WO2024047558A2

WO2024047558A2 - Antigen-binding proteins and chimeric antigen receptors specific for domain 9 of human cd307e

Info

Publication number: WO2024047558A2
Application number: PCT/IB2023/058580
Authority: WO
Inventors: Maria Melina Soares; Dhivya Bharathi; Siddhartha MUKHERJEE; Lakshmikanth S. GANDIKOTA
Original assignee: Immuneel Therapeutics Private Limited
Priority date: 2022-08-31
Filing date: 2023-08-30
Publication date: 2024-03-07
Also published as: WO2024047558A3

Abstract

Disclosed are recombinant antigen-binding proteins that specifically bind human CD307e, and in particular domain 9 of huCD307e, including anti-CD307e antibodies, including antibody fragments such as single-chain fragments, and chimeric receptors including the antibodies, such as chimeric antigen receptors (CARs) or synthetic immune receptors (SIRs). Also disclosed are isolated nucleic acids encoding the antigen-binding proteins, expression vectors comprising them, and host cells, in culture, and genetically engineered cells expressing the antigen-binding proteins and/or the CARs or SIRs, and use of the antigen-binding proteins and cells in methods of treatment and other medical uses, such as, but not limited to, adoptive cell therapy, and diagnostic and prognostic assays.

Description

ANTIGEN-BINDING PROTEINS AND CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR DOMAIN 9 OF HUMAN CD307e

[0001] Sequence Listing

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 29, 2023, is named 1313PCT.xml and is 448 bytes in size.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention. The present disclosure relates in some aspects to antigenbinding proteins that specifically bind human CD307e, in particular, anti-CD307e antibodies, including antibody fragments. The present disclosure further relates to recombinant receptors containing such antibodies, including chimeric antigen receptors (CARs), which contain such antibodies. The disclosure further relates to genetically engineered cells expressing such receptors and antibodies, and use thereof in cell and gene therapy.

[0003] 2. Related Art. Human CD307e (also known as Fc receptor-like 5, FcRL5, FcRH5, or

IRTA2) is a surface protein expressed selectively on naive mature B cells and on bone marrow plasma cells; CD307e binds to all IgG isotypes. CD307e functions as an inhibitory coreceptor on mature B cells in humans via the recruitment of the phosphatase SHP-1 to its two immunoreceptor tyrosine -based inhibitory motifs (ITIM). CD307e can also exist in soluble form and high levels of soluble CD307e have been detected in the blood of patients with multiple myeloma, chronic lymphocytic leukemia, and mantle cell lymphoma. Because surface-bound CD307e is also highly-expressed on malignant B cells from multiple myeloma, chronic lymphocytic leukemia, and mantle cell lymphoma patients, CD307e may be a useful therapeutic target for the treatment of these cancers. Most patients with B cell malignancies are not cured by available therapies, including therapies targeting CD307e and/or other B cell markers. Although various CD307e-binding molecules, including anti-CD307e antibodies, and chimeric antigen receptors containing anti-CD307e antibody portions, and cells expressing such chimeric receptors, are available, improved CD307e-binding molecules and engineered CD307e-targeting cells are needed.

[0004] For example, there is a need for molecules and cells with reduced immunogenicity and/or human antibodies, including antibody fragments that specifically bind to human CD307e and chimeric receptors expressing such human antibodies for use in other antibody-based or antibody-derived therapies. Provided by the present invention are embodiments that meet such needs.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention is directed to an isolated recombinant antigen-binding protein that specifically binds domain 9 of human CD307e, which in situ is a domain proximal to the cell membrane of a cell expressing CD307e. The isolated recombinant antigen-binding protein of the invention includes:

[0006] (A) an immunoglobulin variable light chain (VL) domain comprising a set of three complementarity determining regions: LCDR1, LCDR2, and LCDR3, wherein each LCDR comprises an amino acid sequence, wherein the set of three LCDR amino acid sequences is selected from the group consisting of:

[0007] SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; SEQ ID NO:204 , SEQ ID NO:208, SEQ ID NO: 212, SEQ ID NO:205 , SEQ ID NO: 209 , SEQ ID NO: 213 , SEQ ID NO: 206 , SEQ ID NO: 210, and SEQ ID NO: 214 and

[0008] SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42; SEQ ID NO: 240, SEQ ID NO: 244 , SEQ ID NO: 241 , SEQ ID NO: 245 , SEQ ID NO: 242 and SEQ ID NO: 246 and

[0009] SEQ ID NO:58, SEQ ID NO:40, SEQ ID NO:42; SEQ ID NO: 264, SEQ ID NO: 268 , SEQ ID NO: 272 , SEQ ID NO: 265, SEQ ID NO: 269 , SEQ ID NO: 273 , SEQ ID NO: 266 , SEQ ID NO: 270 , SEQ ID NO: 274, and

[0010] SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79; SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 294, SEQ ID NO: 298, or in which one or more of the LCDR amino acid sequences of the set of three LCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residues; and

[0011] (B) an immunoglobulin variable heavy chain (VH) domain comprising a set of three complementarity determining regions: HCDR1, HCDR2, and HCDR3, wherein each HCDR comprises an amino acid sequence, wherein the set of three HCDR amino acid sequences is selected from the group consisting of:

[0012] SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 198, SEQ ID NO: 202,

[0013] SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50; SEQ ID NO: 216 , SEQ ID NO: 220 , SEQ ID NO:224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 217 , SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO:229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 218 , SEQ ID NO: 222, SEQ ID NO: 226, SEQ ID NO: 230, SEQ ID NO: 234, SEQ ID NO: 238,

[0014] SEQ ID NO:46, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257,

SEQ ID NO: 261, SEQ ID NO: 250, SEQ ID NO: 254, SEQ ID NO: 258, SEQ ID NO: 262, and

[0015] SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ

ID NO: 289, SEQ ID NO: 278 , SEQ ID NO: 282, SEQ ID NO: 286, SEQ ID NO: 290; or in which one or more of the HCDR amino acid sequences of the set of three HCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residue/s, optionally wherein the isolated recombinant antigen-binding protein specifically binds domain 9 of human CD307e with an equilibrium dissociation constant (K or KD) of < 10’⁸ M, more preferably with a K or KD of < 5 x 10’⁹ M, and even more preferably with a Kdor KD of < 5 x 10’¹⁰ M, or even < 10’¹⁰ M, as measured, e.g., by surface plasmon resonance, biolayer interferometry (BLI), and/or by ELISA.

[0016] In some particular examples, the isolated recombinant antigen-binding protein includes a set of six complementarity determining regions comprising three LCDR amino acid sequences: LCDR1, LCDR2, and LCDR3, and three HCDR amino acid sequences: HCDR1, HCDR2, and

HCDR3, the amino acid sequences being selected from the group consisting of:

(a) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16;

(b) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:38, SEQ ID NO:40, and SEQ ID NO:42; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:50;

(c) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:58, SEQ ID NO:40, and SEQ ID NO:42; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:46, SEQ ID NO:65, and SEQ ID NO:67; and

(d) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:83, SEQ ID NO:85, and SEQ ID NO:87; or in which one or more of the three LCDR amino acid sequences and/or one or more of the three HCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residue(s).

(e) Or combinations of the different humanized LCDR1, LCDR2, LCDR3 amino acid sequences and different humanized HCDR1, HCDR2 and HCDR3 amino acid sequences or in which one or more of the three LCDR amino acid sequnces and/or or or more of the three HCDR amino acid sequences comprises a conservative amino acid substitution or one or two, preferably one, amino acid residue (s).

(f)

[0017] The antigen-binding proteins of the present invention include polypeptides, such as anti-CD307e antibodies, including antigen-binding antibody fragments such as single-chain fragments including scFv fragments, and polypeptides containing such antibodies, including fusion proteins, receptors, e.g., recombinant receptors, including chimeric receptors such as chimeric antigen receptors (CARs) containing the antibody as an antigen-recognition component. In particular embodiments, the antibodies are human antibodies, such as human single-chain fragments including a Fab, a Fab', a F(ab')2, a Fv, a scFv, a scFv-Fc, a scFv-CH a scFab, a single chain antibody, a diabody, or a maxibody.

[0018] Provided are antibodies or antigen-binding fragments thereof, including those that specifically bind to CD307e. In some embodiments, the antibodies contain particular complementarity determining regions (CDRs), including heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs).

[0019] Also provided are molecules such as chimeric and/or fusion molecules, including receptors, such as recombinant receptors, that include the inventive antigen-binding protein, e.g., an antibody, of any of the embodiments (e.g., contained in or part of an extracellular domain) and additional domains, such as intracellular signaling domains, spacers, linkers, and/or transmembrane domains. In some embodiments, the receptor is a chimeric antigen receptor, comprising an extracellular portion comprising the antibody or fragment of any of the embodiments and an intracellular signaling domain.

[0020] In some embodiments, the antibody or fragment comprises an scFv. In some embodiments, the intracellular signaling domain comprises an IT AM and/or signaling domain capable of delivering a signal approximating that of natural ligation of an ITAM-containing molecule or receptor complex such as a TCR receptor complex. In some aspects, the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3) chain.

[0021] In some embodiments, the receptor further includes one or more domains, such as a transmembrane domain, linking the antibody transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain comprises a transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule, e.g., CD28, CD27, ICOS, OX-40, and/or 4- IBB. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 4-1BB, and in some embodiments, the receptor includes signaling domains from CD28 and 4- IBB.

[0022] Also provided are nucleic acids encoding the isolated recombinant antigen-binding proteins, e.g., the antibody (including fragments) of any of embodiments or the receptor, e.g., a chimeric antigen receptor (CAR) or synthetic immune receptor (SIR) of any of the embodiments, vectors including such nucleic acids, and cells containing the vectors and/or nucleic acids, for example, for expression of the antibodies and/or molecules.

[0023] Thus, also provided are cells and vectors for producing and expressing the molecules, including the antibodies, antibody fusions, and molecules such as receptors, e.g., chimeric antigen receptors (CARs), including mono-specific, bi-specific, or tri-specific CARs, or synthetic immune receptor (SIRs). For example, provided are engineered cells expressing the CAR or SIR of any of the embodiments. In some aspects, the cell is a T cell. In some aspects, the cell is an immune cell, for example, an NK cell. In some aspects, the cell is a stem cell or other human cell.

[0024] Also provided are compositions comprising the inventive isolated recombinant antigenbinding proteins, as embodied, e.g., in antibodies, receptors, engineered cells, and/or pharmaceutical compositions, e.g., further including pharmaceutically acceptable substances such as carriers or excipients.

[0025] Also provided are methods of administration or medical use (including methods of treatment), carried out by administering the inventive isolated recombinant antigen-binding proteins, as embodied, e.g., in antibodies, receptors, engineered cells, and/or pharmaceutical compositions, to a subject or to a patient in need thereof, for example, in an effective, e.g., a therapeutically effective, amount. In some embodiments, the subject or patient has, or is suspected of having, a disease or disorder associated with CD307e, such as a B cell malignancy, such as B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), pro- lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, Null-acute lymphoblastic leukemias, non-Hodgkin lymphomas, diffuse large B cell lymphomas (DLBCLs), multiple myelomas, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, or Hodgkin lymphoma, or an autoimmune or inflammatory disease in which B cells are implicated, such as, but not limited to, B cell dependent autoimmunity, non- autoimmune inflammatory disease, graft-versus-host disease (GVHD) in transplantation therapy, and in B-cell depletion therapy (BCDT).

[0026] In other aspects of the invention, the inventive isolated recombinant antigen-binding proteins are used in diagnostic and prognostic tests for a patient suffering from any disease or disorder associated with CD307e, e.g., such as a B cell malignancy, such as B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, Null-acute lymphoblastic leukemias, non-Hodgkin lymphomas, diffuse large B cell lymphomas (DLBCLs), multiple myelomas, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, or Hodgkin lymphoma, or an autoimmune or inflammatory disease in which B cells are implicated, such as, but not limited to, B cell dependent autoimmunity, non- autoimmune inflammatory disease, graft-versus-host disease (GVHD) in transplantation therapy, or in connection with B-cell depletion therapy (BCDT).

[0027] Also provided by the present invention is a method of providing anti-disease immunity in a subject comprising administering to the subject an effective amount of the engineered cell, wherein the engineered cell is an autologous or allogeneic immune cell, e.g., an autologous T cell or an allogeneic T cell, or an autologous NKT cell or an allogeneic NKT cell or an autologous or an allogeneic hematopoietic stem cell or an autologous or an allogeneic iPSC that can give rise to an immune effector cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows a schematic illustration of the three dimensional structure of the human CD307e molecule (Uniprot ID Q96RD9; SEQ ID NO: 108), determined by in silico modeling, with a number of domains 5-9 indicated, and with the relative positions shown, within CD307e domain 9, of Peptide 1 (SEQ ID NO: 109), Peptide 2 (SEQ ID NO: 110), and Peptide 3 (SEQ ID NO:111).

[0029] FIG. 2 shows representative results of ELISA assays showing hybridoma supernatant from hybridoma clones #1-44 against Peptide 3 (SEQ ID NO: 111), as described in Example 1 herein. Binding by secondary (“Sec”) antibody alone was used as a negative control. Biolegend anti-huCD307e antibody (Biolegend, Cat. No. 340304), which recognizes a membrane-distal epitope, i.e, an epitope that is not present in CD307e domain 9, did not react to Peptide 3 in the ELISA system.

[0030] FIG. 3 shows representative results of ELISA assays showing hybridoma supernatant from hybridoma clones #1-44 against Peptide 1 (SEQ ID NO: 109), as described in Example 1 herein. Binding by secondary (“Sec”) antibody alone was used as a negative control. Biolegend anti-huCD307e antibody (Biolegend, Cat. No. 340304), which recognizes a membrane-distal epitope, i.e, an epitope not present in CD307e domain 9, did not react to Peptide 1 in the ELISA system.

[0031] FIG. 4A-C shows representative flow cytometry binding data for a commercially available anti-huCD307e antibody (Biolegend, Cat. No. 340304) with RPML8226 cells (FIG. 4A), CD4/CD8 T cells (FIG. 4B), or Jurkat cells (FIG. 4C), as described in Example 2.

[0032] FIG. 5A-C shows representative flow cytometry binding data for Clone 16 with RPMI- 8226 cells (FIG. 5A), CD4/CD8 T cells (FIG. 5B), or Jurkat cells (FIG. 5C), as described in Example 2.

[0033] FIG. 6A-C shows representative flow cytometry binding data for Clone 25 with RPMI- 8226 cells (FIG. 6A), CD4/CD8 T cells (FIG. 6B), or Jurkat cells (FIG. 6C), as described in Example 2.

[0034] FIG. 7A-C shows representative flow cytometry binding data for Clone 26 with RPMI- 8226 cells (FIG. 7A), CD4/CD8 T cells (FIG. 7B), or Jurkat cells (FIG. 7C), as described in Example 2.

[0035] FIG. 8A-C shows representative flow cytometry binding data for Clone 37 with RPMI- 8226 cells (FIG. 8A), CD4/CD8 T cells (FIG. 8B), or Jurkat cells (FIG. 8C), as described in Example 2.

[0036] FIG. 9A-C shows representative flow cytometry binding data for Clone 41 with RPMI- 8226 cells (FIG. 9A), CD4/CD8 T cells (FIG. 9B), or Jurkat cells (FIG. 9C), as described in Example 2. [0037] FIG. 10 shows representative flow cytometry results for the indicated Clones (in separate panels: #2, 9, 10, 13, 15, 16, 17, 18, 22, 37, and 38), as described in Example 3 herein. The left peak in each panel resulted from experiments in which the secondary antibody (goat anti-mouse covalently conjugated to phycoerythrin (PE) or Alexa647) was added, but no hybridoma supernatant was added. The right peak in each panel represents the shift resulting in experiments in which hybridoma supernatant from the indicated Clone was also added.

[0038] FIG. 11A-D shows representative flow cytometry data from the staining of bone marrow mononuclear cells (MNC) collected from a 54-year old female patient diagnosed with multiple myeloma. In FIG. 11 A, the values in each quadrant shown represent the percentage of total cells or percentage of CD45^low ,CD45^low, CD138⁺, BCMA⁺ cells present in the sample. As described in Example 4, mAbs from Clone 15 (Figure 1 IB), Clone 25 (Figure 11C), and Clone 37 (FIG. 1 ID) bound to CD138⁺, BCMA⁺ multiple myeloma tumor cells.

[0039] FIG. 12A-E shows representative flow cytometry data from the staining of bone marrow mononuclear cells (MNC) collected from a 39-year old male patient diagnosed with relapsed multiple myeloma. In FIG. 12A, the values in each quadrant shown represent the percentage of total cells or percentage of CD45^low, CD138⁺, BCMA⁺cells present in the sample. As described in Example 4, mAbs from Clone 16 (FIG. 12B), Clone 25 (FIG. 12C), Clone 26 (FIG. 12D), and Clone 37 (FIG. 12E) bound to CD138⁺, BCMA⁺ multiple myeloma tumor cells.

[0040] FIG. 13A-F shows representative flow cytometry data from the staining of bone marrow mononuclear cells (MNC) collected from a 82-year old male patient diagnosed with multiple myeloma. In FIG. 13 A, the values in each quadrant shown represent the percentage of total cells or percentage of CD45^low, CD138⁺, BCMA⁺cells present in the sample. As described in Example 4, mAbs from Clone 15 (FIG. 13B), Clone 16 (FIG. 13C), Clone 25 (FIG. 13D), Clone 26 (FIG. 13E), and Clone 37 (FIG. 13F) bound to CD138⁺, BCMA⁺ multiple myeloma tumor cells.

[0041] FIG. 14A-L shows representative flow cytometry data from the staining of bone marrow mononuclear cells (MNC) collected from 12 different patients who met the criteria (i.e., >10% plasma cells and CRAB criteria for MM diagnosis), with mAbs from Clone 25, as described in Example 4. [0042] FIG. 15 shows a schematic representation of the vector map of plasmid 3A2D2_Clonel6_CAR_LH_CD8a_hinge, as described in Example 5.

[0043] FIG. 16 shows a schematic representation of the vector map of plasmid 3A2D2_Clonel6_CAR_LH_IgG_hinge, as described in Example 5.

[0044] FIG. 17 shows a schematic representation of the vector map of plasmid Clone25_CAR_LH_CD8a_hinge, as described in Example 5.

[0045] FIG. 18 shows a schematic representation of the vector map of plasmid Clone25_CAR_LH_IgG_hinge, as described in Example 5.

[0046] FIG. 19 shows a schematic representation of the vector map of plasmid 8A4B10_Clone26_CAR_LH_CD8a_hinge, as described in Example 5.

[0047] FIG. 20 shows a schematic representation of the vector map of plasmid 8A4B10_Clone26_CAR_LH_IgG_hinge, as described in Example 5.

