40, ICOS, PD-1, CTLA-4, LAG-3, 2B4, and BTLA. In certain embodiments, the co-stimulatory signaling region of the CCR comprises one co-stimulatory signaling molecule. In certain embodiments, the one co-stimulatory signaling molecule is CD28. In certain embodiments, the one co-stimulatory signaling molecule is 4-1BB. In certain embodiments, the co-stimulatory signaling region of the CCR comprises two co-stimulatory signaling molecules. In certain embodiments, the two co-stimulatory signaling molecules are CD28 and 4-1BB.

[0408] A second antigen is selected so that expression of both the first LAMP5 antigen and the second antigen is restricted to the targeted cells (e.g., cancerous cells). In some 111 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 embodiments, the second antigen is CD19, CD22, CD20, CD21, CD23, CD72, ROR1, BCMA, GPRC5D, CD33, CD123, CD70, CD96, or any suitable secondary antigen such as those taught by Atilla, E.; Benabdellah, K. The Black Hole: CAR T Cell Therapy in AML. Cancers 2023, 15, 2713. / / doi.org / 10.3390 / cancers15102713, which is hereby incorporated by reference in its entirety. Similar to a CAR, the extracellular antigen-binding domain of the CCR can be an scFv, a Fab, a F(ab)2;or a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the CCR comprises an scFv that binds to a B cell antigen (e.g., CD19, CD22, CD20, CD21, CD23, CD72 and ROR1), transmembrane domain comprising a CD28 polypeptide, and a co-stimulatory signaling region comprising two co-stimulatory signaling molecules that are CD28 and 4-1BB.

[0409] In certain embodiments, the antigen recognizing receptor is a truncated CAR. A “truncated CAR” is different from a CAR by lacking an intracellular signaling domain. For example, a truncated CAR comprises an extracellular antigen-binding domain and a transmembrane domain, and lacks an intracellular signaling domain. In accordance with the presently disclosed subject matter, the truncated CAR has a high binding affinity to the second antigen expressed on the targeted cells. The truncated CAR functions as an adhesion molecule that enhances the avidity of a presently disclosed CAR, especially, one that has a low binding affinity to a LAMP5 antigen, thereby improving the efficacy of the presently disclosed CAR or engineered immune cell (e.g., T cell) comprising the same. In certain embodiments, the truncated CAR comprises an extracellular antigen-binding domain that binds to a B cell antigen (e.g., CD19, CD22, CD20, CD21, CD23, CD72, ROR1, BCMA, GPRC5D, CD33, CD123, CD70, or CD96), a transmembrane domain comprising a CD8 polypeptide. A presently disclosed T cell comprises or is transduced to express a presently disclosed CAR targeting LAMP5 antigen and a truncated CAR targeting a B cell antigen (e.g., CD19, CD22, CD20, CD21, CD23, CD72, ROR1, BCMA, GPRC5D, CD33, CD123, CD70, or CD96). In certain embodiments, the targeted cells are AML or B cell malignancies. In some embodiments, the engineered immune cells are further modified to suppress expression of one or more genes. In some embodiments, the engineered immune cells are further modified via genome editing. Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted 112 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 recombination at a predetermined chromosomal locus. See, for example, U.S. Patent Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960, the disclosures of which are incorporated by reference in their entireties. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error born process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration). Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR / Cas system with an engineered crRNA / tracr RNA ('single guide RNA') to guide specific cleavage. In some embodiments, the engineered immune cells are modified to disrupt or reduce expression of an endogenous T-cell receptor gene (see, e.g., WO 2014153470, which is incorporated by reference in its entirety). In some embodiments, the engineered immune cells are modified to result in disruption or inhibition of PD1, PDL-1 or CTLA-4 (see, e.g., U.S. Patent Publication 20140120622), or other immunosuppressive factors known in the art (Wu et al. (2015) Oncoimmunology 4(7): e1016700, Mahoney et al. (2015) Nature Reviews Drug Discovery 14, 561–584). C. Polynucleotides, Polypeptides and Analogs

