CA3173480A1

CA3173480A1 - Modified b cells and methods of use thereof

Info

Publication number: CA3173480A1
Application number: CA3173480A
Authority: CA
Inventors: Kathleen Boyle; Hangil Park; Srinivas Kothakota; Mark Selby; Thomas Brennan; Lewis T. Williams
Original assignee: Walking Fish Therapeutics Inc
Current assignee: Walking Fish Therapeutics Inc
Priority date: 2020-03-31
Filing date: 2021-03-31
Publication date: 2021-10-07
Also published as: CN115552017A; BR112022019797A2; IL296947A; KR20230006821A; JP2023521966A; AU2021249123A1; MX2022012295A; WO2021202810A3; CL2022002667A1; WO2021202810A2

Abstract

The present invention relates to genetically modified B cells and their uses thereof, for example, for the treatment of a variety of diseases and disorders, including cancer, heart disease, inflammatory disease, muscle wasting disease, neurological disease, and the like. In certain embodiments, the invention relates to an isolated modified B cell ("CAR-B cell), capable of expressing a chimeric receptor ("CAR-B receptor"), wherein said chimeric receptor comprises (a) an extracellular domain; (b) a transmembrane domain; and (c) a cytoplasmic domain that comprises at least one signaling domain. In various embodiments, the invention comprises an isolated modified B cell, wherein said B cell is capable of expressing and secreting a payload, wherein the payload is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell. In various embodiments, the payload is an antibody or fragment thereof.

Description

MODIFIED B CELLS AND METHODS OF USE THEREOF
BACKGROUND OF THE INVENTION
[0001] Most cellular immunotherapies to date have focused in T cells. For example, cancer immunotherapies are primarily focused on modification and administration of T
cells ¨ enhancing the killer T cell response to a tumor. Modifying B cells for the treatment of various disease, however, is a technique that has not been extensively studied, despite the critical role of B cells in immune responses.

[0002] B cells, also known as B lymphocytes, are a type of white blood cell responsible for, among other things, helping the body resist infection and diseases. They are part of our adaptive immune system, and are capable of various immune responses, for example, secreting antibodies in response to a recognized antigen. Additionally, B cells are capable of presenting antigens, and can also secret cytokines.

[0003] Many B cells mature into plasma cells that produce antibodies (proteins) capable of fighting off infections. Other B cells mature into memory B cells. All plasma cells descended from a single B cell produce the same antibody that is directed against the antigen that stimulated it to mature. The same principle holds with memory B cells. Thus, all plasma cells and memory cells "remember" the stimulus that led to their formation. The B cell, or B
lymphocyte, is not thymus-dependent, has a short lifespan, and is responsible for the production of immunoglobulins. See e.g., https://www.medicinenet.com/script/mainiartasp?articlekey=2413.
The B cell is thus an immunologically important cell.