[0048] FIG. 21 shows a schematic representation of the vector map of plasmid 3E5D7_Clone37_CAR_LH_CD8a_hinge, as described in Example 5.

[0049] FIG. 22 shows a schematic representation of the vector map of plasmid 3E5D7_Clone37_CAR_LH_IgG_hinge, as described in Example 5.

[0050] FIG. 23A-B shows expression on CD3073 C25-IgG4 hinge CAR-T cells at Day 8 after transduction (FIG. 23A; MOI=23), compared to untransduced control cells (FIG. 23B), as described in Example 5.

[0051] FIG. 24A-C shows expression on CD307e C25-CD8aH CAR-T cells at Day 10 after transduction (FIG. 23A-B; both MOI=1.25), compared to untransduced cells (FIG. 23C), as described in Example 5.

[0052] FIG. 25 shows killing of CD307e-expressing RPMI Multiple Myeloma Cells by CD307e C25 IgG4H-transduced CAR T cells, as described in Example 6. [0053] FIG. 26A-B shows killing of CD307e-expressing U266 Multiple Myeloma Cells by CD307e C25-CD8aH-transduced CAR T cells at MOL2.5 (FIG. 26A) or MOI=1.25 (FIG. 26B), as described in Example 6.

DETAILED DESCRIPTION OF EMBODIMENTS

[0054] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that aspects and variations described herein include "consisting" and/or "consisting essentially of" aspects and variations.

[0055] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0056] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X". [0057] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0058] As used herein, a statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a cell surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.

[0059] As used herein, a statement that a cell or population of cells is "negative" for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0060] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0061] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0062] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0063] Provided are isolated recombinant antigen-binding proteins that specifically bind to domain 9 of human CD307e, or “huCD307e,” which are used interchangeably herein. Human CD307e (also known as Fc receptor-like 5, FcRL5, FcRH5, or IRTA2). The amino acid sequence of human CD307e is known to include 977 amino residues with the following sequence (UniProt ID Q96RD9; SEQ ID NO: 108):

MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFRFY SPQKTKWYHRYLGKEILRETPDNILEVQESGEYRCQAQGSPLSSPVHLDF SSASLILQAPLSVFEGDSVVLRCRAKAEVTLNNTIYKNDNVLAFLNKRTD FHIPHACLKDNGAYRCTGYKESCCPVSSNTVKIQVQEPFTRPVLRASSFQ PISGNPVTLTCETQLSLERSDVPLRFRFFRDDQTLGLGWSLSPNFQITAMW SKDSGFYWCKAATMPYSVISDSPRSWIQVQIPASHPVLTLSPEKALNFEGT KVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENSGNY YCTADNGLGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTLHCEA

QRGSLPILYQFHHEGAALERRSANSAGGVAISFSLTAEHSGNYYCTADNG FGPQRSKAVSLSVTVPVSHPVLTLSSAEALTFEGATVTLHCEVQRGSPQIL YQFYHEDMPLWSSSTPSVGRVSFSFSLTEGHSGNYYCTADNGFGPQRSEV VSLFVTVPVSRPILTLRVPRAQAVVGDLLELHCEAPRGSPPILYWFYHEDV TLGSSSAPSGGEASFNLSLTAEHSGNYSCEANNGLVAQHSDTISLSVIVPVS RPILTFRAPRAQAVVGDLLELHCEALRGSSPILYWFYHEDVTLGKISAPSG GGASFNLSLTTEHSGIYSCEADNGLEAQRSEMVTLKVAVPVSRPVLTLRA

PGTHAAVGDLLELHCEALRGSPLILYRFFHEDVTLGNRSSPSGGASLNLSL TAEHSGNYSCEADNGLGAQRSETVTLYITGLTANRSGPFATGVAGGLLSI AGLAAGALLLYCWLSRKAGRKPASDPARSPSDSDSQEPTYHNVPAWEEL QPVYTNANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGSPIIYSEVKV

ASTPVSGSLFLASSAPHR//SEQ ID NO: 108

[0064] Domain 9 of huCD307e includes amino acid residue positions 752-850 of SEQ ID NO:108.

[0065] The term "antigen-binding protein" (or interchangeably, “antigen binding protein” or “ABP”) includes antibodies or antibody fragments, as defined above, and recombinant peptides or polypeptides or other compounds that contain sequences derived from CDRs having the desired antigen-binding properties such that they specifically bind a target antigen of interest.

[0066] In general, an antigen-binding protein, e.g., an immunoglobulin, such as an antibody or antibody fragment, "specifically binds" to an antigen of interest (e.g., huCD307e domain 9) when it has a significantly higher binding affinity for, and consequently is capable of distinguishing, that antigen, compared to its affinity for other unrelated proteins, under similar binding assay conditions. Typically, an antigen binding protein is said to "specifically bind" its target antigen when the equilibrium dissociation constant (K or KD) is 10’⁸ M. The antigen binding protein, e.g., an antibody, specifically binds antigen with "high affinity" when the K or KD is 5 X 10’⁹ M, and with "very high affinity" when the Kaor KD is 5 x IO ¹⁰ M. In one embodiment, the ABP will bind to the antigen of interest with a Kaor KD of between about 10’⁸ M and IO ¹⁰ M, and in yet another embodiment the antibodies will bind with a KD < 5 x 10’⁹.

[0067] The term "recombinant" indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other well known molecular biological procedures. Examples of such molecular biological procedures are found in Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y(1982). A "recombinant DNA molecule," is comprised of segments of DNA joined together by means of such molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and/or expresses a recombinant nucleic acid.

[0068] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and CD307e-binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0069] The term "polynucleotide" or "nucleic acid" includes both single- stranded and doublestranded nucleotide polymers containing two or more nucleotide residues. The nucleotide residues comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate and phosphoro amidate .

[0070] The term "oligonucleotide" means a polynucleotide comprising 200 or fewer nucleotide residues. In some embodiments, oligonucleotides are 10 to 60 bases in length. In other embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides may be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides may be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides may be used, for example, as PCR primers, cloning primers or hybridization probes.

[0071] A "polynucleotide sequence" or "nucleotide sequence" or "nucleic acid sequence," as used interchangeably herein, is the primary sequence of nucleotide residues in a polynucleotide, including of an oligonucleotide, a DNA, and RNA, a nucleic acid, or a character string representing the primary sequence of nucleotide residues, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence can be determined. Included are DNA or RNA of genomic or synthetic origin which may be single- or double- stranded, and represent the sense or antisense strand. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5 ' end; the left-hand direction of double- stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences;" sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences."

[0072] As used herein, an "isolated nucleic acid molecule" or "isolated nucleic acid sequence" is a nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid or (2) cloned, amplified, tagged, or otherwise distinguished from background nucleic acids such that the sequence of the nucleic acid of interest can be determined. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the immunoglobulin (e.g., antibody) where, for example, the nucleic acid molecule may be in a chromosomal location different from that of natural cells.

[0073] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of ribonucleotides along the mRNA chain, and also determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the RNA sequence and for the amino acid sequence.

[0074] The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Genes typically include coding sequences and/or the regulatory sequences required for expression of such coding sequences. The term "gene" applies to a specific genomic or recombinant sequence, as well as to a cDNA or mRNA encoded by that sequence.

[0075] A "fusion gene" contains a coding region that encodes a fusion polypeptide. Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non- expressed regulatory sequences including transcriptional control elements to which regulatory proteins, such as transcription factors, bind, resulting in transcription of adjacent or nearby sequences.

[0076] "Expression of a gene" or "expression of a nucleic acid" means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent post- translational modification of the polypeptide), or both transcription and translation, as indicated by the context.

[0077] As used herein the term "coding region" or "coding sequence" when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

[0078] The term "control sequence" or "control signal" refers to a polynucleotide sequence that can, in a particular host cell, affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend upon the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. Control sequences for eukaryotes may include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences or elements, poly adenylation sites, and transcription termination sequences. “Control sequences” can include leader sequences and/or fusion partner sequences, and/or insulator elements.

[0079] A “promoter” is a region of DNA including a site at which RNA polymerase binds to initiate transcription of messenger RNA by one or more downstream structural genes. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand). Promoters are typically about 100-1000 bp in length. A common feature of promoter regions in eukaryotes is the TATA box, it is found about 25 bases upstream from the transcription start site and has the ideal sequence of TATAAAT. The closeness of the actual sequence to this ideal sequence effects the ability of the RNA polymerase complex to bind to the DNA and hence initiate the transcription process.

[0080] In some embodiments, the inventive recombinant expression vector includes an expression cassette comprising a weak constitutive promoter, operably linked to an open reading frame encoding a selectable marker. A “constitutive promoter” is: (1) a promoter sequence that initiates mRNA synthesis independent of the influence of regulation, or (2) a promoter sequence that initiates mRNA synthesis independent, or substantially independent, of the influence of regulation, under physiological conditions normally associated with cell culture for the expression of a protein of interest in an industrial protein manufacturing setting. The specific nucleic acid (i.e., nucleotide) sequence of the promoter determines the strength of the promoter (a strong promoter leads to a relatively high rate of transcription initiation). Commonly used constitutive promoters for mammalian cell systems include simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken P-Actin promoter coupled with CMV early enhancer (CAGG). A “weak constitutive promoter” is a constitutive promoter that will not initiate transcription as efficiently as a strong promoter, for example, a modified version of one of the foregoing list of constitutive promoters. Thus, an example of a weak constitutive promoter is a deleted SV40 promoter. (See, e.g., Hartman et al., Vectors and host cells comprising a modified SV40 promoter for protein expression, US 10053720; and Hartman et al., DAC HYP compositions and methods, US9260528B2). Another useful example of a weak constitutive promoter for purposes of the present invention is the PGK promoter described in Qin JY et al. (Qin, JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren B-Z, et al. (2010), Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter, PLoS ONE 5(5): el0611. doi.org/10.1371/journal.pone.0010611; see, also, e.g., Li, J. & Zhang, Y., Relationship between promoter sequence and its strength in gene expression, Eur. Phys. J. E 37: 86 (2014)).

[0081] In general, an “enhancer” is a short (50-1500 bp) region of DNA that can be bound with one or more activator proteins (transcription factors) to activate transcription of a gene. Promoters and enhancers consist of short arrays of DNA that interact specifically with cellular proteins involved in transcription (Maniatis, et al, Science 236: 1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al, Trends Biochem. Sci., 11 :287 (1986) and Maniatis, et al, Science 236: 1237 (1987)).

[0082] “Insulator elements,” or interchangeably, “insulator sequences,” are DNA sequences that protect transcription units from outside regulatory influence. When placed between a transcription unit and an enhancer sequence, these elements can block the action of the enhancer sequence on the transcription unit. In constructs where insulator sequences flank a transcription unit, they can confer position independent expression when transfected in cells. Many insulator elements have been identified and characterized including the chicken HS4, mouse Hl 9, and Xenopus ARS. (See, e.g., Kanduri, C. et al., The 5’ flank of mouse Hl 9 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication, Current Biology 10(8):449-457 (2000); Recillas-Targa, F. et al., Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities. Proc. Nat. Acad. Sci. USA 99(10):6883-6888 (2002); Valenzuela, L., & Kamakaka, R. T., Chromatin Insulators, Annu.

Rev. Genet. 40:107-38 (2006); Watanabe, S. et al., Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells, Genes to Cells, 11(9), 1009-1021 (2006)).

[0083] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein coding information into a host cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." The term "expression vector" or "expression construct" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of the operably linked coding sequence in a particular host cell. An expression vector can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect R A splicing of a coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence of interest, so that the expressed polypeptide can be secreted by the recombinant host cell, for more facile isolation of the polypeptide of interest from the cell, if desired. Such techniques are well known in the art. (E.g., Goodey, Andrew R.; et al, Peptide and DNA sequences, U.S. Patent No. 5,302,697; Weiner et al, Compositions and methods for protein secretion, U.S. Patent No.

6,022,952 and U.S. Patent No. 6,335,178; Uemura et al, Protein expression vector and utilization thereof, U.S. Patent No. 7,029,909; Ruben et al, 27 human secreted proteins, US 2003/0104400 Al; McGrew et al., Hybrid Promoter and Uses Thereof, US 11028410B2; McGrew et a\., Expression from Transposon-Based Vectors and Uses, US 11098310B2; McGrew et al., Inducible Expression From Transposon-Based Vectors and Uses, US 2019/0185881A1).

[0084] The terms "in operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0085] The term "host cell" means a cell that has been transformed, or is capable of being transformed, with a nucleic acid and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present. Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial host cells in culture include bacteria (such as Escherichia coli sp.), yeast (such as Saccharomyces sp.) and other fungal cells, insect cells, plant cells, mammalian (including human) cells, e.g., CHO cells and HEK-293 cells.

[0086] Modifications can be made at the DNA level, as well. The peptide-encoding DNA sequence may be changed to codons more compatible with the chosen host cell. For E. coli, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art.

[0087] The term "transfection" means the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al, 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al, 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0088] The term "transformation" refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been "stably transformed" when the transforming DNA is replicated with the division of the cell.

[0089] By "physiologically acceptable salt" of a composition of matter, for example a salt of the immunoglobulin, such as an antibody, is meant any salt or salts that are known or later discovered to be pharmaceutically acceptable. Some non- limiting examples of pharmaceutically acceptable salts are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; maleate; tartrate; glycolate; gluconate; succinate; mesylate; besylate; salts of gallic acid esters (gallic acid is also known as 3,4, 5 trihydroxybenzoic acid) such as PentaGalloylGlucose (PGG) and epigallocatechin gallate (EGCG), salts of cholesteryl sulfate, pamoate, tannate and oxalate salts.

[0090] " Treatment" or "treating" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Treatment" includes any indicia of success in the amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, self-reporting by a patient, neuropsychiatric exams, and/or a psychiatric evaluation.

[0091] An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with migraine headache. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount or dose sufficient to remedy or mitigate a disease state (e.g., COVID-19 infection), particularly any symptom(s) associated with the disease state of COVID- 19, or otherwise prevent, hinder, retard or reverse the progression of the disease state COVID-19 or any other undesirable symptom associated with the disease in any way whatsoever (i.e. that provides "therapeutic efficacy"). A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of migraine headache or multiple sclerosis symptoms, or reducing the likelihood of the onset (or reoccurrence) of migraine headache, migraine headache symptoms, or multiple sclerosis symptoms. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophy tactically effective amount may be administered in one or more administrations. Useful clinical administration modalities include intravenous injection or infusion, intramuscular, intratumoral, intraperitoneal, or subcutaneous injection.

[0092] " Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, rats, mice, monkeys, etc. Preferably, the mammal is human.

[0093] The term "naturally occurring" as used throughout the specification in connection with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials which are found in nature.

[0094] In some embodiments the antigen-binding protein of the invention is an antibody. The term "antibody", or interchangeably “Ab,” is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies (including human, humanized or chimeric antibodies), monomeric, homodimeric, and heterodimeric antibodies, polyclonal antibodies, multi- specific antibodies (e.g., bispecific antibodies), and antibody fragments that can bind antigen (e.g., Fab, Fab', F(ab')2, Fv, single chain antibodies, diabodies), comprising complementarity determining regions (CDRs) of the foregoing as long as they exhibit the desired biological activity. Multimers or aggregates of intact molecules and/or fragments, including chemically derivatized antibodies, are contemplated. The term “antibody” encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. Antibodies of any isotype class or subclass, including IgG, IgM, IgD, IgA, and IgE, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, or any allotype, are contemplated. Different isotypes have diff erent effector functions; for example, IgGl and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC) activity.

[0095] "Antigen binding region," or interchangeably, "antigen binding site," means a portion of a protein, that specifically binds a specified antigen, e.g., huCD307e domain 9. For example, that portion of an antigen binding protein that contains the amino acid residues that interact with an antigen and confer on the antigen binding protein its specificity and affinity for the antigen is referred to as "antigen binding region." An antigen binding region typically includes one or more "complementarity determining regions" ("CDRs"). Certain antigen binding regions also include one or more "framework" regions ("FRs"). A "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen binding region and an antigen. In a traditional antibody, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions responsible for antigen binding and recognition. A variable region of an immunoglobulin antigen binding protein comprises at least three heavy or light chain CDRs, see, supra (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al, 1989, Nature 342: 877-883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991, supra; see also Chothia and Lesk, 1987, supra).

[0096] An "isolated" antigen-binding protein, for example an antibody or other immunoglobulin molecule, is one which has been separated from one or more components of its natural environment or of a culture medium in which it has been secreted by a producing cell. In some embodiments, an ABP, such as antibody, is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). "Contaminant" components of an ABP’s natural environment or medium are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody, and most preferably more than 99% by weight, or (2) to homogeneity by SDS-PAGE under reducing or nonreducing conditions, optionally using a stain, e.g., Coomassie blue or silver stain. Isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Typically, however, isolated antibody will be prepared by at least one purification step.

[0097] 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. Monoclonal antibodies that are antigen binding proteins are highly specific binders, being directed against an individual antigenic site or epitope, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes. Nonlimiting examples of monoclonal antibodies include murine, rabbit, rat, chicken, chimeric, humanized, or human antibodies, monomeric, homodimeric, and heterodimeric antibodies, fully assembled antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments that can bind an antigen (including, Fab, Fab', F(ab')2, Fv, single chain antibodies, diabodies), maxibodies, nanobodies (i.e., VHH or single domain antibodies), and recombinant peptides comprising CDRs of the foregoing as long as they exhibit the desired biological activity, or variants or derivatives thereof.

[0098] A "domain" or "region" (used interchangeably herein) of a protein is any portion of the entire protein, up to and including the complete protein, but typically comprising less than the complete protein. A domain can, but need not, fold independently of the rest of the protein chain and/or be correlated with a particular biological, biochemical, or structural function or location (e.g., a ligand binding domain, or a cytosolic, transmembrane or extracellular domain).

[0099] 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. For example, the monoclonal antibodies to be used in accordance with the present invention 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 in Clackson et al, Nature, 352:624-628(1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991), for example. [00100] A "multi- specific" binding agent or antigen binding protein or antibody is one that targets more than one antigen or epitope.

[00101] A "bispecific," "dual-specific" or "bifunctional" binding agent or antigen binding protein or antibody is a hybrid having two different antigen binding sites. Biantigen binding proteins, antigen binding proteins and antibodies are a species of multiantigen binding protein, antigen binding protein or multispecific antibody and may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al, 1992, J. Immunol. 148: 1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes, which may reside on the same or different protein targets.