[0410] Also included in the presently disclosed subject matter are polypeptides including extracellular antigen-binding fragments that specifically bind to a LAMP5 antigen (e.g., a human LAMP5 antigen) (e.g., an scFv (e.g., a human scFv), a Fab, or a (Fab)2), CD3ζ, CD8, CD28, etc. or fragments thereof, and polynucleotides encoding the same, that are modified in ways that enhance their biological activity when expressed in an engineered immune cell. The presently disclosed subject matter provides methods for optimizing an amino acid sequence or a nucleic acid sequence by producing an alteration in the sequence. Such alterations may comprise certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further comprises analogs of any naturally-occurring polypeptide of the presently disclosed subject matter. Analogs can differ from a naturally- 113 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 occurring polypeptide of the presently disclosed subject matter by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the presently disclosed subject matter can generally exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity or homology with all or part of a naturally-occurring amino acid sequence of the presently disclosed subject matter. The length of sequence comparison is at least about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100 or more amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3and e-100indicating a closely related sequence. Modifications comprise in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the presently disclosed subject matter by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethyl sulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., beta (β) or gamma (γ) amino acids.

[0411] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptides or peptide domains of the presently disclosed subject matter. A fragment can be at least about 5, about 10, about 13, or about 15 amino acids. In some embodiments, a fragment is at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids. In some embodiments, a fragment is at least about 60 to about 80, about 100, about 200, about 300 or more contiguous amino acids. Fragments of the presently disclosed subject matter can be generated by methods known to those of ordinary skill in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events). 114 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0412] Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein of the present technology. Such analogs are administered according to methods of the presently disclosed subject matter. Such analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the antineoplastic activity of the original polypeptide when expressed in an engineered immune cell. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. The protein analogs can be relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0413] In accordance with the presently disclosed subject matter, the polynucleotides encoding an extracellular antigen-binding fragment that specifically binds to a LAMP5 (e.g., human LAMP5 antigen) (e.g., an scFv (e.g., a human scFv), a Fab, or a (Fab)2), CD3, CD8, CD28 can be modified by codon optimization. Codon optimization can alter both naturally occurring and recombinant gene sequences to achieve the highest possible levels of productivity in any given expression system. Factors that are involved in different stages of protein expression include codon adaptability, mRNA structure, and various cis- elements in transcription and translation. Any suitable codon optimization methods or technologies that are known to ones skilled in the art can be used to modify the polynucleotides of the presently disclosed subject matter, including, but not limited to, OptimumGene™, Encor optimization, and Blue Heron. Vectors

[0414] Many expression vectors are available and known to those of skill in the art and can be used for expression of polypeptides provided herein. The choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational 115 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cells.

[0415] Vectors also can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule, such as, for example, an epitope tag such as for localization, e.g., a hexa-his tag or a myc tag, hemagglutinin tag or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and / or membrane association.

[0416] Expression of antibodies, or antigen binding fragments thereof, and CARs can be controlled by any promoter / enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan. Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310(1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797(1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 75: 1441- 1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Jay et al., Proc. Natl. Acad. Sci. USA 75:5543 (1981)) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 50:21-25(1983)); see also "Useful Proteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrera- Estrella et al., Nature 505:209-213(1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res.9:2871(1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 510: 115-120(1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 55:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409(1986); MacDonald, Hepatology 7:425- 116 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 515: 115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 55:647-658 (1984); Adams et al., Nature 515:533-538 (1985); Alexander et al., Mol. Cell Biol.7: 1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 15:485- 495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes and Devel.1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol.5:1639-403 (1985)); Hammer et al., Science 255:53-58 (1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel.7:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 515:338-340 (1985)); Kollias et al., Cell 5:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 15:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 514:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 254: 1372- 1378 (1986)).

[0417] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of an antibody, or antigen binding fragment thereof, in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the polypeptide chains of interest and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.

[0418] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline antibody chain under the direction of the polyhedron promoter or other strong baculovirus promoter. 117 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0419] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding any of the polypeptides provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.

[0420] Exemplary plasmid vectors useful to produce the polypeptides provided herein contain a strong promoter, such as the HCMV immediate early enhancer / promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 polyA signal.