[0004] B cells appear to be associated with patient outcomes in the treatment of cancer. For example, the presence of tertiary lymphoid structures (TLSs) is associated with better patient outcomes. See, e.g., Helmink, B.A., et at., Nature, 2020, 577(7791), 549-555;
Petitprez F et at., Nature, 2020, 577(7791), 556-560. TLSs are aggregates of immune cells (mostly T and B cells) that arise in response to immunological stimuli. While TLSs that sun-ound tumor cells include B
cells, the role of B cells in antitumor responses have been unclear. B cells found in tumors can produce inhibitory factors that hinder the function of immune cells. See, e.g., Kessel, A., et at., Autoimmun Rev., 2012, 11(9), 670-677; Khan, A.R., et at., Nature Commun., 2015, 6, 5997.
Further, current evidence indicates that B cells impede antitumor responses in most mouse models of cancer. Affara, N.J., et al. Cancer Cell, 2014, 25(6), 809-821; Shalapour, S., et al., Nature, 2017, 551, 340-345; Ammirante, M. et at., Nature, 2010, 464, 302-305. Yet, the presence of B cells in TLS structures is con-elated with positive clinical outcomes to cancer immunotherapy. Petitprez 2020. Intratumoral injection of LPS-activated spleen cells, which include B
cells, in combination with checkpoint inhibitors has been shown to produce anti-tumor responses.
Soldevilla et al., Oncoimmunology, 2018, 7:8, e1450711.
100051 CD79 (also termed "Cluster of Differentiation 79") is a transmembrane protein that forms a complex with the B-cell receptor and is capable of generating a signal following recognition of an antigen by the B-cell receptor.' CD79 is comprised of two different chains known as CD79A
and CD79B (also termed lga and 10). CD79a and CD79b are both members of the immunoglobulin superfamily. These form a heterodimer on the surface of B cells stabilized by disulfide bonding. Both CD79 chains contain an immunoreceptor tyrosine-based activation motif ("ITAM") in their intracellular tail regions that propagate a signal in a B
ce11.2 [0006] Given the natural ability of B cells to present antigens and secrete proteins, there is great potential as a cellular therapy for targeting certain diseased cell types and secreting therapeutic payloads. There thus exists a need for alternative treatments beyond T cell therapies, such as engineered B cells, for the treatment of a variety of diseases and disorders, including cancer, heart disease, inflammatory disease, muscle wasting disease, neurological disease, and the like.
SUMMARY OF THE INVENTION
[0007] It has now been found that engineered B cells can be efficacious in the treatment of various diseases and disorders as recited herein. The invention therefore relates to modified B cells.
[0008] It has also been found that CD79a (Immunoglobulin a) when incorporated into the intracellular signaling domain of the CAR-B constructs of the invention exhibits superior qualities over CD79b (Immunoglobulin 13). Further, it has further been found that when used in the CAR-B
constructs described herein, intracellular CD79b (Immunoglobulin 13) displays no (or even a negative effect) on efficacy. The invention thus relates to, inter alia, CAR-B
constructs comprising the CD79a intracellular signaling domain.
[0009] In certain embodiments, the invention relates to an isolated modified B
cell ("CAR-B
cell), capable of expressing a chimeric receptor ("CAR-B receptor"), wherein said chimeric receptor comprises (a) an extracellular domain; (b) a transmembrane domain;
and (c) a cytoplasmic domain that comprises at least one signaling domain. The cytoplasmic domain preferably comprises CD79a. In various embodiments, the extracellular domain comprises an extracellular binding domain and a hinge domain. In various embodiments, the extracellular binding domain(s) recognizes at least one antigen or protein expressed on the surface of a target cell. In various See Chu PG, Arber DA (June 2001); CD79: a review; Applied Immunohistochemistry & Molecular Morphology.
9(2): 97-106. doi:10.1097/00022744-200106000-00001. PMID 11396639. See also https://en.wikipedia.org/iviki/CD79.
2 See Muller B, Cooper L, Terhorst C (Jan. 1995). Interplay between the human TCR/CD3 epsilon and the B-cell antigen receptor associated Ig-beta (B29); Immunology Letters. 44(2-3): 97-103. doi:10.1016/0165-2478(94)00199-2. PMID 7541024.