[00102] The term "immunoglobulin" encompasses full antibodies comprising two dimerized heavy chains (HC), each covalently linked to a light chain (LC); a single undimerized immunoglobulin heavy chain and covalently linked light chain (HC + LC), or a chimeric immunoglobulin (light chain + heavy chain)-Fc heterotrimer (a so-called "hemibody"). An "immunoglobulin" is a protein, but is not necessarily an antigen binding protein.

[00103] In a typical “antibody,” each tetramer is composed of two identical pairs of polypeptide chains (a homodimer), each pair having one "light" chain of about 220 amino acids (about 25 kDa) and one "heavy" chain of about 440 amino acids (about 50-70 kDa). The amino-terminal portion of each chain includes a "variable" ("V") region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. The variable region differs among different antibodies. The constant region is the same among different antibodies. Within the variable region of each heavy or light chain, there are three hypervariable subregions that help determine the antibody's specificity for antigen in the case of an antibody that is an antigen binding protein. The variable domain residues between the hypervariable regions are called the framework residues and generally are somewhat homologous among different antibodies.

Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. Human light chains are classified as kappa (K) and lambda (1) light chains. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Within the scope of the invention, an "antibody" also encompasses a recombinantly made antibody, and antibodies that are glycosylated or lacking glycosylation.

[00104] The term "light chain" (LC), or interchangeably, "immunoglobulin light chain," includes a full- length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[00105] The term "heavy chain" (HC), or interchangeably, "immunoglobulin heavy chain," includes a full- length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CHI, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxyl- terminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains are classified as mu (p), delta (A), gamma (y), alpha (a), and epsilon (a), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In separate embodiments of the invention, heavy chains may be of any isotype, including IgG (including IgGl , IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE. Several of these may be further divided into subclasses or isotypes, e.g. IgGl , IgG2, IgG3, IgG4, IgAl and IgA2. Different IgG isotypes may have different effector functions (mediated by the Fc region), such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcyRs) on the surface of immune effector cells such as natural killers and macrophages, leading to the phagocytosis or lysis of the targeted cells. In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface.

[00106] An "Fc region", or used interchangeably herein, "Fc domain" or

"immunoglobulin Fc domain", contains two heavy chain fragments, which in a full antibody comprise two complement receptor domains (CR1 and CR2) of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[00107] The term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[00108] "Allotypes" are variations in antibody sequence, often in the constant region, that can be immunogenic and are encoded by specific alleles in humans. Allotypes have been identified for five of the human IGHC genes, the IGHG1, IGHG2, IGHG3, IGHA2 and IGHE genes, and are designated as Glm, G2m, G3m, A2m, and Em allotypes, respectively. At least 18 Gm allotypes are known: nGlm(l), nGlm(2), Glm (1, 2, 3, 17) or Glm (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (bl, c3, b5, bO, b3, b4, s, t, gl, c5, u, v, g5). There are two A2m allotypes A2m(l) and A2m(2).

[00109] For a detailed description of the structure and generation of antibodies, see Roth, D.B., and Craig, N.L., Cell, 94:411-414 (1998), herein incorporated by reference in its entirety. Briefly, the process for generating DNA encoding the heavy and light chain immunoglobulin sequences occurs primarily in developing B-cells. Prior to the rearranging and joining of various immunoglobulin gene segments, the V, D, J and constant (C) gene segments are found generally in relatively close proximity on a single chromosome. During B -cell-differentiation, one of each of the appropriate family members of the V, D, J (or only V and J in the case of light chain genes) gene segments are recombined to form functionally rearranged variable regions of the heavy and light immunoglobulin genes. This gene segment rearrangement process appears to be sequential. First, heavy chain D-to- J joints are made, followed by heavy chain V-to-DJ joints and light chain V-to-J joints. In addition to the rearrangement of V, D and J segments, further diversity is generated in the primary repertoire of immunoglobulin heavy and light chains by way of variable recombination at the locations where the V and J segments in the light chain are joined and where the D and J segments of the heavy chain are joined. Such variation in the light chain typically occurs within the last codon of the V gene segment and the first codon of the J segment. Similar imprecision in joining occurs on the heavy chain chromosome between the D and 1Z2 segments and may extend over as many as 10 nucleotides. Furthermore, several nucleotides may be inserted between the D and 1Z2 and between the VH and D gene segments which are not encoded by genomic DNA. The addition of these nucleotides is known as N-region diversity. The net effect of such rearrangements in the variable region gene segments and the variable recombination which may occur during such joining is the production of a primary antibody repertoire.

[00110] The term "hypervariable" region refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a complementarity determining region or CDR (i.e., residues 24-34 (LI), 50-56 (L2) and S T (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Even a single CDR may recognize and bind antigen, although with a lower affinity than the entire antigen binding site containing all of the CDRs.

[00111] An alternative definition of residues from a hypervariable "loop" is described by Chothia et al, J. Mol.Biol. 196: 901-917 (1987) as residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain.

[00112] " Framework" or "FR" residues are those variable region residues of an antigen binding protein other than the hypervariable region residues.

[00113] "Antibody fragments" comprise a portion of an intact full length antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al, Protein Eng.,8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[00114] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment which contains the constant region. The Fab fragment contains all of the variable domain, as well as the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. The Fc fragment displays carbohydrates and is responsible for many antibody effector functions (such as binding complement and cell receptors), that distinguish one class of antibody from another. [00115] Pepsin treatment yields an F(ab')2 fragment that has two "Single-chain Fv" or "scFv" antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Fab fragments differ from Fab' fragments by the inclusion of a few additional residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[00116] A "Fab fragment" is comprised of one light chain and the CHI and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[00117] " Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[00118] A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[00119] " Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH/VL dimer. A single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site.

[00120] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and United States Patent No. 4,946,778 and No. 5,260,203, the disclosures of which are incorporated by reference in their entireties.

[00121] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain, and optionally comprising a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding (Bird et al, Science 242:423-426, 1988, and Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). An "Fd" fragment consists of the VH and CHI domains.

[00122] 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 (VH VL). 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 antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[00123] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[00124] The term "epitope" is the portion of a target molecule that is bound by an antigen binding protein (for example, an antibody or antibody fragment). The term includes any determinant capable of specifically binding to an antigen binding protein, such as an antibody or to a T-cell receptor. An epitope can be contiguous or non-contiguous (e.g., in a single-chain polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within the context of the molecule are bound by the antigen binding protein). In certain embodiments, epitopes may be mimetic in that they comprise a three-dimensional structure that is similar to an epitope used to generate the antigen binding protein, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antigen binding protein. Most often, epitopes reside on proteins, but in some instances may reside on other kinds of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Generally, antigen binding proteins specific for a particular target will preferentially recognize an epitope on the target in a complex mixture of proteins and/or macromolecules .

[00125] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BEAST or FASTA, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) can be used in conjunction with the computer program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences. In calculating percent identity, the sequences being compared are aligned in a way that gives the largest match between the sequences.

[00126] The GCG program package is a computer program that can be used to determine percent identity, which package includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or two polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3. times, the average diagonal, wherein the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[00127] Recommended parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program include the following:

[00128] Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

[00129] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

[00130] Gap Penalty: 12 (but with no penalty for end gaps)

[00131] Gap Length Penalty: 4

[00132] Threshold of Similarity: 0

[00133] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full- length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

[00134] The term "modification" when used in connection with proteins of interest, include, but are not limited to, one or more amino acid changes (including substitutions, insertions or deletions); chemical modifications; covalent modification by conjugation to therapeutic or diagnostic agents; labeling (e.g., with radionuclides or various enzymes); covalent polymer attachment such as PEGylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of non-natural amino acids. By methods known to the skilled artisan, proteins, can be “engineered” or modified for improved target affinity, selectivity, stability, and/or manufacturability before the coding sequence of the “engineered” protein is included in the expression cassette.

[00135] Conservatively modified forms of the antigen-binding proteins disclosed herein are also contemplated as being embodiments of the present invention. A "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native or non- canonical residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis" (see, for example, MacLennan et al, Acta Physiol. Scand. SuppL, 643:55-67 (1998); Sasaki et al, 1998, Adv. Biophys. 35: 1-24 (1998), which discuss alanine scanning mutagenesis).

[00136] Desired amino acid substitutions (whether conservative or non- conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-conjugated peptide molecules described herein.

[00137] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table A, herein. Amino acid substitutions may be introduced into a binding molecule, e.g., an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. [00138] Naturally occurring residues may be divided into classes based on common side chain properties:

[00139] 1) hydrophobic: norleucine (Nle), Met, Ala, Vai, Leu, He;

[00140] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[00141] 3) acidic: Asp, Glu;

[00142] 4) basic: His, Lys, Arg;

[00143] 5) residues that influence chain orientation: Gly, Pro; and

[00144] 6) aromatic: Trp, Tyr, Phe.

[00145] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues (e.g., norleucine (Nle)), which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.

[00146] In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cy stine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (- 3.9); and arginine (-4.5).

[00147] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al, 1982, J. Mol. Biol.

157: 105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making conservative amino acid substitutions based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included.

[00148] It is also understood in the art that the conservative amino acid substitutions of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[00149] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within +2 is included, in certain embodiments, those that are within +1 are included, and in certain embodiments, those within +0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions."

[00150] Examples of conservative amino acid substitutions include the substitution of one nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, norleucine (Nle), alanine, or methionine for another, the substitution of one polar (hydrophilic) amino acid residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic amino acid residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase "conservative amino acid substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such polypeptide displays the requisite bioactivity. Other exemplary amino acid substitutions that can be useful in accordance with the present invention are set forth in Table A below. [00151] Table A. Some Useful Conservative Amino Acid Substitutions. Conventional three- letter abbreviations are shown.

[00152] Cloning DNA

[00153] Cloning of DNA is carried out using standard techniques (see, e.g., Sambrook et al.

(1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, which is incorporated herein by reference). For example, a cDNA library may be constructed by reverse transcription of polyA+ mRNA, preferably membrane-associated mRNA, and the library screened using probes specific for human immunoglobulin polypeptide gene sequences. In one embodiment, however, the polymerase chain reaction (PCR) is used to amplify cDNAs (or portions of full-length cDNAs) encoding an immunoglobulin gene segment of interest (e.g., a light or heavy chain variable segment). The amplified sequences can be readily cloned into any suitable vector, e.g., expression vectors, minigene vectors, or phage display vectors. It will be appreciated that the particular method of cloning used is not critical, so long as it is possible to determine the sequence of some portion of the protein of interest.

[00154] One source for antibody nucleic acids is a hybridoma produced by obtaining a B cell from an animal immunized with the antigen of interest and fusing it to an immortal cell. Alternatively, nucleic acid can be isolated from B cells (or whole spleen) of the immunized animal. Yet another source of nucleic acids encoding antibodies is a library of such nucleic acids generated, for example, through phage display technology. Polynucleotides encoding peptides of interest, e.g., variable region peptides with desired binding characteristics, can be identified by standard techniques such as panning.

[00155] Sequencing of DNA is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which is incorporated herein by reference). By comparing the sequence of the cloned nucleic acid with published sequences of genes and cDNAs, one of skill will readily be able to determine, depending on the region sequenced. One source of gene sequence information is the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD. Gene sequencing can also be done, for example, by standard methods or by so-called “Nextgeneration” sequencing of engineered DNA constructs prior to transfection. (See, e.g., Buermans, H. P. J., & den Dunnen, J. T., Next generation sequencing technology: Advances and applications, Biochimica et Biophysica Acta - Molecular Basis of Disease 7542(10): 1932-1941 (2014)).

[00156] Chemical synthesis of parts or the whole of a coding region containing codons reflecting desires protein changes can be cloned into an expression vector by either restriction digest and ligation of 5' and 3' ends of fragments or the entire open reading frame (ORF), containing nucleotide overhangs that are generated by restriction enzyme digestion and which are compatible to the destination vector. The fragments or inserts are typically ligated into the destination vector using a T4 ligase or other common enzyme. Other useful methods are similar to the above except that the cut site for the restriction enzyme is at location different from the recognition sequence. Alternatively, isothermal assembly (i.e., “Gibson Assembly”) can be employed, in which nucleotide overhangs are generated during synthesis of fragments or ORFs; digestion by exonucleases is employed. Alternatively, nucleotide overhangs can be ligated ex vivo by a ligase or polymerase or in vivo by intracellular processes.

[00157] Alternatively, homologous recombination can be employed, similar to isothermal assembly, except exonuclease activity of T4 DNA ligase can used on both insert and vector and ligation can be performed in vivo.

[00158] Another useful cloning method is the so-called “TOPO” method, in which a complete insert containing a 3' adenosine overhang (generated by Taq polymerase) is present, and Topoisomerase I ligates the insert into a TOPO vector.

[00159] Another useful cloning method is degenerate or error-prone PCR exploiting degenerate primers and/or a thermally stable low-fidelity polymerase caused by the polymerase within certain reaction conditions. Fragments or inserts are then cloned into an expression vector.The above are merely examples of known cloning techniques, and the skilled practitioner knows how to employ any other suitable cloning techniques.

[00160] Isolated DNA can be operably linked to control sequences or placed into expression vectors, which are then transfected into host cells that do not otherwise produce immunoglobulin protein, to direct the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is well known in the art.

[00161] Nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[00162] Many vectors are known in the art. Vector components may include one or more of the following: a signal sequence (that may, for example, direct secretion of the expressed protein by the recombinant host cells, i.e., the engineered cells); an origin of replication, one or more selective marker genes (that may, for example, confer antibiotic or other drug resistance, complement auxotrophic deficiencies, or supply critical nutrients not available in the media), an enhancer element, a promoter, and a transcription termination sequence, all of which are well known in the art.

[00163] Protein expression and Cell Culture

[00164] The inventive method for manufacturing a purified protein of interest (e.g., but not limited to, a protein drug substance) involves culturing protein-secreting mammalian cells. Such cultured mammalian cells are typically made by recombinant DNA technology involving transient or stable transfection, e.g., the pooled plasmid constructs (expression vectors) from the cloning step can be transfected into a plurality of host cells (e.g., mammalian, e.g., HEK 293 or CHO, bacterial, insect, yeast cells) for expression using a cationic lipid, polyethylenimine, Lipof ectamine™, or ExpiFectamine™, or electroporation. The skilled practitioner is aware of numerous suitable means for transfecting to achieve expression of recombinant antibodies. Alternatively, methods for stable genomic integration of expressions cassettes encoding the protein of interest can be employed to make a production cell line of protein-secreting mammalian cells. (See, e.g., Zhang, Crispr-Cas Systems and Methods for Altering Expression Of Gene Products, WO2014093661 A2; Frendewey et al., Methods and Compositions for the Targeted Modification of a Genome, US9228208 B2; Church et al., Multiplex Automated Genome Engineering, W02008052101A2, US8153432 B2; Bradley et al., Methods Cells and Organisms, US2015/0079680 Al; Begemann et al., Compositions and Methods for Modifying Genomes, WO2017141173A2; Gill et al., Nucleic acid-guided nucleases, US9982279 Bl; Minshull et al., Enhanced nucleic acid constructs for eukaryotic gene expression, US9428767B2, US9580697B2, US9574209B2; Minshull et al., DNA Vectors, Transposons And. Transposases For Eukaryotic Genome Modification, US10041077B2; McGrew et al., Hybrid Promoter and Uses Thereof, US 11028410B2; McGrew et al., Expression from Transposon-Based Vectors and Uses, US 11098310B2; McGrew et al., Inducible Expression From Transposon-Based Vectors and Uses, US 2019/0185881A1).

[00165] Optionally, the transfectant or transformant cells will be provided with a recombinant expression cassette for a selectable marker, for example, but not limited to, one or more of the following: glutamine synthase (or glutamine synthetase; GS), dihydrofolate reductase (DHFR), puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acetyl transferase, or a protein that binds to zeocin.

[00166] The protein of interest is typically obtained by culturing the transfected or transformed host cells under physiological conditions allowing the cells to express recombinant proteins.

Most conveniently, the expressed recombinant proteins are directly secreted into the extracellular culture medium (by employing appropriate secretory-directing signal peptides) and are harvested therefrom; otherwise, additional steps will be needed to isolate the expressed antibodies from a cell extract.

[00167] Useful secretory signal peptide (SP) sequences are known in the art, and these can be added, adjacent or distal, to any of the sequences shown in Table 1, herein, for the purpose of facilitating secretion of the inventive antigen-binding protein. An example of a useful SP sequence is the IGKVl-39*01 SP signal peptide:

[00168] MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 112). Other examples of useful SP sequences include:

[00169] MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 113),

[00170] MEWTWRVLFLVAAATGAHS (SEQ ID NO: 114),

[00171] METPAQLLFLLLLWLPDTTG (SEQ ID NO: 115), [00172] MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 116),

[00173] MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 117), but any other suitable signal peptide sequence may be employed within the scope of the invention.

[00174] The desired scale of the recombinant expression will be dependent on the type of expression system and the desired quantity of protein production. Some expression systems such as ExpiCHO™ usually produce higher yields as compared to some earlier HEK293 technologies. A smaller scale ExpiCHO™ might then suffice as compared to an HEK293 system. Efficiency of transfection can also be a consideration in choosing an appropriate expression system. Electroporation can be a suitable method given its effectiveness, relative low cost and the fact that high-throughput during this step is not critical. Additionally, the ratio of immunoglobulin light chain to heavy chain can be varied during the co-transfection to improve expression of certain variants. The product yield for a given variant has to be sufficient to survive numerous handling steps and produce a signal high enough to be detected by the chosen fluorescence detector.

[00175] In general, the transfected or transformed host cells are typically cultured by any conventional type of culture, such as batch, fed-batch, intensified fed-batch, or continuous. Suitable continuous cultures included repeated batch, chemostat, turbidostat or perfusion culture with product and cell retention or solely cell retention. However, for purposes of the invention, culturing is carried out in one or more single-use perfusion bioreactors, each of which can contain a volume of liquid culture medium of about 50 L to about 4000 L (e.g., 50 L, 60 L, 75 L, 100 L, 250 L, 500 L, 650 L, 750 L, 1000 L, 1250 L, 1500 L, 1750 L, 2000 L, 2250 L, 2500 L, 2750 L, 3000 L, 3250 L, 3500 L, 3750 L, or 4000 L), as desired. The number of single-use bioreactors employed to culture the cells is one, two, three, four, five, or six single-use perfusion bioreactors of the desired volume(s).