[0421] Any of the reagents, cell lines, plasmids, vectors, or regulatory sequences or elements, or methods of use or synthesis or implementation thereof, that are discussed with regards to the engineering of cells are equally applicable to the expression of antibodies. Similarly, any of the reagents, cell lines, plasmids, vectors, or regulatory sequences or elements, or methods of use or synthesis or implementation thereof, that are discussed with regards to expression of antibodies are equally applicable to the engineering of cells. A. Engineered Cells for Adoptive Cell Therapy

[0422] Genetic modification of engineered immune cells (e.g., T cells, NK cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA or RNA construct. The vector can be a retroviral vector (e.g., gamma retroviral), which is employed for the introduction of the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding the LAMP5-specific receptor (e.g., a LAMP5-specific chimeric antigen receptor) can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter. 118 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0423] Non-viral vectors or RNA may be used as well. Random chromosomal integration, or targeted integration (e.g., using a nuclease, transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs), or transgene expression (e.g., using a natural or chemically modified RNA) can be used.

[0424] For initial genetic modification of the cells to provide LAMP5-specific receptor (e.g., a LAMP5-specific chimeric antigen receptor) expressing cells, a retroviral vector is generally employed for transduction, however any other suitable viral vector or non-viral delivery system can be used. For subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) likewise proves effective. Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al., Mol. Cell. Biol.5:431-437 (1985)); PA317 (Miller, et al., Mol. Cell. Biol.6:2895-2902 (1986)); and CRIP (Danos, et al. Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Non-amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0425] Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al., Blood 80: 1418-1422(1992), or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al., Exp. Hemat.22:223-230 (1994); and Hughes, et al., J. Clin. Invest.89: 1817 (1992).

[0426] Transducing viral vectors can be used to express a co-stimulatory ligand and / or secretes a cytokine (e.g., 4-1BBL and / or IL-12) in an engineered immune cell. In some embodiments, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263267 (1996); and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A.94: 10319, (1997)). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma 119 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, (1990); Friedman, Science 244: 1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614, (1988); Tolstoshev et al., Current Opinion in Biotechnology 1:55- 61(1990); Sharp, The Lancet 337: 1277-1278 (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Pat. No.5,399,346).

[0427] In certain non-limiting embodiments, the vector expressing a presently disclosed LAMP5-specific receptor (e.g., a LAMP5-specific chimeric antigen receptor) is a retroviral vector, e.g., an oncoretroviral vector. In some instances, the retroviral vector is a SFG retroviral vector or murine stem cell virus (MSCV) retroviral vector. In certain non-limiting embodiments, the vector expressing a presently disclosed LAMP5-specific receptor (e.g., a LAMP5-specific chimeric antigen receptor) is a lentiviral vector or a transposon vector.

[0428] Non-viral approaches can also be employed for the expression of a protein in a cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Nat'l. Acad. Sci. U.S.A.84:7413, (1987); Ono et al., Neuroscience Letters 17:259 (1990); Brigham et al., Am. J. Med. Sci. 298:278, (1989); Staubinger et al., Methods in Enzymology 101 :512 (1983)), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263 : 14621 (1988); Wu et al., Journal of Biological Chemistry 264: 16985 (1989)), or by micro- injection under surgical conditions (Wolff et al., Science 247: 1465 (1990)). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., Zinc finger nucleases, meganucleases, or TALEN nucleases). Transient expression may 120 4860-7760-7933.2Atty. Dkt. No.: 642631-0168 be obtained by RNA electroporation.

[0429] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g., the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0430] The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes. B. Antibodies and antigen binding fragments thereof

[0431] As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide constructs encoding an anti-LAMP5 antibody of the present technology typically include an expression control sequence operably-linked to the coding sequences of anti-LAMP5 antibody chains, including naturally-associated or heterologous promoter regions. As such, another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding an anti- LAMP5 antibody of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, the nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-LAMP5 antibody is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. Nos.6,291,160 and 6,680,192. 121 4860-7760-7933.2Atty. Dkt. No.: 642631-0168

[0432] In general, expression vectors useful in recombinant DNA techniques are often in the form of pla...

Claims

Atty. Dkt. No.: 642631-0168 WHAT IS CLAIMED IS 1. An antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein: the VHcomprises a VH-CDR1 sequence of GYTFTDFTI (SEQ ID NO: 5), a VH-CDR2 sequence of IIPNNGNA (SEQ ID NO: 6), and a VH-CDR3 sequence of ARARYYFDY (SEQ ID NO: 7); and the VLcomprises a VL-CDR1 sequence of RSSSGAVTTSNSAN (SEQ ID NO: 12), a VL-CDR2 sequence of GTNNRPS (SEQ ID NO: 13), and a VL- CDR3 sequence of ALWNSNHWV (SEQ ID NO: 14) 2. The antibody or antigen binding fragment of claim 1, wherein (a) the VHcomprises an amino acid sequence of SEQ ID NO: 4; and / or (b) the VLcomprises an amino acid sequence of SEQ ID NO:

11.