embodiments, the target cell is selected from the group consisting of a tumor cell, cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell, and an endothelial cell. In various embodiments, the B cell expresses more than one CAR-B receptor construct. In various embodiments, the CAR-B receptor comprises more than one extracellular binding domain. In various embodiments, the extracellular binding domain is a single chain variable fragment (scFv), or a full-length antibody, or the extracellular domain of a receptor or ligand. In various embodiments, the extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GPC3, ASGR1, ASGR2, Sarcoglycan, Corin, FAP (fibroblast activation protein) and Her2. In various embodiments, the hinge domain is derived from the group consisting of IgG, CD28 and CD8. In various embodiments, the hinge domain is comprised of a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 27, 29, 31. In various embodiments, the cytoplasmic domain comprises at least one signaling domain native to B cell receptors. In various embodiments, the cytoplasmic domain comprises a domain that is selected from the group consisting of: CD79a (Immunoglobulin a), CD79b (Immunoglobulin 13), CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, C:03(,, and BLNK. In various embodiments, the cytoplasmic domain further comprises a costimulatory domain.
[0010] In various embodiments, the invention comprises an isolated modified B
cell, wherein said B cell is capable of expressing and secreting a payload, wherein the payload is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell. In various embodiments, the payload is an antibody or fragment thereof In various embodiments, the antibody is a secreted antibody and can include blocking antibodies (eg anti-PD-1) or agonist antibodies (anti-CD137, GITR, 0X40) engineered to contain native or engineered Fc regions and can be soluble or membrane-bound In various embodiments, the payload(s) can be immune modifiers such as chemokines or cytokines. . In various embodiments, the payload is selected from the group consisting of: IL-1, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL18, IL-21, interferon a, interferon 13, interferon y, TSLP, CCL21, FLT3L, XCL1, LIGHT(TNFSF14), OX4OL, CD137L, CD4OL, ICOSL, anti-CD3 antibody, CD47, TIM4-FC, CXCL13, CCL21, CD80, CD4OL, IFNa A2, LIGHT, 4-1BBL, MDGF (C19orf10), FGF10, PDGF, agrin, TNF-a, GM-CSF, an anti-FAP
antibody, an anti-TGF-I3 antibody; a TGF-I3 trap, decoy or other inhibitory molecule; an anti-BMP
antibody; a BMP trap, decoy or other inhibitory molecule. In various embodiments, the B cell is capable of expressing more than one payload. In various embodiments, the B
cell is capable of expressing more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 payloads.
[0011] In various embodiments, the invention relates to a method of treating a patient comprising administering the modified B cell of the present invention. In various embodiments, the modified B cell is administered intra-tumorally, intravenously, subcutaneously, or intradermally. In various embodiments, the method further comprises administering a checkpoint inhibitor. In various embodiments, the checkpoint inhibitor to a checkpoint molecule that is selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG3, TIM-3 and NKG2A proteins. In various embodiments, the checkpoint inhibitor is a monoclonal antibody.
100121 In various embodiments, the invention relates to an isolated modified B
cell, capable of expressing a chimeric receptor, wherein said chimeric receptor comprises (a) an extracellular domain, wherein the extracellular domain comprises an extracellular binding domain and a hinge domain; (b) a transmembrane domain; and (c) a cytoplasmic domain that comprises at least one signaling domain, wherein said modified B cell is further capable of expressing a payload, wherein the payload is not naturally expressed on the surface of a cell. In various embodiments, the extracellular binding domain recognizes an antigen or protein expressed on the surface of a target cell. In various embodiments, the target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell and an endothelial cell. In various embodiments, the B cell expresses more than one CAR-B
receptor construct. In various embodiments, the CAR-B receptor comprises more than one extracellular binding domain. In various embodiments, the extracellular binding domain is a single chain variable fragment (scFv), an antibody, or the extracellular domain of a receptor or ligand. In various embodiments, the extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of PSMA, GP3, ASGR1, ASGR2, Sarcoglycan, Corin, FAP and Her2. In various embodiments, the hinge domain is derived from the group consisting of IgG, CD28 and CD8. In various embodiments, the hinge domain is comprised of a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 27, 29, and 31. In various embodiments, the cytoplasmic domain comprises at least one signaling domain native to B cells.
In various embodiments, the cytoplasmic domain comprises a domain selected from the group consisting of: CD79a (Immunoglobulin a), CD79b (Immunoglobulin 13), CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, CD3. and BLNK. In various embodiments, the cytoplasmic domain further comprises a costimulatory domain.
In various embodiments, the payload is a secreted or membrane bound antibody or fragment thereof In various embodiments, the payload is selected from the group consisting of: IL-1, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, IL-21, interferon a, interferon f, interferon 7, TSLP, CCL21, FLT3L, XCL1, LIGHT(TNFSF14), OX4OL, CD137L, CD4OL, ICOSL, anti-CD3 antibody, CD47, FC, CXCL13, CCL21, CD80, CD4OL, IFNa A2, LIGHT, 4-1BBL, MDGF (C19orf10), FGF10, PDGF, agrin, TNF-a, GM-CSF, an anti-FAP antibody, an anti-TGF-I3 antibody; a TGF-I3 trap, decoy or other inhibitory molecule; an anti-BMP antibody; a BMP trap, decoy or other inhibitory molecule. In various embodiments, the B cell is capable of expressing more than one payload In various embodiments, the B cell is capable of expressing more than 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, 11, or 12 payloads. In various embodiments, the modified B cell further encodes at least one protein selected from the group consisting of: the cytoplasmic domains of CD79a, CD79b, CD40, CD19, CD137, Fcyr2a, (21)3( and MyD88. In various embodiments, the intention relates to a method of treating a patient comprising administering the modified B cell. In various embodiments, the method further comprises administering a checkpoint inhibitor. In various embodiments, the checkpoint inhibitor is selected from inhibitors to one or more checkpoint molecules from the group consisting of: PD-1, PD-L1, CTLA-4, LAG3, TIM-3 and NKG2A. In various embodiments, the checkpoint inhibitor is a monoclonal antibody. In various embodiments, the present invention relates to an isolated modified B cell, capable of expressing a chimeric receptor, wherein said chimeric receptor comprises an extracellular domain, wherein said extracellular domain comprises a hinge domain and an extracellular binding domain, wherein said extracellular binding domain is not naturally expressed on a B cell; and wherein said extracellular binding domain is capable of recognizing a target of interest. In various embodiments, the binding domain is a single chain variable fragment (scFv), antibody, ligand or receptor. In various embodiments, the binding domain is an scFv. In various embodiments, the binding domain is a receptor, a ligand, or a fragment thereof In various embodiments, the B cell is further capable of expressing a payload. In various embodiments, the invention comprises a method of treating a patient comprising administering the modified B cell to a patient.
[0013] In various embodiments, the present invention comprises a nucleic acid capable of expressing a chimeric B cell receptor, wherein said chimeric receptor comprises: (a) an extracellular domain, wherein said extracellular domain comprises an extracellular binding domain and a hinge domain; (b) a transmembrane domain; and (c) a cytoplasmic domain that comprises at least one signaling domain. In various embodiments, the extracellular binding domain, recognizes an antigen or protein expressed on the surface of a target cell. In various embodiments, the extracellular binding domain is a single chain variable fragment (scFv), antibody, receptor or ligand. In various embodiments, the target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell and an endothelial cell. In various embodiments, the vector expresses more than one CAR-B receptor. In various embodiments, the CAR-B receptor expresses more than one extracellular binding domain.
In various embodiments, the extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of PSMA, GP3, ASGR1, ASGR2, Sarcoglycan, Corin, Her2, FAP, MUC1, CEA153, JAM-1, and LFA-1. In various embodiments, the hinge domain is derived from the group consisting of IgG, CD28 and CD8. In various embodiments, the hinge domain is comprised of a nucleic acid sequence selected from the group consisting of SEQ ID Nos.
27, 29, and 31. In various embodiments, the cytoplasmic domain comprises at least one signaling domain native to B cell receptors. In various embodiments, the cytoplasmic domain comprises a domain selected from the group consisting of: CD79a (Immunoglobulin u), CD79b (Immunoglobulin (3), CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, CD3C and BLNK. In various embodiments, the cytoplasmic domain further comprises a costimulatory domain.
[0014] In various embodiments, the invention relates to a vector comprising a nucleic acid capable of expressing a chimeric B cell receptor, wherein said chimeric receptor comprises: (a) an extracellular domain, wherein said extracellular domain comprises an extracellular binding domain and a hinge domain; (b) a transmembrane domain; and (c) a cytoplasmic domain that comprises at least one signaling domain. In various embodiments, the extracellular binding domain recognizes an antigen or protein. In various embodiments, the target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell and an endothelial cell. In various embodiments, the vector expresses more than one CAR-B receptor. In various embodiments, the CAR-B expresses more than one extracellular binding domain. In various embodiments, the extracellular binding domain is a single chain variable fragment (scFv), antibody, receptor or ligand. In various embodiments, the extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GPC3, ASGR1, AGSR2, Sarcoglycan, Corin, Her2, FAP, MUCI, CEA153, JAM-1, and LFA-1. In various embodiments, the hinge domain is derived from the group consisting of IgG, CD28 and CDR. In various embodiments, the hinge domain is comprised of a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 27, 29, and 31. In various embodiments, the cytoplasmic domain comprises at least one signaling domain native to B cells.
In various embodiments, the cytoplasmic domain comprises a signaling domain selected from the group consisting of: CD79a (Immunoglobulin a), CD79b (Immunoglobulin 13), CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, CD3C and BLNK. In various embodiments, the cytoplasmic domain further comprises a costimulatory domain.
The various embodiments, the cytoplasmic region is comprised of multiple, 2 or more, domains, being either identical or unique.
[0015] In various embodiments, the invention relates to an isolated modified B
cell, capable of expressing an integrin, a homing antibody, protein, a receptor, or combinations thereof, wherein said integrin, homing antibody, protein, or receptor is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell; and wherein said integrin, homing antibody, protein, receptor, or combinations thereof is attracted to a site or target of interest. In