[00176] The host cells used to produce the protein of interest or “POI” (e.g., non-glycosylated or glycosylated proteins) in the invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; W090103430; WO 87/00195; or U.S. Patent Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source, such that the physiological conditions of the cell in, or on, the medium promote expression of the protein of interest by the host cell; any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[00177] The culture conditions, to be predetermined, such as temperature (for mammalian cells, typically, but not necessarily, about 37° ± 1°C), pH (typically, but not necessarily, the cell culture medium is maintained within the range of about pH 6.5-7.5), oxygenation, and the like, will be apparent to the ordinarily skilled artisan. By "culturing at" or "maintaining at" a predetermined culture condition, is meant that the process control systems are set at a particular value for that condition, in other words the intended, volume, target temperature, pH, oxygenation level, or the like, maintained at predetermined set points for each parameter, within a narrow range (i.e., “narrow deadband”) most optimal for the cell line and protein product of interest. Clearly, there will be small variations of the temperature, pH, or other culture condition over time, and from location to location through the culture vessel (i.e., the bioreactor). (See, also, e.g., Oguchi et al., pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture, Cytotechnology. 52(3): 199-207 (2006); Al-Fageeh et al., The coldshock response in cultured mammalian cells: Harnessing the response for the improvement of recombinant protein production, Biotechnol. Bioeng. 93:829-835 (2006); Marchant, R.J. et al., Metabolic rates, growth phase, and mRNA levels influence cell-specific antibody production levels from in vitro cultured mammalian cells at sub-physiological temperatures, Mol.

Biotechnol. 39:69-77 (2008)).

[00178] Digital control units and sensory monitors are available commercially or can be constructed by the skilled artisan for use with cell culture bioreactors. Alternative digital control units (DCU) control and monitor the cell culture process are available commercially, made by companies such as B. Braun, New Brunswick, Sartorius, or Thermo Fisher Scientific. Other on- line or off-line analyses can include off-gas measurements by mass spectrometry, in-depth determination of media composition (amino acids, vitamins, trace minerals) and expanded examination of cellular metabolites other than CO2 and lactic acid.

[00179] Examples of epitope tags include the flu HA tag polypeptide and its antibody 12CA5 (Field et al, Mol. Cell. Biol. 8: 2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al, Mol. Cell. Biol. 5(12): 3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al, Protein Engineering 3(6): 547-553 (1990)). Other exemplary tags are a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, NY) are well known and routinely used in the art.

[00180] Some particular, non-limiting, embodiments of amino acid substitution variants of the inventive immunoglobulins, including antibodies and antibody fragments are exemplified below.

[00181] Any cysteine residue not involved in maintaining the proper conformation of the immunoglobulin also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the immunoglobulin to improve its stability (particularly where the immunoglobulin is an antibody fragment such as an Fv fragment).

[00182] In certain instances, immunoglobulin variants are prepared with the intent to modify those amino acid residues which are directly involved in epitope binding in a starting sequence. In other embodiments, modification of residues which are not directly involved in epitope binding or residues not involved in epitope binding in any way, is desirable, for purposes discussed herein. Mutagenesis within any of the CDR regions and/or framework regions is contemplated.

[00183] In order to determine which antigen binding protein amino acid residues are important for epitope recognition and binding, alanine scanning mutagenesis can be performed to produce substitution variants. See, for example, Cunningham et al, Science, 244: 1081-1085 (1989), the disclosure of which is incorporated herein by reference in its entirety. In this method, individual amino acid residues are replaced one-at-a-time with an alanine residue and the resulting antibody is screened for its ability to bind its specific epitope relative to the unmodified polypeptide. Modified antigen binding proteins with reduced binding capacity are sequenced to determine which residue was changed, indicating its significance in binding or biological properties.

[00184] Substitution variants of antigen binding proteins can be prepared by affinity maturation wherein random amino acid changes are introduced into the parent polypeptide sequence. See, for example, Ouwehand et al, Vox Sang 74 (Suppl 2):223-232, 1998; Rader et al, Proc. Natl. Acad. Sci. USA 95 :8910-8915, 1998; DaU'Acqua et al, Curr. Opin. Struct. Biol. 8:443-450, 1998, the disclosures of which are incorporated herein by reference in their entireties. Affinity maturation involves preparing and screening the antigen binding proteins, or variants thereof and selecting from the resulting variants those that have modified biological properties, such as increased binding affinity relative to the parent antigen binding protein. A convenient way for generating substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites are mutated to generate all possible amino substitutions at each site. The variants thus generated are expressed in a monovalent fashion on the surface of filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). See e.g., WO 92/01047, WO 93/1 12366, WO 95/15388 and WO 93/19172.

[00185] Current antibody affinity maturation methods belong to two mutagenesis categories: stochastic and nonstochastic. Error prone PCR, mutator bacterial strains (Low et al, J. Mol. Biol. 260, 359-68, 1996), and saturation mutagenesis (Nishimiya et al, J. Biol. Chem. 275: 12813-20, 2000; Chowdhury, P. S. Methods Mol. Biol. 178, 269-85, 2002) are typical examples of stochastic mutagenesis methods (Rajpal et al, Proc Natl Acad Sci U S A. 102:8466-71, 2005). Nonstochastic techniques often use alanine- scanning or site-directed mutagenesis to generate limited collections of specific muteins. Some methods are described in further detail below.

[00186] Affinity maturation via panning methods — Affinity maturation of recombinant antibodies is commonly performed through several rounds of panning of candidate antibodies in the presence of decreasing amounts of antigen. Decreasing the amount of antigen per round selects the antibodies with the highest affinity to the antigen thereby yielding antibodies of high affinity from a large pool of starting material. Affinity maturation via panning is well known in the art and is described, for example, in Huis et al. (Cancer Immunol Immunother. 50: 163-71, 2001). Methods of affinity maturation using phage display technologies are described elsewhere herein and known in the art (see e.g., Daugherty et al, Proc Natl Acad Sci USA. 97:2029-34, 2000).

[00187] Look- through mutagenesis — Look- through mutagenesis (LTM) (Rajpal et al, Proc Natl Acad Sci U S A. 102:8466-71, 2005) provides a method for rapidly mapping the antibodybinding site. For LTM, nine amino acids, representative of the major side-chain chemistries provided by the 20 natural amino acids, are selected to dissect the functional side-chain contributions to binding at every position in all six CDRs of an antibody. LTM generates a positional series of single mutations within a CDR where each "wild type" residue is systematically substituted by one of nine selected amino acids. Mutated CDRs are combined to generate combinatorial single- chain variable fragment (scFv) libraries of increasing complexity and size without becoming prohibitive to the quantitative display of all muteins. After positive selection, clones with improved binding are sequenced, and beneficial mutations are mapped.

[00188] Error-prone PCR — Error-prone PCR involves the randomization of nucleic acids between different selection rounds. The randomization occurs at a low rate by the intrinsic error rate of the polymerase used but can be enhanced by error- prone PCR (Zaccolo et al, J. Mol. Biol. 285:775-783, 1999) using a polymerase having a high intrinsic error rate during transcription (Hawkins et al., J Mol Biol. 226:889-96, 1992). After the mutation cycles, clones with improved affinity for the antigen are selected using routine methods in the art.

[00189] Techniques utilizing gene shuffling and directed evolution may also be used to prepare and screen antigen binding proteins, or variants thereof, for desired activity. For example, Jermutus et al, Proc Natl Acad Sci U S A., 98(1) :75-80 (2001) showed that tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of scFvs; Fermer et al., Tumour Biol. 2004 Jan- Apr;25(l-2):7-13 reported that use of phage display in combination with DNA shuffling raised affinity by almost three orders of magnitude. Dougherty et al., Proc Natl Acad Sci U S A. 2000 Feb. 29; 97(5):2029-2034 reported that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (ii) gain- of-function mutants are well represented in such libraries, and (iii) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site. [00190] Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen, or to use computer software to model such contact points. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, they are subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[00191] Immunoglobulins with modified carbohydrate

[00192] Immunoglobulin variants can also be produced that have a modified glycosylation pattern relative to the parent polypeptide, for example, adding or deleting one or more of the carbohydrate moieties bound to the immunoglobulin, and/or adding or deleting one or more glycosylation sites in the immunoglobulin.

[00193] Glycosylation of polypeptides, including antibodies is typically either N- linked or O- linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X- serine and asparagine -X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Thus, N-linked glycosylation sites may be added to an immunoglobulin by altering the amino acid sequence such that it contains one or more of these tripeptide sequences. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5 -hydroxyproline or 5 -hydroxy lysine may also be used. O-linked glycosylation sites may be added to an immunoglobulin by inserting or substituting one or more serine or threonine residues to the sequence of the original immunoglobulin or antibody. [00194] Altered Effector Function

[00195] Cysteine residue(s) may be removed or introduced in the Fc region of an antibody or Fc-containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in this region. A homodimeric immunoglobulin thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibodydependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric immunoglobulins or homodimeric antibodies (i.e., antibodies having two copies of a single LC monomer species and a single HC monomer species) may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, an immunoglobulin can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-CancerDrug Design 3: 219- 230 (1989).

[00196] It is also contemplated that one or more of the N-terminal 20 amino acid residues (e.g., a signal sequence) of the heavy and/or light chain are removed, or amino acid residues are deleted from the C-terminal, for example, amino acid sequences from which one, two, three, four or five amino acid residues are lacking from the N-terminal or C-terminal, or from both.

[00197] Modifications to increase serum half- life also may desirable, for example, by incorporation of or addition of a salvage receptor binding epitope (e.g., by mutation of the appropriate region or by incorporating the epitope into a peptide tag that is then fused to the immunoglobulin at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., W096/32478) or adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers.

[00198] The salvage receptor binding epitope preferably constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the immunoglobulin or fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CHI, CH3, or VH region, or more than one such region, of the immunoglobulin or antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the immunoglobulin fragment. See also International applications WO 97/34631 and WO 96/32478 which describe Fc variants and their interaction with the salvage receptor.

[00199] Other sites and amino acid residue(s) of the constant region have been identified that are responsible for complement dependent cytotoxicity (CDC), such as the Clq binding site, and/or the antibody-dependent cellular cytotoxicity (ADCC) (see, e.g., Molec. Immunol. 29 (5): 633-9 (1992); Shields et al, J. Biol. Chem., 276(9):6591-6604 (2001); Lazar et al, Proc. Nat'l. Acad. Sci. 103(11): 4005 (2006) which describe the effect of mutations at specific positions, each of which is incorporated by reference herein in its entirety). Mutation of residues within Fc receptor binding sites can result in altered (i.e. increased or decreased) effector function, such as altered affinity for Fc receptors, altered ADCC or CDC activity, or altered half-life. As described above, potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different subclass (e.g. replacing an IgGl residue with a corresponding IgG2 residue at that position).

[00200] The invention also encompasses production of immunoglobulin molecules, including antibodies and antibody fragments, with altered carbohydrate structure resulting in altered effector activity, including antibody molecules with absent or reduced fucosylation that exhibit improved ADCC activity. A variety of ways are known in the art to accomplish this. For example, ADCC effector activity is mediated by binding of the antibody molecule to the FcyRIII receptor, which has been shown to be dependent on the carbohydrate structure of the N-linked glycosylation at the Asn-297 of the CH2 domain. Non-fucosylated antibodies bind this receptor with increased affinity and trigger FcyRIII -mediated effector functions more efficiently than native, fucosylated antibodies. For example, recombinant production of non-fucosylated antibody in CHO cells in which the alpha- 1,6-fucosyl transferase enzyme has been knocked out results in antibody with 100-fold increased ADCC activity (Yamane-Ohnuki et al, Biotechnol Bioeng. 2004 Sep 5;87(5):614- 22). Similar effects can be accomplished through decreasing the activity of this or other enzymes in the fucosylation pathway, e.g., through siRNA or antisense RNA treatment, engineering cell lines to knockout the enzyme(s), or culturing with selective glycosylation inhibitors (Rothman et al., Mol Immunol. 1989 Dec;26(12): 1113-23). Some host cell strains, e.g. Lecl3 or rat hybridoma YB2/0 cell line naturally produce antibodies with lower fucosylation levels. Shields et al, J Biol Chem. 2002 Jul 26;277(30):26733-40; Shinkawa et al, J Biol Chem. 2003 Jan 31;278(5):3466-73. An increase in the level of bisected carbohydrate, e.g. through recombinantly producing antibody in cells that overexpress GnTIII enzyme, has also been determined to increase ADCC activity. Umana et al., Nat Biotechnol. 1999 Feb; 17(2): 176- 80. It has been predicted that the absence of only one of the two fucose residues may be sufficient to increase ADCC activity. (Ferrara et al., J Biol Chem. 2005 Dec 5).

[00201] Variable Domains of Antigen-Binding Proteins, e.g., Antibodies

[00202] Exemplary amino acid sequences of light chain variable regions (VL) and heavy chain variable regions (VH) and encompassed CDR sequences of the inventive antigen-binding proteins are provided in Table 1 herein. Each of the disclosed variable regions may be attached to heavy and light chain constant regions to form a complete antibody light chain (LC) and heavy chain (HC), respectively. Non-limiting examples of light chain and heavy chain sequences of the invention are provided in Table 1, respectively (see, below). Further, each of the so-generated heavy and light chain sequences may be combined to form a complete antibody structure. It should be understood that the light chain (VL) and heavy chain (VH) variable regions provided herein can also be attached to other constant domains having different sequences than the exemplary sequences listed below in Table 1.

[00203] Ordinarily, amino acid sequence variants of the immunoglobulin sequences disclosed herein will have an amino acid sequence having at least 60% amino acid sequence identity with the original or reference immunoglobulin domain (e.g., VL, VH, or VHH) amino acid sequence. Amino acid variant sequences of a VL, VH, or VHH domain sequence of the invention can also have at least 65%, or at least 70%, or at least 75% or at least 80% amino acid sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90%) sequence identity, and most preferably at least 95% sequence identity, including for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the sequence that are identical with the original or reference sequence, after aligning the sequences and candidate introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative amino acid substitutions as part of the sequence identity. None of N-terminal, C- terminal, or internal extensions, deletions, or insertions into the immunoglobulin or antibody sequence shall be construed as affecting sequence identity or homology.

[00204] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an immunoglobulin with an N- terminal methionyl residue or the immunoglobulin (including antibody or antibody fragment) fused to an epitope tag or a salvage receptor binding epitope. Other insertional variants of the immunoglobulin or antibody molecule include the fusion to a polypeptide which increases the serum half-life of the immunoglobulin, e.g. at the N-terminus or C-terminus.

[00205] huCD307e Binding Molecules

[00206] Provided in some aspects are CD307e antigen-binding molecules, such as CD307e- binding polypeptides. Such binding molecules include antibodies that specifically bind to CD307e, such as a human CD307e molecule, including antigen-binding fragments thereof. Also among the binding molecules are recombinant receptors such as chimeric antigen receptors containing such antibodies.

[00207] Among the antigen-binding proteins (including antibodies) provided are anti-CD307e ABPs that specifically bind domain 9 of human CD307e (or “huCD307e”) or the membrane proximal region of the human Cd307e molecule. The molecules include isolated recombinant molecules.

[00208] The terms "complementarity determining region," and "CDR," synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). "Framework regions" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full- length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).

[00209] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme), Lefranc M P et al., "IM GT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January; 27(l):55-77 ("IMGT" numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70, ("Aho" numbering scheme).

[00210] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[00211] Thus, unless otherwise specified, a "CDR" or "complementary determining region," or individual specified CDRs (e.g., "HCDR1, HCDR2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a HCDR3) contains the amino acid sequence of a corresponding CDR in a given VH or VL amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., HCDR3) within the variable region, as defined by any of the aforementioned schemes. In some embodiments, specified CDR sequences are specified.

[00212] Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given.

[00213] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[00214] Among the provided antibodies are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multi- specific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs. [00215] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.

[00216] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.

[00217] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[00218] Among the provided anti-CD307e antibodies are human antibodies. A "human antibody" is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigenbinding regions, such as those in which all or substantially all CDRs are non-human.

[00219] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extra-chromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.

[00220] Among the provided antibodies are monoclonal antibodies, including monoclonal antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.

[00221] Exemplary Anti-CD307e Antibodies

[00222] In some embodiments, the anti-CD307e antibody, e.g., antigen-binding antibody fragment, contains particular heavy and/or light chain CDR sequences and/or heavy and/or light chain variable (VH or VL) region sequences. It should be appreciated that the heavy and light chains can be arranged in different configurations. For example, the heavy chain can be first and the light chain can be second, or the light chain can be first and the heavy chain can be second. Also among the provided antibodies are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence, as disclosed in Table 1, below. [00223] Table 1. Sequences

[00224] Also provided are vectors containing the nucleic acids, host cells containing the vectors, e.g., for producing the antibodies. Also provided are methods for producing the antibodies. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In some embodiments, a method of making the anti-CD307e antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[00225] Also provided are methods of making the anti-CD307e antibodies (including antigenbinding fragments). For recombinant production of the anti-CD307e antibody, nucleic acid encoding an antibody, e.g., as described above, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[00226] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[00227] Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, the antibody heavy chains and/or light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[00228] In some embodiments, the antibody is produced in a cell-free system. Exemplary cell- free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009);

Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003). [00229] The provided embodiments further include vectors and host cells and other expression systems for expressing and producing the antibodies and other binding proteins, including eukaryotic and prokaryotic host cells, including bacteria, filamentous fungi, and yeast, as well as mammalian cells such as human cells, as well as cell-free expression systems.

[00230] Exemplary Features

[00231] In some aspects, the provided isolated recombinant antigen-binding proteins are isolated antibodies, including antigen-binding fragments. Theses embodiments of the isolated recombinant antigen-binding protein include an immunoglobulin variable light chain (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO: 101, and SEQ ID NO: 105; and an immunoglobulin variable heavy chain (VH) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO: 103, and SEQ ID NO: 107; or in which the V_L domain and/or the VH domain is an amino acid sequence variant of one of these members, and the antigen-binding protein, comprises the amino acid sequence variant VL domain and/or the amino acid sequence variant VH domain, which specifically binds domain 9 of human CD307e. In some embodiments the VL domain comprises an amino acid sequence with at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:93, and the VH domain comprises an amino acid sequence with at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:95; or the VL domain comprises an amino acid sequence with at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:97, and the VH domain comprises an amino acid sequence with at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:99; or the VL domain comprises an amino acid sequence with at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 101, and the VH domain comprises an amino acid sequence with at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 103; or the VL domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 105, and the VH domain comprises an amino acid sequence with at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 107. In some of these embodiments of the isolated recombinant antigen-binding protein, the molecule has one or more specified functional features, such as binding properties, including binding to particular epitopes, such as epitopes that are similar to or overlap with those of other antibodies, the ability to compete for binding with other antibodies, and/or particular binding affinities.