3. The antibody or antigen binding fragment of claim 1 or 2, further comprising a Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

4. The antibody of claim 3, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

5. The antibody of claim 3, comprising an IgG4 constant region comprising a S228P mutation.

6. The antigen binding fragment of claim 1 or 2, wherein the antigen binding fragment is selected from the group consisting of Fab, F(ab’)2, Fab’, scFv, and Fv.

7. The antibody or antigen binding fragment of any one of claims 1-6, wherein the antibody or antigen binding fragment binds to a LAMP5 polypeptide.

8. The antibody of any one of claims 1-2 or 7, further comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO: 2, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:

9.

9. An antibody comprising (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain -183--7760-7933.2Atty. Dkt. No.: 642631-0168 sequence of SEQ ID NO: 11; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO:

4.

10. An antibody comprising: (a) a LC sequence that is at least 95% identical to the LC sequence present in SEQ ID NO: 9; and / or (b) a HC sequence that is at least 95% identical to the HC sequence present in SEQ ID NO:

2.

11. The antibody of any one of claims 9-10, wherein the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

12. The antibody of any one of claims 9-10, wherein the antibody comprises an IgG4 constant region comprising a S228P mutation.

13. The antibody of any one of claims 1-12, wherein the antibody lacks α-1,6-fucose modifications.

14. The antibody or antigen binding fragment of any one of claims 1-13, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or multi-specific antibody.

15. The multi-specific antibody of claim 14, wherein the multi-specific antibody or antigen binding fragment binds to T cells, B-cells, myeloid cells, plasma cells, or mast-cells.

16. The multi-specific antibody or antigen binding fragment of claim 14, wherein the multi-specific antibody or antigen binding fragment binds to CD3, GPA33, HER2 / neu, GD2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, MUM-1, CDK4, N- acetylglucosaminyltransferase, p15, gp75, beta-catenin, ErbB2, cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), RAGE, MART (melanoma antigen), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, tyrosinase, Pmel 17 (gp100), GnT-V intron V sequence (N- acetylglucoaminyltransferase V intron V sequence), Prostate cancer psm, PRAME (melanoma antigen), β-catenin, EBNA (Epstein-Barr Virus nuclear antigen) 1-6, LMP2, p53, lung resistance protein (LRP), Bcl-2, prostate specific antigen (PSA), Ki-67, CEACAM6, colon-specific antigen-p (CSAp), HLA-DR, CD40, CD74, CD138, EGFR, EGP-1, EGP-2, VEGF, PlGF, insulin-like growth factor (ILGF), tenascin, platelet-derived growth -184--7760-7933.2Atty. Dkt. No.: 642631-0168 factor, IL-6, CD20, CD19, PSMA, CD33, CD123, MET, DLL4, Ang-2, HER3, IGF-1R, CD30, TAG-72, SPEAP, CD45, L1-CAM, Lewis Y (Ley) antigen, E-cadherin, V-cadherin, GPC3, EpCAM, CD4, CD8, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, CD56, DLL3, PD-1, PD-L1, CD28, CD137, CD99, GloboH, CD24, STEAP1, B7H3, Polysialic Acid, OX40, OX40-ligand, or peptide MHC complexes (with peptides derived from TP53, KRAS, MYC, EBNA1-6, PRAME, MART, tyronsinase, MAGEA1-A6, pmel17, LMP2, or WT1).

17. A recombinant nucleic acid sequence encoding the antibody or antigen binding fragment of any one of claims 1-16.

18. A recombinant nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1 and 8.

19. A host cell or vector comprising the recombinant nucleic acid sequence of claim 17 or claim 18.

20. A composition comprising the antibody or antigen binding fragment of any one of claims 1-16 and a pharmaceutically-acceptable carrier, wherein the antibody or antigen binding fragment is optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof.

21. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding fragment of any one of claims 1-16 or the composition of claim 20.