6 various embodiments, the integrin, homing antibody, protein, and receptor is selected from CLA
(PSGL-1 glycoform), CLA (PSGL-1 glycoform), CCR10, CCR3, CCR4, CCR5, CCR6, CCR9, CD43E, CD44, c-Met, CXCR3, CXCR4, LFA-1, LFA-1 (aLi32), selectin ligands, VLA-4, VLA-4 (a4131), and a4137, or combinations thereof. In various embodiments, the site of interest is a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment, where delivery of payloads is desirable. in various embodiments, the homing or target tissue is selected from skin, gut (intestine, colon, mesenteric lymph nodes (mLN), Peyer's Patch (PP), small intestine), liver, lung, bone marrow, heart, peripheral lymph node (LN), CNS, thymus, and bone marrow. In various embodiments, the target of interest is selected from CXCL16, CCL17, CCL17(22), CCL20 (MIP-3a), CCL21, CCL25, CCL27, CCL28, CCL4, CCL5, CD62E, CD62P, CXCL10, CXCL12, CXCL13, CXCL16, CXCL9/CXCL10, CXCR3, E/P-selectin, E-selectin, GPR15L, HGF, Hyaluronate, ICAM-1, ligands for CCR1, 2, 5, MAdCAM, MAdCAM-1, PNAd, VAP-1, VCAM, and VCAM-1, or combinations thereof In various embodiments, the method comprises treating a patient by administering the isolated modified B
cell. In various embodiments, the method involves further administering a compound or a derivative thereof, wherein the compound or derivative thereof is capable of increasing the expression of the integrin, homing antibody, protein, and receptor, or combinations thereof In various embodiments, the compound or a derivative thereof is capable of altering trafficking of B cells to a site or target of interest in the patient. In various embodiments, the compound is all-trans-retinoic acid (ATRA) or a derivative thereof.
[0016] In various embodiments, the invention relates to an isolated modified B
cell, capable of expressing an immune inhibitory molecule, wherein said immune inhibitory molecule is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B
cell. In various embodiments, said immune inhibitory molecule is selected from IL-10, TGF-13, PD-L1, PD-L2, LAG-3, and TIM-3, or combinations thereof In various embodiments, said immune inhibitory molecule is capable of decreasing inflammation and autoimmune activity of B
cells at a site or target of interest in a patient. In various embodiments, the invention relates to a method of treating a patient comprising administering said isolated modified B
cell. In various embodiments, said immune inhibitory molecule is selected from IL-10, TGF-I3, PD-L1, PD-L2, LAG-3, and TIM-3, or combinations thereof. In various embodiments, said immune inhibitory molecule is capable of decreasing inflammation and autoimmune activity of B
cells at a site or target of interest in the patient. In various embodiments, the invention relates to further administering a compound or a derivative thereof capable of increasing the expression of an integrin, a homing antibody, a protein, a receptor, or combinations thereof in the B cell. In various embodiments, said compound or derivative thereof is capable of altering trafficking of B cells to a