[00232] In particular embodiments, an immunoglobulin variable light chain (VL) domain and an immunoglobulin variable heavy chain (VH) domain, wherein the VL domain comprises an amino acid sequence and the VH domain comprises an amino acid sequence, wherein the amino acid sequences of the VL domain and the VH domain, respectively, are a pair selected from the group consisting of:

[00233] SEQ ID NO:93 and SEQ ID NO:95;

[00234] SEQ ID NO:97 and SEQ ID NO:99;

[00235] (c) SEQ ID NO: 101 and SEQ ID NO: 103; and

[00236] (d) SEQ ID NO: 105 and SEQ ID NO: 107; or

[00237] in which the VL domain and/or the VH domain is an amino acid sequence variant of one of these members, wherein the antigen-binding protein, comprising the amino acid sequence variant VL domain and/or the amino acid sequence variant VH domain, specifically binds domain 9 of human CD307e.

[00238] Generally, the observation that an antibody or other binding molecule binds to huCD307e or specifically binds to huCD307e does not necessarily mean that it binds to CD307e of every species. For example, in some embodiments, features of binding to CD307e, such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to human CD307e protein and the antibody may not have this feature with respect to a CD307e of another species, such as monkey or mouse.

[00239] In some exemplary embodiments, the isolated recombinant antigen-binding protein includes: (a) an immunoglobulin light chain (LC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:36, SEQ ID NO:56, and SEQ ID NO:73, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 291, SEQ ID NO: 295, or comprising any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are lacking from the N-terminal or C-terminal, or from both; and

(b) an immunoglobulin heavy chain (HC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:44, SEQ ID NO:62, and SEQ ID NO:81, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, or comprises any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are lacking from the N-terminal or C-terminal, or from both. In some examples of such isolated recombinant antigen-binding proteins, the LC and HC, respectively, comprise the amino acid sequence pairs of:

[00240] SEQ ID NO:2 and SEQ ID NO: 10;

[00241] SEQ ID NO:36 and SEQ ID NO:44;

[00242] SEQ ID NO:56 and SEQ ID NO:62; or

[00243] SEQ ID NO:73 and SEQ ID NO:81;

[00244] or pairs of any of the humanized LC and HC in different combinations

[00245] or they can have any one of the foregoing amino acid sequences lacking one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s from its N-terminal or C-terminal, or from both.

[00246] In some embodiments, the epitope contains one or more amino acids within, is within, or includes a full or partial domain 9 portion of CD307e corresponding to aminos acid residues 757-776 of the human CD307e sequence set forth in SEQ ID NO: 108, such as a portion having the sequence set forth in SEQ ID NO: 109; or amino acid residues 820-839 of SEQ ID NO: 108, e.g., SEQ ID NO:111.

[00247] In some embodiments, the epitope is the same as, similar to, or overlapping with, or contains one or more of the same amino acids as an epitope that is specifically bound to by a reference antibody. In some embodiments, the same one or more amino acids is important for the binding of the provided antibody and the reference antibody.

[00248] In some embodiments, the extent of binding of an anti-CD307e antibody to an unrelated, non-CD307e protein, such as non-human CD307e or other non-CD307e protein, is less than about 40% of the binding of the antibody to human CD307e as measured, for example, by an ELISA or a radioimmunoassay (RIA). In some embodiments, among provided antibodies are antibodies in which binding to a non-human CD307e or other non-CD307e protein is less than or about 30%, less than or about 20% or less than or about 10% of the binding of the antibody to human CD307e.

[00249] In some embodiments, such properties of provided antibodies, including antigenbinding fragments, are described in relation to properties observed for another antibody, e.g., a reference antibody. In some embodiments, a reference antibody is a non-human anti-CD307e antibody, such as a murine or chimeric or humanized anti-CD307e antibody and/or a fragment derived therefrom, such as an scFv fragment thereof, and/or an antibody containing the VH and VL sequences of such an antibody and/or the heavy and light chain CDRs of such an antibody.

[00250] In some embodiments, the antibodies display a binding preference for CD307e- expressing cells as compared to CD307e-negative cells, such as particular cells known in the art and/or described herein. In some embodiments, the binding preference is observed where a significantly greater degree of binding is measured to the CD307e-expressing, as compared to the non-expressing, cells. In some embodiments, the fold change in degree of binding detected, for example, as measured by mean fluorescence intensity in a flow cytometry-based assay and/or dissociation constant or EC50, to the CD307e-expressing cells as compared to the non-CD307e- expressing cells, is at least at or about 1.5, 2, 3, 4, 5, 6, or more, and/or is about as great, about the same, at least as great or at least about as great, or greater, than the fold change observed for a reference antibody. In some cases, the total degree of observed binding to CD307e or to the CD307e-expressing cells is approximately the same, at least as great, or greater than that observed for a reference antibody. In any of the provided embodiments, comparison of binding properties, such as affinities or competition, may be via measurement by the same or similar assay. (See, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).

[00251] In some embodiments, two antibodies specifically bind to the same epitope if all or essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. In some embodiments, two antibodies specifically bind to an overlapping epitope if at least some of the amino acid mutations in the antigen that reduce binding or eliminate binding to the antigen by one antibody also reduce or eliminate binding to the antigen by the other antibody.

[00252] In some embodiments, the provided antibodies are capable of binding CD307e, such as human CD307e, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by an equilibrium dissociation constant (KD or Kd); in some embodiments, the affinity is represented by EC50. In certain embodiments, the binding affinity (EC50) and/or the equilibrium dissociation constant of the antibody to CD307e is at or about or less than at or about 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM, such as between at or about 1 nM and at or about 15 nM, e.g., between at or about 5 and at or about 10 nM. In one embodiment, the extent of binding of an anti-CD307e antibody to an unrelated, non-CD307e protein is less than at or about 10% of the binding of the antibody to CD307e as measured, e.g., by ELISA or a radioimmunoassay (RIA).

[00253] Anti-CD307e antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various known assays. In one aspect, the antibody is tested for its antigen binding activity, e.g., by known methods such as ELISA, RIA, Western blotting, and/or flow cytometric assays, including cellbased binding assays, for example, assessing binding of the antibody (e.g., conjugated to a fluorescent marker or tagged) to a cell expressing the target antigen, e.g., CD307e, in some cases compared to results using cells that do not express the target antigen, e.g., CD307e. Binding affinity may be measured as or KD or Kd or EC50. [00254] Competition assays may be used to identify an antibody that competes with any of the antibodies described herein. Assays for mapping epitopes bound by the antibodies and reference antibodies also may be used and are known.

[00255] Immunoconjugates

[00256] In some embodiments, the antibody is or is part of an immunoconjugate, in which the antibody is conjugated to one or more heterologous molecule(s), such as, but not limited to, a cytotoxic agent, an imaging agent, a detectable moiety a multimerization domain or other heterologous molecule. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins. In some embodiments, the antibody is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[00257] Among the immunoconjugates are antibody-drug conjugates (ADCs), in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0425 235 B l); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529- 1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

[00258] Also among the immunoconjugates are those in which the antibody is conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[00259] Also among the immunoconjugates are those in which the antibody is conjugated to a radioactive atom to form a radioconjugate. Exemplary radioactive isotopes include At-211, 1-131, 1-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, Pb-212 and radioactive isotopes of Lu.

[00260] Conjugates of an antibody and cytotoxic agent may be made using any of a number of known protein coupling agents, e.g., linkers, (see Vitetta et al., Science 238:1098 (1987)), WO94/11026. The linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell, such as acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, and disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[00261] Conjugates may also include fusion proteins such as Fc-fusions and chimeric molecules.

[00262] Multispecific Antibodies

[00263] In certain embodiments, the CD307e-binding molecules, e.g., antibodies are multispecific. Among the multispecific binding molecules are multispecific antibodies, including, e.g. bispecific. Multi specific binding partners, e.g., antibodies, have binding specificities for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is for CD307e and the other is for another antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CD307e. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD307e. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Among the bispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Also provided are multispecific chimeric receptors, such as multispecific CARs, containing the antibodies.

[00264] Exemplary additional antigens include other B cell specific antigens and antigens expressed on T cells. Exemplary antigens include CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD56, CD74, CD80, CD126, CD138, B7, MUC-1, la, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, li, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL- R2 (DR5).

[00265] Variants

[00266] In certain embodiments, the antibodies include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of an antibody described herein. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, 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 can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

[00267] In certain embodiments, the antibodies include one or more amino acid substitutions, e.g., as compared to an antibody sequence described herein and/or compared to a sequence of a natural repertoire, e.g., human repertoire. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, improved half-life, and/or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, the variant antibody exhibits retained or improved binding to CD307e.

[00268] In some embodiments, one or more residues within a CDR of a parent antibody (e.g. a humanized or human antibody) is/are substituted. In some embodiments, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human subject.

[00269] In some embodiments, alterations are made in CDR "hotspots," residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. HCDR3 and LCDR3 in particular are often targeted.

[00270] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[00271] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C -terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[00272] Modifications

[00273] In certain embodiments, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and/or by modifying the oligosaccharide(s) attached to the glycosylation sites, e.g., using certain cell lines. Glycosylation sites include asparagine 297 of the heavy chain (according to Kabat numbering).

[00274] Exemplary modifications, variants, and cell lines are described, e.g., in Patent Publication Nos. US 2003/0157108, US 2004/0093621, US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003/085107); WO 2003/011878 (Jean- Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.); WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

[00275] Among the modified antibodies are those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

[00276] Such modifications can be made, e.g., to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector functions. [00277] Also among the variants are cysteine engineered antibodies such as "thioMAbs" and other cysteine engineered variants, in which one or more residues of an antibody are substituted with cysteine residues, in order to generate reactive thiol groups at accessible sites, e.g., for use in conjugation of agents and linker- agents, to produce immunoconjugates. Cysteine engineered antibodies are described, e.g., in U.S. Pat. Nos. 7,855,275 and 7,521,541.

[00278] In some embodiments, the antibodies are modified to contain additional non- proteinaceous moieties, including water soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3 -dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[00279] Recombinant Receptors

[00280] Among the provided CD307e binding molecules are recombinant receptors, such as antigen receptors and other chimeric receptors, that specifically bind to CD307e, such as receptors containing the provided anti-CD307e antibodies, e.g., antibody fragments. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing the recombinant receptors and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with CD307e expression. [00281] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al. Exemplary of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, e.g., and in which the antigen-binding portion, e.g., scFv, is replaced by an antibody, e.g., as provided herein.

[00282] Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that includes, is, or is comprised within, one of the provided anti-CD307e antibodies. Thus, the chimeric receptors, e.g., CARs, typically include in their extracellular portions one or more CD307e- binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules, such as those described herein. In some embodiments, the CAR includes a CD307e-binding portion or portions of the antibody molecule, such as a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.

[00283] CD307e-targeting CARs are described, for example, by Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282. [00284] In some embodiments, the recombinant receptor, such as a CAR, such as the antibody portion thereof, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Pat. No. 8,822,647 or published app. No. US2014/0271635.

[00285] In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some embodiments, the spacer has the sequence ESKYGPPCPPCP// (SEQ ID NO: 118, or a variant thereof. In some embodiments, the constant region or portion is of IgD.

[00286] The antigen recognition domain generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the CD 307 e- specific binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[00287] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, and/or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, e.g., CD8alpha, or functional variant thereof. In some embodiments the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).

[00288] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[00289] The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the CD307e-binding antibody is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor .gamma., CD8, CD4, CD25, or CD 16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor- y and CD8, CD4, CD25 or CD16.

[00290] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immuno stimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.

[00291] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

[00292] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

[00293] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[00294] In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40, DAP10, or ICOS, or CD27. In some aspects, the same CAR includes both the activating and costimulatory components.

[00295] In some embodiments, the activating domain (e.g. CD3 zeta) is included within one CAR, whereas the costimulatory component (e.g. CD28 or 4- IBB) is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the CD307e-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than CD307e, whereby an activating signal delivered through the CD307e- targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[00296] In some embodiments, the intracellular signaling component of the recombinant receptor, such as CAR, comprises a CD3 zeta intracellular domain and a costimulatory signaling region. In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta; e.g., SEQ ID NO:30) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and/or CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.

[00297] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB. [00298] In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR) or a functional variant thereof. In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.

[00299] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.

[00300] In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells will be adoptively transferred.

[00301] In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.

[00302] In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3 -chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.

[00303] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv and the intracellular domain contains an IT AM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB (e.g., SEQ ID NO:28).

[00304] For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof (e.g., SEQ ID NO:28) and a signaling portion of CD3 zeta or functional variant thereof (e.g., SEQ ID NO:30). In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.

[00305] In some embodiments, the transmembrane domain of the recombinant receptor, e.g., the CAR, is or includes a transmembrane domain of human CD28 (e.g. Accession No. P01747.1) or variant thereof. [00306] In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g. the CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4- IBB (e.g. (Accession No.

Q07011.1) or functional variant or portion thereof.

[00307] In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g. the CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or 8,911,993.

[00308] In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4- derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only. In some embodiments, the spacer is or comprises a glycine- serine rich sequence or other flexible linker such as known flexible linkers.

[00309] For example, in some embodiments, the CAR includes an anti-CD307e antibody such as an anti-CD307e antibody fragment, such as any of the provided human anti-CD307e antibodies, e.g., single-chain antibodies including scFvs, described herein, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an anti-CD307e antibody or fragment, such as any of the human anti-CD307e antibodies, including scFvs described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

[00310] In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR. [00311] In other embodiments, the inventive antigen-binding proteins can also be included in synthetic immune receptors, as exemplified in Chaudhary et al., WO2018/102795A2. The term "Synthetic Immune Receptor" or alternatively, "SIR", refers to a set of polypeptides, typically two, which when expressed in an effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In a typical embodiment, a SIR comprises one or more antigen binding domains (e.g., antibody or antibody fragment, a ligand or a receptor) that bind to antigens as described herein, and are joined to one or more T cell receptor constant chains or regions via an optional linker. In some embodiments, the set of polypeptides are contiguous with each other. In some embodiments, a SIR comprises two or more sets of two or more polypeptides. The polypeptides of each set of SIR are contiguous with each other (functional polypeptide unit 1) but are not contiguous with the polypeptides of the other set (functional polypeptide unit 2). In some aspects, the T cell receptor constant chains (or regions) of the SIR is chosen from the constant chain of human T cell receptor- alpha (TCR-alpha or TCRa or TCRa or hTCR-alpha or hTCRa or hTCRa or Ca), human T cell receptor-betal(TCR-betal or TCR i or TCRbl or hTCR-betal or hTCR i or hTCRbl or cpi), human T cell receptor-beta 2 (TCR-beta2 or TCR 2 or TCRb2 or hTCR-beta2 or hTCR 2 or hTCRb2 or C 2 also designated TCR-beta, TCR or TCRb or OP), human Pre-T cell receptor alpha ((preTCR-alpha or preTCRa or preTCRa or preCa), human T cell receptor-gamma (TCR-gamma or TCRy or TCRg or or hTCR-gamma or hTCRy or hTCRg or hTCRyl or hTCRgammal), or human T cell receptor-delta (TCR-delta or TCRd or TCR5 or hTCR-delta or hTCRd or hTCR5). In some embodiments, the TCR constant chains of SIR are encoded by their wild-type nucleotide sequences while in other aspects the TCR constant chains of SIR are encoded by the nucleotide sequences that are not wild-type. In some embodiments, the TCR constant chains of SIR are encoded by their codon optimized sequences. In some embodiments, the TCR constant chains of SIR encode for the wild-type polypeptide sequences while in other embodiments the TCR constant chains of SIR encoded for polypeptides that carry one or more mutations. In some embodiments, the TCR constant chains of SIR are encoded by their codon optimized sequences that carry one or more mutations. A SIR that comprises an antigen binding domain (e.g. , a scFv, or VHH) that targets a specific tumor maker "X", such as those described herein, is also referred to as X-SIR or XSIR. For example, a SIR that comprises an antigen binding domain that targets CD 19 is referred to as CD19-SIR or CD19SIR. The TCR constant chain/domain of a SIR can be derived from the same species in which the SIR will ultimately be used. For example, for use in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or comprised of human TCR constant chains. However, in some instances, it is beneficial for the TCR constant chain to be derived from the same species in which the SIR will ultimately be used in, but modified to carry amino acid substitutions that enhance the expression of the TCR constant chains. For example, for use in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or comprised of human TCR constant chains but in which certain amino acids are replaced by the corresponding amino acids from the murine TCR constant chains. Such murinized TCR constant chains provide increased expression of the SIR.

[00312] Recombinantly Engineered Cells

[00313] Also provided are cells, cell populations, and compositions containing the cells or cell populations, e.g., the engineered cells, e.g. that contain an engineered antigen receptor, e.g., that contains an extracellular domain including the anti-CD307e antibody or fragment, described herein. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[00314] Thus also provided are genetically engineered cells expressing the recombinant receptors containing the antibodies, e.g., cells containing the CARs or SIRs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4⁺ cells, CD8⁺ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.

[00315] Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MATT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and gamma delta T cells.

[00316] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

[00317] In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[00318] Vectors and Methods for Genetic Engineering

[00319] Also provided are methods, nucleic acids, compositions, and kits, for expressing the binding molecules, including receptors comprising the antibodies, and for producing the genetically engineered cells expressing such binding molecules. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.

[00320] In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.

[00321] In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[00322] In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs or SIRs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.

[00323] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al.

(2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550- 557.

[00324] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Uawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[00325] Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637- 1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[00326] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PEoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome- mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et ah, Mol. Cell Biol., 7: 2031-2034 (1987)).

[00327] Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.

[00328] Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US 94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

[00329] Preparation of Cells for Engineering

[00330] In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the huCD307e-binding molecule, e.g., CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.

[00331] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[00332] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[00333] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig.

[00334] In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.

[00335] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.

[00336] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca⁺⁺/Mg⁺⁺ -free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[00337] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[00338] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[00339] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[00340] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[00341] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[00342] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺, CD4⁺, CD8⁺,CD3⁺ , CD45RA⁺, and/or CD45RO⁺ T cells, are isolated by positive or negative selection techniques.

[00343] For example, CD3⁺, CD28⁺ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).

[00344] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker⁺) at a relatively higher level (marker¹¹¹⁸¹¹) on the positively or negatively selected cells, respectively.

[00345] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4⁺ or CD8⁺ selection step is used to separate CD4⁺ helper and CD8⁺ cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. [00346] In some embodiments, CD8⁺ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining Tcw-enriched CD8⁺ T cells and CD4⁺ T cells further enhances efficacy.

[00347] In embodiments, memory T cells are present in both CD62L⁺ and CD62L’ subsets of CD8⁺ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L’ CD8⁺ and/or CD62L⁺ CD8⁺ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[00348] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8⁺ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8⁺ cell population or subpopulation, also is used to generate the CD4⁺ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[00349] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4⁺ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD 14 and CD45RA or CD307e, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[00350] CD4⁺ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4⁺ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4⁺T lymphocytes are CD45RO’, CD45RA⁺, CD62L⁺, CD4⁺ T cells. In some embodiments, central memory CD4⁺ cells are CD62L⁺ and CD45RO⁺. In some embodiments, effector CD4⁺ cells are CD62L’ and CD45RO’.