22. The method of claim 21, wherein the cancer is selected from the group consisting of myeloma, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, multiple myeloma, relapsed / refractory multiple myeloma, leukemia, plasma cell leukemia, mixed lineage leukemia, acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, and glioma and metastases thereof. -185--7760-7933.2Atty. Dkt. No.: 642631-0168 23. The method of claim 21 or 22, wherein the antibody or antigen binding fragment or the composition is administered to the subject separately, sequentially or simultaneously with an additional therapeutic agent.

24. The method of claim 23, wherein the additional therapeutic agent is one or more of alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, and bisphosphonate therapy agents.

25. A method for detecting cancer in a subject in vivo comprising (a) administering to the subject an effective amount of the antibody or antigen binding fragment of any one of claims 1-16, wherein the antibody or antigen binding fragment is configured to localize to a cancer cell expressing LAMP5 and is labeled with a radioisotope; and (b) detecting the presence of a cancer in the subject by detecting radioactive levels emitted by the antibody or antigen binding fragment that are higher than a reference value.

26. The method of claim 25, wherein the subject is diagnosed with or is suspected of having cancer.

27. The method of claim 25 or 26, wherein the radioactive levels emitted by the antibody or antigen binding fragment are detected using positron emission tomography or single photon emission computed tomography.

28. The method of any one of claims 25-27, further comprising administering to the subject an effective amount of an immunoconjugate comprising the antibody or antigen binding fragment of any one of claims 1-16 conjugated to a radionuclide.

29. The method of claim 28, wherein the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger-emitter, or any combination thereof.

30. The method of claim 29, wherein the beta particle-emitting isotope is selected from the group consisting of86Y,90Y,89Sr,165Dy,186Re,188Re,177Lu, and67Cu. -186--7760-7933.2Atty. Dkt. No.: 642631-0168 31. A kit comprising the antibody or antigen binding fragment of any one of claims 1-16 and instructions for use.

32. The kit of claim 31, wherein the antibody or antigen binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

33. The kit of claim 31 or 32, further comprising a secondary antibody that specifically binds to the antibody of any one of claims 1-16.

34. A method for detecting LAMP5 protein expression levels in a biological sample comprising contacting the biological sample with the antibody or antigen binding fragment of any one of claims 1-16, and detecting binding to LAMP5 protein in the biological sample.

35. The method of claim 34, wherein the biological sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsied body tissue.

36. The method of claim 34 or 35, wherein the LAMP5 protein expression levels are detected via enzyme linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay, FACS, immunohistochemistry, FRET, or immunofluorescence.

37. A method for determining the prognosis of an individual of African American descent at risk for myeloma or leukemia comprising (a) detecting LAMP5 mRNA and / or protein expression levels in a sample obtained from the individual; and (b) determining that the prognosis of the individual is negative when the expression levels of LAMP5 mRNA and / or protein is elevated compared to a control sample obtained from a healthy individual of African American descent or a predetermined threshold.

38. The method of claim 37, wherein the LAMP5 protein expression levels are detected with the antibody or antigen binding fragment of any one of claims 1-16.

39. The method of claim 37 or 38, further comprising administering to the individual an effective amount of an anti-cancer therapy. -187--7760-7933.2Atty. Dkt. No.: 642631-0168 40. The method of claim 39, wherein the anti-cancer therapy is selected from among radiation therapy, hormonal therapy, chemotherapy, immunotherapy or combinations thereof.

41. A chimeric antigen receptor (CAR) comprising the antibody or antigen binding fragment of any one of claims 1-16.

42. The CAR of claim 41, wherein the chimeric antigen receptor comprises (i) an extracellular antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain.

43. The CAR of claim 42, wherein the extracellular antigen binding domain is an scFv, a Fab, or a F(ab)2.

44. The CAR of claim 42 or 43, wherein the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO:

60.

45. The CAR of claim 42 or 43, wherein the extracellular antigen binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

60.

46. The CAR of any one of claims 42-45, wherein the extracellular antigen binding domain comprises a signal peptide that is covalently joined to the N-terminus of the extracellular antigen binding domain.