7 site or target of interest in the patient In various embodiments, said compound is all-trans-retinoic acid (ATRA) or a derivative thereof In various embodiments, the invention relates to an isolated modified B cell, wherein the isolated modified B cell is treated with a compound or a derivative thereof, wherein said compound or derivative thereof is capable of increasing the expression of an integrin, a homing antibody, a protein, a receptor, or combinations thereof in B cells. In various embodiments, said compound or derivative thereof is capable of altering trafficking of B cells to a site or target of interest in the patient. In various embodiments, said compound is all-trans-retinoic acid (ATRA) or a derivative thereof In various embodiments, said compound or derivative thereof is capable of (i) increasing the expression of an integrin, a homing antibody, a protein, a receptor, or combinations thereof in B cells, and (ii) altering trafficking of B cells to a site or target of interest in the patient. In various embodiments, said compound is all-trans-retinoic acid (ATRA) or a derivative thereof [0017] In various embodiments, the invention relates to an isolated modified B
cell, capable of expressing at least one or more of a constitutively active Toll-like receptor (TLR), wherein said TLR is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell. In various embodiments, said TLR is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and TLR13, or combinations thereof In various embodiments, said TLR is capable of potentiating B cells for increasing immune responses in a patient. In various embodiments, said TLR is capable of producing potent effector B cells for increasing immune responses in a patient. In various embodiments, said immune inhibitory molecule is capable of decreasing inflammation and autoimmune activity of B
cells at a site or target of interest in a patient. In various embodiments, said TLR is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, or combinations thereof In various embodiments, said TLR is capable of (i) potentiating B cells, and (ii) producing potent effector B cells, for increasing immune responses in a patient. In various embodiments, at least one or more of a TLR agonist is administered to the patient. In various embodiments, the isolated modified B cell is treated with at least one or more of a TLR agonist. In various embodiments, said TLR agonist is capable of (i) potentiating B cells, and (ii) producing potent effector B cells, for increasing immune responses in a patient. In various embodiments, said TLR agonist binds ic.3 one or more 11_,Rs selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, or combinations thereof In various embodiments, said TLR agonist is selected from CpG-rich oligonucleotides, double-stranded RNA
mimic, polyinosinic acid:polycytidylic acid (poly-I: C). In various embodiments, said TLR agonist comprises CpG oligonucleotides. In various embodiments, said TLR agonist is capable of is capable of (i) potentiating B cells, and (ii) producing potent effector B
cells, for increasing immune