[00351] In one example, to enrich for CD4⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U.

Schumacher.COPYRGT. Humana Press Inc., Totowa, N.J.).

[00352] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[00353] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples. [00354] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[00355] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[00356] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[00357] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[00358] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[00359] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.

[00360] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[00361] In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

[00362] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a precolumn and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

[00363] In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother 35(9):689-701.

[00364] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5) :355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[00365] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[00366] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80. degree. C. at a rate of 1. degree, per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[00367] In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.

[00368] Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.

[00369] In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[00370] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[00371] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL- 15, for example, an IL-2 concentration of at least about 10 units/mL.

[00372] In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

[00373] In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[00374] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[00375] In embodiments, antigen-specific T cells, such as antigen- specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen- specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

[00376] Pharmaceutical Compositions and Formulations

[00377] Also provided are compositions including the CD307e binding molecules and engineered cells, including pharmaceutical compositions and formulations, and methods of using and uses of the molecules and compositions, such as in the treatment of diseases, conditions, and disorders in which CD307e is expressed, and/or detection, diagnostic, and prognostic methods.

[00378] For example, provided are pharmaceutical formulations including the CD307e-binding molecule, e.g., an ABP, antibody, or CAR or SIR, and/or the engineered cells expressing the molecules. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[00379] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. [00380] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[00381] In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[00382] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins;

21st ed. (May 1, 2005).

[00383] Formulations of the antibodies can include lyophilized formulations and aqueous solutions.

[00384] The formulation or composition may also contain more than one active ingredients useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.

[00385] Active ingredients may be entrapped in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[00386] The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

[00387] The pharmaceutical composition in some embodiments contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactic ally effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[00388] In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject.

[00389] The may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[00390] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[00391] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[00392] Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[00393] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00394] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

[00395] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[00396] Therapeutic and Prophylactic Methods and Uses

[00397] Also provided are methods for using and uses of the CD307e binding molecules, including the anti-CD307e antibodies, e.g., antibody fragments, and/or engineered cells expressing the recombinant receptors. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder expressing or associated with CD307e expression, and/or in which cells or tissues express CD307e. In some embodiments, the molecule, cell, and/or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the antibodies and cells in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the antibodies or cells, or compositions comprising the same, to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. [00398] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[00399] As used herein, "delaying development of a disease" means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[00400] "Preventing," as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.

[00401] As used herein, to "suppress" a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody or composition or cell.

[00402] An "effective amount" of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, or cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result. [00403] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation, antibody, or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the molecules, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.

[00404] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[00405] As used herein, a "subject" is a mammal, such as a human or other animal, and typically is human. The diseases and disorders include B cell malignancies, such as B cell leukemias and lymphomas, including B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, Null-acute lymphoblastic leukemias, non-Hodgkin lymphomas, diffuse large B cell lymphomas (DLBCLs), multiple myelomas, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, Hodgkin lymphoma. Also among the diseases and conditions are autoimmune and inflammatory diseases, including those associated with inappropriate or enhanced B cell numbers and/or activation. Exemplary diseases and conditions include multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (SLE).

[00406] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another CD307e-specific antibody and/or cells expressing a CD307e-targeting chimeric receptor and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another CD307e-targetetd therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.

[00407] In some embodiments, the treatment does not induce an immune response by the subject to the therapy, and/or does not induce such a response to a degree that prevents effective treatment of the disease or condition. In some aspects, the degree of immunogenicity and/or graft versus host response is less than that observed with a different but comparable treatment. For example, in the case of adoptive cell therapy using cells expressing CARs including the provided anti-CD307e antibodies, the degree of immunogenicity is reduced compared to CARs including a different antibody that binds to a similar, e.g., overlapping epitope and/or that competes for binding to CD307e with the provided antibody, such as a mouse antibody.

[00408] In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided anti-CD307e-containing receptors (e.g., CD307e- targeted CARs or SIRs) are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in a CD307e-targeted manner, such that the cells of the disease or disorder are targeted for destruction.

[00409] Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in a CD307e-expressing cancer.

[00410] Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[00411] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[00412] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[00413] In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject or patient can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).

[00414] The CD307e-binding molecules, such as antibodies and chimeric receptors containing the antibodies and cells expressing the same, can be administered by any suitable means, for example, by injection, e.g., intratumoral, intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.

[00415] For the prevention or treatment of disease, the appropriate dosage of the binding molecule or cell may depend on the type of disease to be treated, the type of binding molecule, the severity and course of the disease, whether the binding molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the binding molecule, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.

[00416] Depending on the type and severity of the disease, dosages of antibodies may include about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg), about 1 .mu.g/kg to 100 mg/kg or more, about 0.05 mg/kg to about 10 mg/kg, 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg. Multiple doses may be administered intermittently, e.g. every week or every three weeks. An initial higher loading dose, followed by one or more lower doses may be administered.

[00417] In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.

[00418] In some embodiments, the cells or antibodies are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.

[00419] The cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after to the one or more additional therapeutic agents.

[00420] Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and/or antibodies in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFN. gamma., IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[00421] In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3:111 (1995), and U.S. Pat. No. 5,087,616. [00422] Diagnostic, Prognostic, and Detection Methods

[00423] Also provided are methods involving use of the provided binding molecules, e.g., antibodies, including antibody fragments, and molecules (such as conjugates and complexes) containing one or more of such antibodies, for detection, prognosis, diagnosis, staging, determining binding of a particular treatment to one or more tissues or cell types, and/or informing treatment decisions in a subject, such as by the detection of CD307e and/or the presence of an epitope thereof recognized by the antibody. In some embodiments, the methods are diagnostic and/or prognostic methods in association with a CD307e-expressing disease or condition. The methods in some embodiments include incubating and/or probing a biological sample with the antibody and/or administering the antibody to a subject. In certain embodiments, a biological sample includes a cell or tissue or portion thereof, such as tumor or cancer tissue or biopsy or section thereof. In certain embodiments, the contacting is under conditions permissive for binding of the anti-CD307e antibody to CD307e present in the sample. In some embodiments, the methods further include detecting whether a complex is formed between the anti-CD307e antibody and CD307e in the sample, such as detecting the presence or absence or level of such binding. Such a method may be an in vitro or in vivo method. In one embodiment, an anti- CD307e antibody is used to select subjects eligible for therapy with an anti-CD307e antibody or engineered antigen receptor, e.g. where CD307e is a biomarker for selection of patients.

[00424] In some embodiments, a sample, such as a cell, tissue sample, lysate, composition, or other sample derived therefrom is contacted with the inventive antigen-binding protein, e.g., an anti-CD307e antibody, and binding or formation of a complex between the antibody and the sample (e.g., CD307e in the sample) is determined or detected. When binding in the test sample is demonstrated or detected as compared to a reference cell of the same tissue type, it may indicate the presence of an associated disease or condition, and/or that a therapeutic containing the antibody (e.g., antibody fragment) will specifically bind to a tissue or cell that is the same as or is of the same type as the tissue or cell or other biological material from which the sample is derived. In some embodiments, the sample is from human tissues and may be from diseased and/or normal tissue, e.g., from a subject having the disease or condition to be treated and/or from a subject of the same species as such subject but that does not have the disease or condition to be treated. In some cases, the normal tissue or cell is from a subject having the disease or condition to be treated but is not itself a diseased cell or tissue, such as a normal tissue from the same or a different organ than a cancer that is present in a given subject or patient.

[00425] Various methods known in the art for detecting specific ABP-antigen binding can be used. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (EEISA), and radioimmunoassay (RIA). An indicator moiety, or label group, can be attached or covalently conjugated to the inventive antigen-binding protein, e.g., an antibody, and is selected so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. Exemplary labels include radionuclides (e.g. ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P and/or chromium (⁵¹Cr), cobalt (⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ⁱⁿIn), iodine (¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum ("Mo), palladium (¹⁰³Pd), phosphorous (³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), rutheroium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), (⁸⁵Sr), sulphur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Ti) tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or P-galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot.TM. nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). Various general techniques to be used in performing the various immunoassays noted above are known.

[00426] For purposes of diagnosis, the inventive antigen-binding protein, e.g., an antibody, can be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to an antibody are known in the art.

[00427] In some embodiments, antibodies need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to any of the antibodies.

[00428] The inventive antigen-binding protein, e.g., an antibody, provided herein can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[00429] The inventive antigen-binding protein, e.g., an antibody, can also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody is labeled with a radionuclide (such as ⁱⁿIn, "Tc, ¹⁴C, ¹³¹I, ¹²⁵I, or ³H) so that the cells or tissue of interest can be localized in vivo following administration to a subject.

[00430] The inventive antigen-binding protein, e.g., an antibody, can also be used as staining reagent in pathology, e.g., using known techniques.

[00431] Articles Of Manufacture

[00432] Also provided are articles of manufacture containing the provided binding molecules, e.g., antibodies and CARs and/or genetically engineered cells, and/or compositions. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection. The label or package insert may indicate that the composition is used for treating the CD307e-expressing or -associated disease or condition. The article of manufacture may include (a) a first container with a composition contained therein, wherein the composition includes the antibody or engineered antigen receptor; and (b) a second container with a composition contained therein, wherein the composition includes a further agent, such as a cytotoxic or otherwise therapeutic agent. The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes. [00433] As used herein, reference to a "corresponding form" of an antibody means that when comparing a property or activity of two antibodies, the property is compared using the same form of the antibody. For example, if it is stated that an antibody has greater activity compared to the activity of the corresponding form of a first antibody, that means that a particular form, such as a scFv of that antibody, has greater activity compared to the scFv form of the first antibody.

[00434] As used herein, recitation that nucleotides or amino acid positions "correspond to" nucleotides or amino acid positions in a disclosed sequence, such as set forth in the sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

[00435] " Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

[00436] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[00437] The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[00438] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[00439] By way of further illustration, the following embodiments of the present invention are enumerated:

[00440] Embodiment 1 : An isolated recombinant antigen-binding protein that specifically binds domain 9 of human CD307e, comprising:

(A) an immunoglobulin variable light chain (VL) domain comprising a set of three complementarity determining regions: LCDR1, LCDR2, and LCDR3, wherein each LCDR comprises an amino acid sequence, wherein the set of three LCDR amino acid sequences is selected from the group consisting of:

(a) SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; SEQ ID NO:204, SEQ ID NO:208, SEQ ID NO: 212, SEQ ID NO:205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO: 206, SEQ ID NO: 210, and SEQ ID NO: 214 and

(b) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42; SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NO: 241, SEQ ID NO: 245, SEQ ID NO: 242 and SEQ ID NO: 246 and

(c) SEQ ID NO:58, SEQ ID NO:40, SEQ ID NO:42; SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 266, SEQ ID NO: 270, SEQ ID NO: 274, and (d) SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79; SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 294, SEQ ID NO: 298, or wherein one or more of the LCDR amino acid sequences of the set of three LCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residues; and

(B) an immunoglobulin variable heavy chain (VH) domain comprising a set of three complementarity determining regions: HCDR1, HCDR2, and HCDR3, wherein each HCDR comprises an amino acid sequence, wherein the set of three HCDR amino acid sequences is selected from the group consisting of: a) SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 198, SEQ ID NO: 202 b) SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50; SEQ ID NO: 216 , SEQ ID NO: 220 , SEQ ID NO:224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 217 , SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO:229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 218 , SEQ ID NO: 222, SEQ ID NO: 226, SEQ ID NO: 230, SEQ ID NO: 234, SEQ ID NO: 238 c) SEQ ID NO:46, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 250, SEQ ID NO: 254, SEQ ID NO: 258, SEQ ID NO: 262 d) SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 278 , SEQ ID NO: 282, SEQ ID NO: 286, SEQ ID NO: 290 or wherein one or more of the HCDR amino acid sequences of the set of three HCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residue/s, optionally wherein the isolated recombinant antigen-binding protein specifically binds domain 9 of human CD307e with an equilibrium dissociation constant (Kd or KD) of < 10’⁸ M, more preferably with a Kd or KD of < 5 x 10’⁹ M, and even more preferably with a Kd or KD of < 5 X IO ¹⁰ M, or even < 10'

¹⁰ M, as measured, e.g., by surface plasmon resonance, biolayer interferometry (BLI) or ELISA.

[00441] Embodiment 2: The isolated recombinant antigen-binding protein according to Embodiment 1, comprising:

(a) an immunoglobulin variable light chain (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO: 101, and SEQ ID NO: 105; and

(b) an immunoglobulin variable heavy chain (VH) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO: 103, and SEQ ID NO: 107; or wherein the VL domain and/or the VH domain is an amino acid sequence variant of one of these members, wherein the antigen-binding protein, comprising the amino acid sequence variant VL domain and/or the amino acid sequence variant VH domain, specifically binds domain 9 of human CD307e.

[00442] Embodiment 3: The isolated recombinant antigen-binding protein according to any of Embodiments 1-2, comprising: an immunoglobulin monomer comprising a variable light chain (VL) domain and an immunoglobulin variable heavy chain (VH) domain, wherein the VL domain comprises an amino acid sequence and the VH domain comprises an amino acid sequence, wherein the amino acid sequences of the VL domain and the VH domain, respectively, are a pair selected from the group consisting of:

(a) SEQ ID NO:93 and SEQ ID NO:95;

(b) SEQ ID NO:97 and SEQ ID NO:99;

(c) SEQ ID NO: 101 and SEQ ID NO: 103; and

(d) SEQ ID NO: 105 and SEQ ID NO: 107;

(e) or different combinations of the humanized VL and VH sequences listed in Table 1 or wherein the VL domain and/or the VH domain is an amino acid sequence variant of one of these members, wherein the antigen-binding protein, comprising the amino acid sequence variant VL domain and/or the amino acid sequence variant VH domain, specifically binds domain 9 of human CD307e.

[00443] Embodiment 4: The isolated recombinant antigen-binding protein according to any of Embodiments 1-3, comprising a set of six complementarity determining regions comprising three LCDR amino acid sequences: LCDR1, LCDR2, and LCDR3, and three HCDR amino acid sequences: HCDR1, HCDR2, and HCDR3, the amino acid sequences being selected from the group consisting of:

LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, ; SEQ ID NO:204 , SEQ ID NO:208, SEQ ID NO: 212, SEQ ID NO:205 , SEQ ID NO: 209 , SEQ ID NO: 213 , SEQ ID NO: 206 , SEQ ID NO: 210, and SEQ ID NO: 214; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 198, SEQ ID NO: 202;

LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:38, SEQ ID NO:40, and SEQ ID NO:42; SEQ ID NO: 240, SEQ ID NO: 244 , SEQ ID NO: 241 , SEQ ID NO: 245 , SEQ ID NO: 242 and SEQ ID NO: 246 and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:50, SEQ ID NO: 216 , SEQ ID NO: 220 , SEQ ID NO:224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 217 , SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO:229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 218 , SEQ ID NO: 222, SEQ ID NO: 226, SEQ ID NO: 230, SEQ ID NO: 234, SEQ ID NO: 238

LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:58, SEQ ID NO:40, and SEQ ID NO:42, SEQ ID NO: 264 , SEQ ID NO: 268 , SEQ ID NO: 272 , SEQ ID NO: 265, SEQ ID NO: 269 , SEQ ID NO: 273 , SEQ ID NO: 266 , SEQ ID NO: 270 , SEQ ID NO: 274, and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:46, SEQ ID NO:65, and SEQ ID NO:67; SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 250, SEQ ID NO: 254, SEQ ID NO: 258, SEQ ID NO: 262, and

LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79, SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 294, SEQ ID NO: 298; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 278 , SEQ ID NO: 282, SEQ ID NO: 286, and SEQ ID NO: 290; or wherein one or more of the three LCDR amino acid sequences and/or one or more of the three HCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residue(s).

[00444] Embodiment 5: The isolated recombinant antigen-binding protein according to any of Embodiments 1-4, comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein:

(a) the VL domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:93, and the VH domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:95;

(b) the VL domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:97, and the VH domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:99;

(c) the VL domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 101, and the VH domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 103; or

(d) the VL domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 105, and the VH domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 107. [00445] Embodiment 6: The isolated recombinant antigen-binding protein according to any of Embodiments 1-5, comprising an antibody or an antibody fragment.

[00446] Embodiment 7 : The isolated recombinant antigen-binding protein according to any of Embodiments 1-6, comprising a Fab, a Fab', a F(ab')2, a Fv, a scFv, a scFv-Fc, a scFv-CH a scFab, a single chain antibody, a diabody, a maxibody, or a Bispecific T cell Engager molecule.

[00447] Embodiment 8: The isolated recombinant antigen-binding protein of Embodiments 6-7, wherein the antibody or antibody fragment is or comprises an IgGl, IgG2, IgG3 or IgG4 antibody or antibody fragment, preferably an IgG2, or and IgG4 antibody or antibody fragment.

[00448] Embodiment 9: The isolated recombinant antigen-binding protein according to any of Embodiments 1-8, wherein the recombinant antigen-binding protein is or comprises a monoclonal antibody.

[00449] Embodiment 10: The isolated recombinant antigen-binding protein according to any of Embodiments 1-9, wherein the recombinant antigen-binding protein is or comprises a human antibody.

[00450] Embodiment 11 : The isolated recombinant antigen -binding protein according to any of Embodiments 1-10, comprising:

(a) an immunoglobulin light chain (LC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:73, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 291, and SEQ ID NO: 295, or comprising any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are lacking from the N-terminal or C-terminal, or from both; and

(b) an immunoglobulin heavy chain (HC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:44, SEQ ID NO:62, SEQ ID NO:81, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283 and SEQ ID NO: 287, or comprising any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are lacking from the N-terminal or C-terminal, or from both.

[00451] Embodiment 12: The isolated recombinant antigen -binding protein of any of Embodiments 1-11, wherein the LC and HC, respectively, comprise the amino acid sequence pairs of:

(a) SEQ ID NO:2 and SEQ ID NO: 10;

(b) SEQ ID NO:36 and SEQ ID NO:44;

(c) SEQ ID NO:56 and SEQ ID NO:62;

(d) SEQ ID NO:73 and SEQ ID NO:81; or

(e) any combinations included the humanized LCs and HCs listed in Table 1 or wherein any one of the foregoing amino acid sequences lacks one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s from its N-terminal or C-terminal, or from both.

[00452] Embodiments 1-12 include, inter alia, monomeric, homodimeric, and heterodimeric antibodies.

[00453] Embodiment 13: An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin variable light chain (VL) domain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO: 101, and SEQ ID NO: 105; or encoding an amino acid sequence variant of any of these members.