47. The CAR of any one of claims 42-46, wherein the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain.

48. The CAR of any one of claims 42-47, wherein the intracellular domain comprises one or more costimulatory domains.

49. The CAR of claim 48, wherein the one or more costimulatory domains are selected from among a CD28 costimulatory domain, a 4-1BB costimulatory domain, an OX40 costimulatory domain, an ICOS costimulatory domain, a DAP-10 costimulatory domain, a PD- 1 costimulatory domain, a CTLA-4 costimulatory domain, a LAG-3 costimulatory domain, a 2B4 costimulatory domain, a BTLA costimulatory domain, a CD3ζ-chain, or any combination thereof. -188--7760-7933.2Atty. Dkt. No.: 642631-0168 50. An engineered immune cell comprising the CAR of any one of claims 41-49, wherein the engineered immune cell is a lymphocyte.

51. The engineered immune cell of claim 50, wherein the engineered immune cell is a B cell, a T cell, a tumor infiltrating lymphocyte, a CD4+ T cell, a CD8+ T cell or a natural killer (NK) cell.

52. The engineered immune cell of claim 50 or 51, wherein the engineered immune cell is derived from an autologous donor or an allogenic donor.

53. A nucleic acid encoding the CAR of any one of claims 41-49.

54. A vector comprising the nucleic acid of claim 53.

55. A host cell comprising the nucleic acid of claim 53 or the vector of claim 54.

56. A kit comprising the engineered immune cell of any one of claims 50-52, and instructions for use.

57. A method for preparing immune cells for cancer therapy comprising isolating immune cells from a donor subject; and transducing the immune cells with (a) the nucleic acid of claim 53 or (b) the vector of claim 54.

58. A method of treatment comprising isolating immune cells from a donor subject; transducing the immune cells with (a) the nucleic acid of claim 53 or (b) the vector of claim 54; and administering the transduced immune cells to a recipient subject.

59. The method of claim 58, wherein the donor subject and the recipient subject are the same or different.

60. The method of claim 58 or 59, wherein the immune cells isolated from the donor subject comprise one or more lymphocytes.

61. The method of claim 60, wherein the one or more lymphocytes is a T cell, a B cell, or a natural killer (NK) cell.

62. The method of claim 61, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

63. The method of any one of claims 58-62, wherein the immune cells isolated from the donor subject comprise tumor infiltrating lymphocytes. -189--7760-7933.2Atty. Dkt. No.: 642631-0168 64. A method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the engineered immune cell of any one of claims 50-52.

65. The method of claim 64, further comprising administering to the subject a cancer specific monoclonal antibody.

66. A method for treating or inhibiting tumor growth or metastasis in a subject with cancer comprising contacting a tumor cell with an effective amount of the engineered immune cell of any one of claims 50-52.

67. The method of any one of claims 64-66, wherein the engineered immune cell is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratumorally.

68. The method of any one of claims 64-67, further comprising administering an additional cancer therapy.

69. The method of claim 68, wherein the additional cancer therapy is selected from among chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti-cancer viruses or microorganisms, and any combinations thereof.

70. The method of any one of claims 64-69, further comprising administering a cytokine to the subject.

71. The method of claim 70, wherein the cytokine is selected from the group consisting of interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2. -190--7760-7933.2Atty. Dkt. No.: 642631-0168 72. The method of any one of claims 64-71, wherein the cancer or tumor is selected from among myeloma, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, multiple myeloma, relapsed / refractory multiple myeloma, leukemia, plasma cell leukemia, mixed lineage leukemia, acute myeloid leukemia, neuroblastoma, B-cell acute lymphoblastic leukemia, B-cell lymphoma, osteosarcoma, medulloblasatoma, Burkitt lymphoma, small cell lung carcinoma, and glioma and metastases thereof.

73. A composition comprising the antibody or antigen binding fragment of any one of claims 1-16, conjugated to an agent selected from the group consisting of drugs, toxins, or any combination thereof.

74. The composition of claim 73, wherein the drugs comprise one or more of Tesirine or MMAE.

75. A method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the composition of claim 73 or 74 and an additional cancer therapy.

76. A method for treating or inhibiting tumor growth or metastasis in a subject with cancer comprising administering to the subject an effective amount of the composition of claim 73 or 74 and an additional cancer therapy.

77. The method of claim 75 or 76, wherein the additional cancer therapy comprises one or more of Venetoclax or Idarubicin. -191--7760-7933.2

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