8 responses in the patient In various embodiments, said TLR agonist binds to one or more TLRs selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRIO, TLR11, TLR12, and TLR13, or combinations thereof In various embodiments, said TLR
agonist is selected from CpG-rich oligonucleo tides, double-stranded RNA mimic, polyinosinic acid:poly cy tidylic acid (poly-1:C). In various embodiments, said TLR agonist comprises CpG
oligonucleotides.
[0018] In various embodiments, the invention relates to an isolated modified B
cell, wherein said B cell is electroporated with an mRNA encoding at least one or more of an antigen fused to a targeting signal. In various embodiments, said antigen is (i) not naturally presented by a B cell, (ii) not presented by a B cell simultaneously in both HLA class I and class II
molecules naturally, or (iii) not presented by a B cell with high efficiencies in both HLA class I
and class II molecules naturally. In various embodiments, said targeting signal is targeting signal of a lysosomal protein.
In various embodiments, said targeting signal is a targeting signal of lysosome-associated membrane protein-1 (LAMP1). In various embodiments, said antigen is capable of being targeted to the lysosomes and presented simultaneously and efficiently in both HLA
class I and class II
molecules. In various embodiments, said B cells is capable of increasing antigen-specific immune responses in a patient. In various embodiments, said antigen is (i) not naturally presented by a B
cell, (ii) not presented by a B cell simultaneously in both HLA class 1 and class 11 molecules naturally, or (iii) not presented by a B cell with high efficiencies in both HLA class I and class II
molecules naturally. In various embodiments, said targeting signal is targeting signal of a lysosomal protein. In various embodiments, said targeting signal is a targeting signal of lysosome-associated membrane protein-1 (LAMP1). In various embodiments, said antigen is capable of being targeted to the lysosomes and presented simultaneously and efficiently in both HLA class I and class II molecules. In various embodiments, said B cells is capable of increasing antigen-specific immune responses in the patient.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 sets forth an example of a chimeric B Cell Receptor (CAR-B) of the present invention. In certain embodiments, the CAR-B construct will comprise an extracellular domain, a transmembrane domain, and a cytoplasmic domain. As depicted in FIG. 1, the extracellular domain may in certain embodiments comprise a binding domain and a hinge region. In certain embodiments, the binding region may be an scF v. CAR-B constructs are cloned into a vector for expression.
[0020] FIGS. 2A-2C show examples of engineered B cells with homing domains. In various embodiments, the engineered B cells may comprise (a) an scFv binding domain and optional hinge