[00454] Embodiment 14: An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin variable light chain (VH) domain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO: 103, and SEQ ID NO: 107; or encoding an amino acid sequence variant of any of these members.

[00455] Embodiment 15: An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin light chain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:36, SEQ ID NO:56, and SEQ ID NO:73, or that encodes any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are amino acid residues are lacking from the N-terminal or C-terminal, or from both.

[00456] Embodiment 16: An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin heavy chain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:44, SEQ ID NO:62, and SEQ ID NO:81, or that encodes any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are amino acid residues are lacking from the N-terminal or C-terminal, or from both.

[00457] Embodiment 17: An expression vector, comprising the isolated nucleic acid of any of Embodiments 13-16.

[00458] Embodiment 18: An expression vector, comprising the isolated nucleic acid of Embodiment 15 or Embodiment 16, or both.

[00459] Embodiment 19: A host cell, in culture, comprising the expression vector according to any of Embodiments 17-18.

[00460] Embodiment 20: The host cell, in culture, according to Embodiment 19, where the host cell is a mammalian cell.

[00461] Embodiment 21: The host cell, in culture, according to any of Embodiments 19-20, being derived from a Chinese Hamster Ovary (CHO) cell or a human embryonic kidney (HEK) cell.

[00462] Embodiment 22: A method of producing an antigen-binding protein that specifically binds human CD307e, comprising:

(a) culturing the host cell according to any of Embodiments 19-21, in an aqueous medium under physiological conditions permitting expression of the antigenbinding protein; and

(b) recovering the antigen-binding protein from the medium. [00463] Embodiment 23: A composition, comprising the antigen-binding protein according to any of Embodiments 1-12; and a pharmaceutically acceptable carrier or excipient.

[00464] Embodiment 24: An engineered cell expressing a receptor comprising the antigenbinding protein according to any of Embodiments 1-12 on its cell surface.

[00465] Embodiment 25: The engineered cell of Embodiment 24, being an immune effector cell or a stem cell that can give rise to an immune effector cell.

[00466] Embodiment 26: An immune effector cell population, comprising a plurality of the engineered cell or the stem cell that can give rise to an immune effector cell according to Embodiment 25.

[00467] Embodiment 27: A composition comprising the engineered cell or the immune effector cell population according to any of Embodiments 24-26, and a pharmaceutically acceptable carrier or excipient.

[00468] Embodiment 28: A method of treatment, comprising administering the composition of Embodiment 23 or Embodiment 27 to a subject or patient having a disease or disorder associated with CD307e.

[00469] Embodiment 29: A method of providing anti-disease immunity in a subject comprising administering to the subject an effective amount of the engineered cell according to any of Embodiments 24-25, wherein the engineered cell is an autologous or allogeneic immune cell.

[00470] Embodiment 30: The method of providing anti-disease immunity in a subject of Embodiment 29, wherein the engineered cell is selected from the group consisting of an autologous T cell, an allogeneic T cell, an autologous NKT cell, an allogeneic NKT cell, an autologous hematopoietic stem cell, an allogeneic hematopoietic stem cell, an autologous iPSC that can give rise to an immune effector cell, and an allogeneic iPSC that can give rise to an immune effector cell.

[00471] Embodiment 31: The composition of Embodiment 23 or Embodiments 27, for use in the treatment of a disease or disorder associated with CD307e. [00472] Embodiment 32: The composition of Embodiment 31, wherein the disease or disorder associated with CD307e is a B cell malignancy, B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), a pro-lymphocytic leukemia, a hairy cell leukemia, a common acute lymphocytic leukemia, a Null-acute lymphoblastic leukemia, a non-Hodgkin lymphoma, a diffuse large B cell lymphoma (DLBCL), a multiple myeloma, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, Hodgkin lymphoma, an autoimmune or inflammatory disease in which B cells are implicated, B cell dependent autoimmunity, non- autoimmune inflammatory disease, or graft-versus-host disease (GVHD), or in connection with B-cell depletion therapy (BCDT).

[00473] Embodiment 33: The composition of Embodiment 23 or Embodiment 27, for use in the diagnosis or prognosis of a patient suffering from a disease or disorder associated with CD307e.

[00474] Embodiment 34: The composition of Embodiment 33, wherein the disease or disorder associated with CD307e is a B cell malignancy, B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), a pro-lymphocytic leukemia, a hairy cell leukemia, a common acute lymphocytic leukemia, a Null-acute lymphoblastic leukemia, a non-Hodgkin lymphoma, a diffuse large B cell lymphoma (DLBCL), a multiple myeloma, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, Hodgkin lymphoma, an autoimmune or inflammatory disease in which B cells are implicated, B cell dependent autoimmunity, non- autoimmune inflammatory disease, or graft-versus-host disease (GVHD), or in connection with B-cell depletion therapy (BCDT).

[00475] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES

[00476] Example 1 : Generation and Assessment of Anti-CD307e Antigen-binding Proteins, e.g., Antibodies [00477] Exemplary anti-CD307e antibodies that specifically bind to human CD307e-expressing cells were generated and assessed.

[0462] As illustrated schematically in Figure 1, Balb/c mice (4-6 weeks old) were immunized with one of three human CD307e domain 9 peptides (and CFA and IFA adjuvants): Peptide 1 (RAPGTHAAVGDLLELHCEAL//SEQ ID NO: 109), from amino acid residue positions 757-776 of SEQ ID NO: 108; Peptide 2 (EALRGSPLILYRFFHEDVTL//SEQ ID NO: 110), from amino acid residue positions 774-793 of SEQ ID NO: 108; or Peptide 3 (EADNGLGAQRSETVTLYITG//SEQ ID NO: 111), from amino acid residue positions 820-839 of SEQ ID NO: 108. The mice were boosted two (2) times, following which their spleens were harvested and used for the generation of hybridoma clones. Only Peptide 1 and 3 hybridoma clones were used for further screening.

[0463] Hybridoma supernatants were screened for binding specificity using ELISA to determine antibody binding to the immunizing peptides (i.e., confirmation of binding to epitope); binding to huCD307e (i.e., human FCRL5) protein, and binding to the other FCRL family members (i.e., evaluation of cross reactivity). The source for each of the human FCRL family members used in the ELISA experiments was the following:

[0464] FCRL1: Mouse myeloma cell line (NSO)-derived human FCRLl/FcRHl protein Alal7- Asn3O3, with a C-terminal hexa-histidine tag (UniProt Accession No. Q96LA6).

[0465] FCRL2: Mouse myeloma cell line (NSO)-derived human FCRL2/FcRH2 protein Leu20- Asp395, with a C-terminal hexa-histidine tag (UniProt Accession No. Q96LA5).

[0466] FCRL3: Mouse myeloma cell line (NSO)-derived human FCRL3/FcRH3 protein Arg 14- Arg569, with a C-terminal hexa-histidine tag (UniProt Accession No. NP_443171).

[0467] FCRL4: Chinese Hamster Ovary cell line (CHO)-derived human FCRL4/FcRH4 protein Alal8-Arg385, with a C-terminal hexa-histidine tag (UniProt Accession No. Q96PJ5).

[0468] FCRL5: Mouse myeloma cell line (NSO)-derived human FCRL5/FcRH5 protein Glnl6- Arg844, with a C-terminal hexa-histidine tag (UniProt Accession No. NP_AAK93971). [0469] Briefly, the ELISA protocol was run in 96-well plates and included:

[0470] Peptide coating: lOOpl of a CD307e (FCRL5) peptide, i.e., either Peptide 1 (SEQ ID NO: 109), Peptide 3 (SEQ ID NO: 111), or CD307e (FCRL5) polypeptide, respectively, coated at 4pg/ml in 0.5M bicarbonate buffer at 4°C overnight.

[0471] Blocking: Plates were washed thrice with wash buffer (phosphate buffered saline (PBS) and 0.05% Tween-20) and were then blocked with 200pl of 1% bovine serum albumin (BSA) in PBS at room temperature (RT) for 1 hour.

[0472] Primary: In individual wells, lOOpl of each the separate hybridoma supernatants was added and incubated at RT for 2 hours.

[0473] Secondary: lOOpl of goat-anti-mouse-HRP-conjugated antibody (Jackson) was added to each well and incubated at RT for 1 hour.

[0474] Substrate: lOOpl of 3,3',5,5'-tetramethylbenzidine (TMB) liquid peroxidase substrate (Sigma- Aldrich, Cat. No. T0440) was added per well, and the plates were incubate at RT for 10 mins. The ELISA reactions were stopped with IN HC1 (50pl/well).

[0475] Flow cytometry was subsequently performed to determine binding to known CD307e⁺ cell lines; binding to known CD307e“ cell lines; binding to K562 cells transduced to express Domain 9 of human CD307e; and binding to patient multiple myeloma (MM) cells.

The CD307e-specific TotalSeq™ C0829 anti-human CD307e (FcRL5) Mouse IgG2a, K antibody (Biolegend, Cat. No. 340304) antibody was used as a positive control, as necessary. Negative control was secondary antibody alone.

[0476] As shown in Figure 2, only hybridoma mAb Clones 1 through 24 bound to Peptide 3 (SEQ ID NO: 111), which was used to generate them. As expected, mAbs of Clones 25 through 44, generated using Peptide 1 (SEQ ID NO: 109) as the immunogen, did not bind to Peptide 3 (SEQ ID NO: 111). This confirmed that the mAbs in the supernatants of Clones 1 through 24 are capable of binding to immunizing Peptide 3 (SEQ ID NO: 111).

[0477] As shown in Figure 3, only hybridoma mAb Clones 25 through 44 bound to Peptide 1 (SEQ ID NO: 109), which was used to generate them. As expected, mAbs of Clones 1 through 24, generated using Peptide 3 (SEQ ID NO: 111), did not bind to Peptide 1 (SEQ ID NO: 109), although some relatively mild cross -reactivity with Peptide 1 was seen in Clones 2, 11, 13, 21 and 22, as shown by Figure 3, which would be expected due to some polypeptide sequences in the different domains of the CD307e molecule being conserved across the different FCRL family members. This result confirmed that the mAbs in the supernatants of Clones 25 through 44 are capable of binding to immunizing Peptide 1 (SEQ ID NO: 109). In the ELISA experiments, binding to CD307e was observed with anti-huCD307e antibody positive control (Biolegend, Cat. No. 340304), and with 18/24 clones produced by immunogen Peptide 3 (SEQ ID NO: 111), and with 3/20 clones produced by immunogen Peptide 1 (SEQ ID NO: 109). Representative results are shown in Table 2, below, which shows absorbance readings in the ELISA. It is possible that the clones that did not bind to FCRL5 in the ELISA experiment bind to linear epitopes that are not formed within the tertiary structure of recombinant FCRL5 protein.

[0478] Table 2. Representative ELISA results for Clones 1-44 in wells coated with huCD307e (FCRL5) polypeptide, in a 96-well plate. “BL anti-CD307e” means CD307e-specific TotalSeq™ C0829 anti-human CD307e (FcRL5) Mouse IgG2a, K antibody (Biolegend, Cat. No. 340304) antibody was used as a positive control, in the indicated quantity. “Sec alone” means no hybridoma supernatant was added as primary, but lOOpl of secondary goat-anti-mouse-HRP- conjugated antibody (Jackson). “OD” means optical density at 450-570 nm wavelength corresponding to each of Rows A-E.

[0479] Binding to other recombinant protein FCRLs and cross -reactivity. To evaluate crossreactivity of the hybridoma clones with other FCRL family members, the ability of the 44 hybridoma clone supernatants to bind to recombinant FCRL1, FCRL2, FCRL3 and FCRL4 proteins was evaluated by ELISA. For this, ELISA plates were coated with the respective recombinant FCRL proteins and then incubated with the individual hybridoma clone supernatants. Binding was detected using goat anti-mouse HRP conjugated secondary antibody and TMB substrate. The ELISA protocol was run in 96-well plates and included:

[0480] Coating: 1 OOp I of Recombinant Human FCRL1 or FCRL2 or FCRL3 or FCRL4, coated at Ipg/mL concentration in 0.5M bicarbonate buffer for 2 hours at 37°C.

[0481] Blocking: Plates were washed thrice with wash buffer (PBS + 0.05% Tween-20) and blocked with 200pl of 1% BSA in PBS at RT for 1 hour.

[0482] Primary: In individual wells, lOOp I of hybridoma supernatant (1:1 dilution) was added and incubated at RT for 2 hours.

[0483] Secondary: lOOpl of goat-anti-mouse-HRP-conjugated antibody (1:3000 dilution) was added to each well and incubated at RT for 1 hour.

[0484] Substrate: lOOpl of TMB/well was incubated at RT for 10 min.

[0485] The ELISA reactions were stopped with IN HC1, 5 O 1/well.

[0486] Representative results are shown in Table 3 (FCRL1), Table 4 (FCRL2), Table 5 (FCRL3), and Table 6 (FCRL4), respectively (below).

[0487] The ELISA showed that 14/24 clones (i.e., Clones 1, 2, 3, 4, 5, 6, 9, 10, 11, 13, 14, 21, 22, and 23) produced by immunogen Peptide 3 (SEQ ID NO: 111) bind to FCRL1, and 3/20 clones (i.e., Clones 26, 27, and 28) produced by immunogen Peptide 1 (SEQ ID NO: 109) were seen to bind to FCRL1. The ELISA showed that 13/24 clones (i.e., Clones 1, 2, 3, 4, 5, 6, 9, 10, 11, 13, 14, 21, and 22) produced by immunogen Peptide 3 (SEQ ID NO: 111) bind to FCRL2, and 1/20 clones (i.e., Clone 26) produced by immunogen Peptide 1 (SEQ ID NO: 109) bound to FCRL2. [0488] The ELISA showed that 6/24 clones (i.e., Clones 2, 15, 16, 17, 18 and 22) produced by immunogen Peptide 3 (SEQ ID NO: 111) bind to FCRL3, and 0/20 clones produced by immunogen Peptide 1 (SEQ ID NO: 109) bound to FCRL3. The ELISA showed that 3/24 clones (i.e., Clone 2, 13 and 22) produced by immunogen Peptide 3 (SEQ ID NO: 111) bind to FCRL4, and 0/20 clones produced by immunogen Peptide 1 (SEQ ID NO: 109) bound to FCRL4.

[0489] Table 3. Representative ELISA results for Clones 1-44 in wells coated with human FCRL1 polypeptide, in a 96-well plate. “Sec alone” means no hybridoma supernatant was added as primary, but lOOpl of secondary goat-anti-mouse-HRP-conjugated antibody (Jackson). “OD” means optical density at 450-570 nm wavelength corresponding to each of Rows A-D.

[0490] Table 4. Representative ELISA results for Clones 1-44 in wells coated with human

FCRL2 polypeptide, in a 96-well plate. “Sec alone” means no hybridoma supernatant was added as primary, but lOOpl of secondary goat-anti-mouse-HRP-conjugated antibody (Jackson). “OD” means optical density at 450-570 nm wavelength corresponding to each of Rows A-D.

[0491] Table 5. Representative ELISA results for Clones 1-44 in wells coated with human

FCRL3 polypeptide, in a 96-well plate. “Sec alone” means no hybridoma supernatant was added as primary, but lOOpl of secondary goat-anti-mouse-HRP-conjugated antibody (Jackson). “OD” means optical density at 450-570 nm wavelength corresponding to each of Rows A-D.

[0492] Table 6. Representative ELISA results for Clones 1-44 in wells coated with human

FCRL4 polypeptide, in a 96-well plate. “Sec alone” means no hybridoma supernatant was added as primary, but lOOpl of secondary goat-anti-mouse-HRP-conjugated antibody (Jackson). “OD” means optical density at 450-570 nm wavelength corresponding to each of Rows A-D.

[0493] Example 2: Binding of Clones to FCRL5 (CD307e)-expressing Multiple Myeloma Cell Line RPMI-8226

[0494] We next used flow cytometry to evaluate the ability supernatants of four of the hybridoma clones of interest (Clones 16, 25, 26, and 37) to bind to CD307e-positive and CD307e-negative cells. Cells were incubated with the individual hybridoma clone supernatants. Binding was detected with a goat anti-mouse IgG covalently conjugated to fluorescent dye Alexa Fluor 488 (Thermo Fisher Scientific; “AF488”). A commercially available anti-CD307e antibody (Biolegend Cat. No. 340304) was used to confirm whether the selected cell lines were positive or negative for CD307e expression.

[0495] As seen in Figure 4A-C, RPMI-8226 was CD307e positive (Figure 4A), whilst human CD4/CD8 T-cells (Figure 4B) and Jurkat cells (Figure 4C) were negative for CD307e expression.

[0496] Clone 16 (Figure 5A-C), Clone 25 (Figure 6A-C), Clone 26 (Figure 7A-C), and Clone 37 (Figure 8A-C) were shown by flow cytometry to bind to RPMI-8226 cells and not to bind to CD4/CD8 T cells and the Jurkat cells. Thus, their binding correlated with the expression, or nonexpression, of CD307e, as shown in Figure A-C).

[0497] Representative flow cytometry data is shown, in Figure 9A-C, for Clone 41, which did not bind to the CD307e-positive RPMI-8226 cells. This implies that the binding of some clones to CD307e is conformation-dependent. [0498] Example 3: Binding to huCD307e Domain 9

[0499] Further experiments demonstrated that the peptide epitopes targeted by the CD307e clones lie in the membrane proximal region of the huCD307e molecule within domain 9. The clones were evaluated for their ability to bind to K562 cells transduced to express Domain 9 by flow cytometry. K562 cells were transduced with a lentiviral vector for expressing CD307e domain 9. The domain 9-expressing K562 cells were incubated with individual hybridoma clone supernatants. Hybridoma supernatant was diluted 1:1 with FACS buffer (Biolegend FACS Buffer, Cat. No. 420201). A 100-pl aliquot of diluted supernatant from selected hybridoma clones was incubated with K562 cells for 30 min on ice and washed. Binding was detected by staining with a secondary antibody (goat anti-mouse covalently conjugated to phycoerythrin (PE) or Alexa647; diluted 1:300 in FACS buffer), which was added to the K562 cells, which were then incubated for 30 min on ice and washed with FACS buffer, after which the cells were ready for flow cytometry analysis.

[0500] Flow cytometry results showed that Clone 25 and Clone 26 did not bind to huCD307e domain 9 (data not shown), implying that their target epitope(s) is conformation-dependent and that huCD307e domain 9 expressed on the surface of the K562 cells did not attain the required conformation for binding of these antibodies. Clone 16 and Clone 37 bound to huCD307e domain 9, as indicated by the shift of the peak to the right (Figure 10).

[0501] Example 4: Binding of Clones to bone marrow BCMA-positive plasma cells from human multiple myeloma (MM) patients

[0502] None of the supernatants from any of the anti-CD307e hybridoma clones exhibited binding to normal donor human peripheral blood mononuclear cells (PBMCs; data not shown), nor to any immune cell subpopulation thereof (e.g., B cells, T cells, CD45⁺).

[0503] To evaluate whether the mAbs produced by the hybridoma clones recognize CD45^low, BCMA⁺ cells from the bone marrow aspirates of multiple myeloma patients, bone marrow mononuclear cells (MNC) were isolated on a Ficoll density gradient. Isolated bone marrow MNC were washed twice with PBS and IxlO⁶ cells were used for staining. The bone marrow MNC were incubated with individual hybridoma supernatants and secondary antibody. The cells were then stained with a cocktail of antibodies specifically targeting CD45, CD19 and BCMA. The cells were then washed and analyzed on a flow cytometer.

[0504] CD45^lowCD138⁺BCMA⁺cells in the multiple myeloma bone marrow samples are thought to be multiple myeloma tumor cells. As shown in Figure 11 A-D, Figure 12A-E, and Figure 13A-F, Clones 16, 25, 26 and 37 each bound to CD45^low, CD138⁺, BCMA⁺ tumor cells from the bone marrow of at least three different multiple myeloma patients. This finding implies that the clones tested can be used to target the destruction of multiple myeloma tumor cells in patients, i.e., used to design different therapies for multiple myeloma. The clones can also be used in diagnostic and prognostic tests concerning patients suffering from multiple myeloma.

[0505] Binding to immune cells from normal human donors. We evaluated whether selected clones can specifically bind to immune cells that are known to express FCRL proteins. Normal donor human peripheral blood mononuclear cells were isolated from peripheral blood using a Ficoll gradient. The cells were stained with antibodies to different cell surface specific markers CD3 (T cells), CD20 (B cells), CD16, CD56 (NK cells), CD14 (macrophages and granulocytes) and 7-AAD for live cell gating. The binding data are presented in Table 7, below, as percentage (%) live cells double positive for the marker and Clones 16, 25, 26 or 37. Briefly, none of the supernatants from any of the anti-CD307e hybridoma clones exhibited binding to normal donor human peripheral blood mononuclear cells, from which result we concluded that the Clone 16, 25, 26 and 37 monoclonal antibodies do not cross-react with human peripheral blood mononuclear cells.

[0506] Table 7. Binding of Clone 16, 25, 26, and 37 monoclonal antibodies to various immune cell types as % live cells double positive for the marker and respective clonesl6, 25, 26, and 37. The control was cells stained with secondary antibody alone.

[0507] Binding to MM Plasma cells from Patients. We assessed whether selected clones bind to CD45^low CD138⁺CD38⁺ Multiple Myeloma Plasma cells from human patients. Bone marrow mononuclear cells (MNC) were isolated on a Ficoll density gradient from bone marrow aspirates of multiple myeloma patients. (Patients meet the CRAB criteria for multiple myeloma diagnosis.) Isolated bone marrow MNC were washed twice with PBS and 1 x 10⁶ cells were used for staining. The bone marrow MNC were incubated with individual hybridoma supernatants and secondary antibody. The cells were then stained with a cocktail of antibodies specifically targeting CD45, CD138, CD38 and BCMA. The cells were then washed and analyzed on a flow cytometer.

[0508] As seen in Figure 14A-L, Clone 25 supernatant bound to plasma cells from all of the 12 multiple myeloma patient samples that met the criteria (i.e., >10% plasma cells and CRAB criteria for MM diagnosis). As seen in Table 8, below, Clone 25 mAbs bound to the patient plasma cells whether they were BCMA⁺ or BCMA’. The observations that Clone 25 mAbs bind to plasma cells from all multiple myeloma patients irrespective of BCMA expression implies that the Clone 25-derived CAR T cells would recognize plasma cells from most multiple myeloma patients and would be expected to have therapeutic activity in patients.

[0509] Table 8. Clone 25 mAbs bound to MM Plasma Cells irrespective of BCMA Expression.

[0510] Example 5. Generation of CD307e-Specific CAR T cells

[0511] We next produced CD307e CAR T cells and confirmed CAR expression on their surface. The CD307e CARs were designed, synthesized and then cloned into the backbone of a lentiviral (LV) plasmid (See, e.g., plasmid maps in Figure 15, Figure 16, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, and Figure 22). To generate the CD307e CAR LV, the respective CD307e CAR lentiviral plasmids were co-transfected along with GAG-POL, REV and VSVG helper plasmids into 293T cells. The cell supernatant was harvested, concentrated and frozen in aliquotes. The functional titer of the LV was determined by titration on HEK293 cells. To produce CD307e-specific CAR T cells, normal healthy donor T cells were selected, activated and transduced with CD307e LV at different MOIs. The T cells were expanded in vitro using TexMacs medium supplemented with IL-7 and IL- 15. The expression of CAR on the surface of the transduced cells was evaluated using flow cytometry. The transduced T cells were stained with APC-conjugated anti-mouse IgG Fab2 fragment (H+L) antibody (Jackson Immunolabs).

[0512] As seen in Figure 23A-B and Figure 24A-C, when selected T cells from normal healthy donors are transduced with CD307e LV, CAR can be detected on the surface of transduced cells (Figure 23 A), but not untransduced controls (Figure 23B). Figure 24A-B shows representative data from multiple donors transduced with different CD307eCAR LVs at different MOIs, compared to untransduced control.

[0513] These data show that the CD307e C25 LV can be used to generated CD307e CAR T cells that express CARs on their surface.

[0514] Example 6. Functional Evaluation of CD307e CAR T cells against CD307e-expressing Multiple Myeloma Cells

[0515] We next evaluated the functional potency of CD307e CAR T cells, i.e., their ability to recognize and kill CD307e-expressing multiple myeloma tumor cells; the CD307e-specific CAR T cells were tested for their ability to kill CD307-expressing multiple myeloma cells using a flow cytometry-based CTL assay. CD307e CAR T cells were co-incubated with multiple myeloma target cells at different effector: target ratios for 16 hours. CD307e-expressing U266 or RPML 8226 multiple myeloma cells were used as targets; LentiX-293T or K562 cells were used as negative controls. After the 16-h incubation, the cells were stained with antibodies to CD3, BCMA (to mark the target cells) and 7-AAD (to distinguish between live and dead cells). Relative binding data are presented in Table 9, below.

[0516] Table 9. Clone 25 (“C25”) epitope exposed on MM Tumor Cells Used as Targets for the CTL Assay.

[0517] Representative results show killing of CD307e-expressing RPMI Multiple Myeloma cells (Figure 25) or U266 Multiple Myeloma cells (Figure 26A-B), as a percent (%) viable tumor cells. Clone 25 bound to U266 and RPMI-8226 Multiple myeloma cells, implying that the epitope is exposed on these cells, but not on the negative control cell types. The CTL evaluation results (Figure 25 and Figure 26A-B) implied that there was significant lysis of CD307- expressing U266 and RPMI-8226 multiple myeloma cells by CD307 CAR T cells. As shown in Figure 26A, on day 10 (at MOI = 2.5), there was 21 % cytotoxicity observed at 5:1 E:T ratio. Data in Figure 26B show that on day 11 (MOI = 1.25), there was 20 % cytotoxicity observed at 5:1 and 10:1 E:T ratios.

[0518] From these results we conclude that the CD307e CAR T cells have functional activity and are capable of killing multiple myeloma cells that express CD307e.

[0519] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

WHAT IS CLAIMED IS:

1. An isolated recombinant antigen-binding protein that specifically binds domain 9 of human CD307e, comprising:

(c) SEQ ID NO:58, SEQ ID NO:40, SEQ ID NO:42; SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 266, SEQ ID NO: 270, SEQ ID NO: 274, and

(d) SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79; SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 294, SEQ ID NO: 298, or wherein one or more of the LCDR amino acid sequences of the set of three LCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residues; and

(B) an immunoglobulin variable heavy chain (VH) domain comprising a set of three complementarity determining regions: HCDR1, HCDR2, and HCDR3, wherein each HCDR comprises an amino acid sequence, wherein the set of three HCDR amino acid sequences is selected from the group consisting of:

(a) SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 198, SEQ ID NO:

(b) SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50; SEQ ID NO: 216 , SEQ ID NO: 220 , SEQ ID NO:224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 217 , SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO:229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 218 , SEQ ID NO: 222, SEQ ID NO: 226, SEQ ID NO: 230, SEQ ID NO: 234, SEQ ID NO: 238

(c) SEQ ID NO:46, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 250, SEQ ID NO: 254, SEQ ID NO: 258, SEQ ID NO: 262

(d) SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 277, SEQ ID NO:

281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 278 , SEQ ID NO:

282, SEQ ID NO: 286, SEQ ID NO: 290 or wherein one or more of the HCDR amino acid sequences of the set of three HCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residue/s, optionally wherein the isolated recombinant antigen-binding protein specifically binds domain 9 of human CD307e with an equilibrium dissociation constant (Kd or KD) of < 10’⁸ M, more preferably with a Kd or KD of < 5 x 10’⁹ M, and even more preferably with a Kd or KD of < 5 X IO ¹⁰ M, or even < 10' ¹⁰ M, as measured, e.g., by surface plasmon resonance, biolayer interferometry, or ELISA. The isolated recombinant antigen-binding protein according to Claim 1, comprising:

3. The isolated recombinant antigen-binding protein according to any of Claims 1-2, comprising: an immunoglobulin monomer comprising a variable light chain (VL) domain and an immunoglobulin variable heavy chain (VH) domain, wherein the VL domain comprises an amino acid sequence and the VH domain comprises an amino acid sequence, wherein the amino acid sequences of the VL domain and the VH domain, respectively, are a pair selected from the group consisting of:

(a) SEQ ID NO:93 and SEQ ID NO:95;

(b) SEQ ID NO:97 and SEQ ID NO:99;

(c) SEQ ID NO: 101 and SEQ ID NO: 103; and

(d) SEQ ID NO: 105 and SEQ ID NO: 107; or wherein the VL domain and/or the VH domain is an amino acid sequence variant of one of these members, wherein the antigen-binding protein, comprising the amino acid sequence variant VL domain and/or the amino acid sequence variant VH domain, specifically binds domain 9 of human CD307e.

4. The isolated recombinant antigen-binding protein according to any of Claims 1-3, comprising a set of six complementarity determining regions comprising three LCDR amino acid sequences: LCDR1, LCDR2, and LCDR3, and three HCDR amino acid sequences: HCDR1, HCDR2, and HCDR3, the amino acid sequences being selected from the group consisting of:

(a) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, SEQ ID NO:204, SEQ ID NO:208, SEQ ID NO: 212, SEQ ID NO:205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO: 206, SEQ ID NO: 210, and SEQ ID NO: 214; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 197, SEQ ID NO: 201,

SEQ ID NO: 198, SEQ ID NO: 202

(b) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:38, SEQ ID NO:40, and SEQ ID NO:42, SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NO: 241, SEQ ID NO: 245, SEQ ID NO: 242 and SEQ ID NO: 246; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:50, SEQ ID NO: 216 , SEQ ID NO: 220 , SEQ ID NO:224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 217 , SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO:229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 218 , SEQ ID NO: 222, SEQ ID NO: 226, SEQ ID NO: 230, SEQ ID NO: 234, SEQ ID NO: 238;

(c) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:58, SEQ ID NO:40, and SEQ ID NO:42, SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 266, SEQ ID NO: 270, SEQ ID NO: 274; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:46, SEQ ID NO:65, and SEQ ID NO:67, SEQ ID NO: 248, SEQ ID NO: 252, SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 250, SEQ ID NO: 254, SEQ ID NO: 258, SEQ ID NO: 262; and

(d) LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, being: SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79, SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 294, SEQ ID NO: 298; and HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, being: SEQ ID NO:83, SEQ ID NO:85, and SEQ ID NO:87, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 278 , SEQ ID NO: 282, SEQ ID NO: 286, SEQ ID NO: 290; or wherein one or more of the three LCDR amino acid sequences and/or one or more of the three HCDR amino acid sequences comprises a conservative amino acid substitution of one or two, preferably one, amino acid residue(s).

5. The isolated recombinant antigen-binding protein according to any of Claims 1-4, comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein:

(d) the VL domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 105, and the VH domain comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 107.

6. The isolated recombinant antigen-binding protein according to any of Claims 1-5, comprising an antibody or an antibody fragment.

7. The isolated recombinant antigen-binding protein according to any of Claims 1-6, comprising a Fab, a Fab', a F(ab')2, a Fv, a scFv, a scFv-Fc, a scFv-CH a scFab, a single chain antibody, a diabody, a maxibody, or a Bispecific T cell Engager molecule.

8. The isolated recombinant antigen-binding protein of Claims 6-7, wherein the antibody or antibody fragment is or comprises an IgGl, IgG2, IgG3 or IgG4 antibody or antibody fragment, preferably an IgG2, or and IgG4 antibody or antibody fragment.

9. The isolated recombinant antigen-binding protein according to any of Claims 1-8, wherein the recombinant antigen-binding protein is or comprises a monoclonal antibody.

10. The isolated recombinant antigen-binding protein according to any of Claims 1-9, wherein the recombinant antigen-binding protein is or comprises a human antibody.

11. The isolated recombinant antigen-binding protein according to any of Claims 1-10, comprising:

(a) an immunoglobulin light chain (LC) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:36, SEQ ID NO:56, and SEQ ID NO:73, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 291, SEQ ID NO: 295, or comprising any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are lacking from the N-terminal or C-terminal, or from both; and

(b) an immunoglobulin heavy chain (HC) comprising an amino acid sequence selected from the group consisting of SEQ ID NOTO, SEQ ID NO:44, SEQ ID NO:62, and SEQ ID NO:81, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, or comprising any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are lacking from the N-terminal or C-terminal, or from both.

12. The isolated recombinant antigen-binding protein of any of Claims 1-11, wherein the LC and HC, respectively, comprise the amino acid sequence pairs of:

(a) SEQ ID NO:2 and SEQ ID NO: 10;

(b) SEQ ID NO:36 and SEQ ID NO:44;

(c) SEQ ID NO:56 and SEQ ID NO:62; or

(d) SEQ ID NO:73 and SEQ ID NO:81;

(e) Or any combinations with the humanized LC and HC listed in Table 1 or wherein any one of the foregoing amino acid sequences lacks one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s from its N-terminal or C-terminal, or from both.

13. An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin variable light chain (VL) domain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO: 101, and SEQ ID NO: 105; or encoding an amino acid sequence variant of any of these members.

14. An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin variable light chain (VH) domain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO: 103, and SEQ ID NO: 107; or encoding an amino acid sequence variant of any of these members.

15. An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin light chain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:36, SEQ ID NO:56, and SEQ ID NO:73, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 291, SEQ ID NO: 295, or that encodes any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are amino acid residues are lacking from the N-terminal or C-terminal, or from both.

16. An isolated nucleic acid, comprising a nucleotide sequence encoding an immunoglobulin heavy chain that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:44, SEQ ID NO:62, and SEQ ID NO:8, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, or that encodes any one of the foregoing amino acid sequences from which one, two, three, four or five, preferably one, two or three, most preferable one or two, and even more preferably one amino acid residue/s is/are amino acid residues are lacking from the N-terminal or C-terminal, or from both.

17. An expression vector, comprising the isolated nucleic acid of any of Claims 13-16.

18. An expression vector, comprising the isolated nucleic acid of Claim 15 or Claim 16, or both.

19. A host cell, in culture, comprising the expression vector according to any of Claims 17-

18.

20. The host cell, in culture, according to Claim 19, where the host cell is a mammalian cell.

21. The host cell, in culture, according to any of Claims 19-20, being derived from a Chinese Hamster Ovary (CHO) cell or a human embryonic kidney (HEK) cell.

22. A method of producing an antigen-binding protein that specifically binds human CD307e, comprising:

(a) culturing the host cell according to any of Claims 19-21, in an aqueous medium under physiological conditions permitting expression of the antigen-binding protein; and

(b) recovering the antigen-binding protein from the medium.

23. A composition, comprising the antigen-binding protein according to any of Claims 1-12; and a pharmaceutically acceptable carrier or excipient.

24. An engineered cell expressing a receptor comprising the antigen-binding protein according to any of Claims 1-12 on its cell surface.

25. The engineered cell of Claim 24, being an immune effector cell or a stem cell that can give rise to an immune effector cell.

26. An immune effector cell population, comprising a plurality of the engineered cell or the stem cell that can give rise to an immune effector cell according to Claim 25.

27. A composition comprising the engineered cell according to any of Claims 24-25, and a pharmaceutically acceptable carrier or excipient.

28. A method of treatment, comprising administering the composition of Claim 23 or Claim

27 to a subject having a disease or disorder associated with CD307e.

29. A method of providing anti-disease immunity in a subject comprising administering to the subject an effective amount of the engineered cell according to any of Claims 24-25, wherein the engineered cell is an autologous or allogeneic immune cell.

30. The method of providing anti-disease immunity in a subject of Claim 29, wherein wherein the engineered cell is selected from the group consisting of an autologous T cell, an allogeneic T cell, an autologous NKT cell, an allogeneic NKT cell, an autologous hematopoietic stem cell, an allogeneic hematopoietic stem cell, an autologous iPSC that can give rise to an immune effector cell, and an allogeneic iPSC that can give rise to an immune effector cell.

31. The composition of Claim 23 or Claim 27, for use in the treatment of a disease or disorder associated with CD307e.

32. The composition of Claim 31, wherein the disease or disorder associated with CD307e is a B cell malignancy, B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), a pro-lymphocytic leukemia, a hairy cell leukemia, a common acute lymphocytic leukemia, a Null-acute lymphoblastic leukemia, a non-Hodgkin lymphoma, a diffuse large B cell lymphoma (DLBCL), a multiple myeloma, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, Hodgkin lymphoma, an autoimmune or inflammatory disease in which B cells are implicated, B cell dependent autoimmunity, non-autoimmune inflammatory disease, or graft-versus-host disease (GVHD), or in connection with B-cell depletion therapy (BCDT).

33. The composition of Claim 23 or Claim 27, for use in the diagnosis or prognosis of a patient suffering from a disease or disorder associated with CD307e.

34. The composition of Claim 33, wherein the disease or disorder associated with CD307e is a B cell malignancy, B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), a pro-lymphocytic leukemia, a hairy cell leukemia, a common acute lymphocytic leukemia, a Null-acute lymphoblastic leukemia, a non-Hodgkin lymphoma, a diffuse large B cell lymphoma (DLBCL), a multiple myeloma, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, Hodgkin lymphoma, an autoimmune or inflammatory disease in which B cells are implicated, B cell dependent autoimmunity, non-autoimmune inflammatory disease, or graft-versus-host disease (GVHD), or in connection with B-cell depletion therapy (BCDT).