9

10 region; (b) an scFv binding domain directly linked to the cell through a transmembrane domain, or (c) a ligand/receptor binding domain directly linked to a cell through a transmembrane domain.
100211 FIG. 3 shows examples of certain CAR-B constructs of the present invention. (A) CAR-B that binds GPC3. (B) CAR-B that binds PSMA.
[0022] FIG. 4 shows examples of CAR-B receptors of the present invention capable of binding (A) GPC3 and (B) PSMA. The -C" domain corresponds to the native BCR C-terminus.
[0023] FIG. 5 sets forth expression of various anti-PSMA CARs on the surface of HEK-293 cells.
[0024] FIGS. 6A-6C set forth a FACS Plot illustrating interrogation of binding of anti-PSMA
CAR and of anti-sarcoglycan CAR to PSMA. B cells expressing anti-PSMA CAR-B
constructs pWF396 and pWF397 bound PSMA whereas the B cells expressing pWF398 (anti-sarcoglycan CAR-B) did not bind PSMA.
[0025] FIG. 7 illustrates the ability of adenovirus F35 encoding GFP to transduce human B cells.
Human B cells were isolated from peripheral blood. The B cells were infected with adenovirus encoding GFP. 0, 1, 3, 10 ul, representing the microliter volume of the adenovirus preparation used to infect human B cells. The titer of the adenovirus preparations were approximately 1 x particles/ml.
[0026] FIG. 8 describes an experiment where BALB/c mice were injected with CT26 bilateral tumors at day zero. At day 12 and day 16, tumor-bearing mice were injected intra-tumorally with payload-expressing cells at a volume of 106 in 50 L.
100271 FIG. 9 illustrates the effect of 12 different combinations of payloads injected intra-tumorally on tumor volume over 30-35 days as compared to saline and 3T3 cells (without a payload).
[0028] FIG. 10 illustrates the effect of 12 different combinations of payloads injected intra-tumorally on tumor volume over 30-35 days as compared to saline and 3T3 cells (without a payload).
[0029] FIGS. 11A-11C illustrate the effect of the top three combinations of payloads injected intra-tumorally on tumor volume over 30 days as compared to saline and 3T3 cells (without a payload).
[0030] FIG. 12 illustrates the abscopal effect of intratumorally injected B
cells. B cells were then injected either (i) fresh or (ii) first stimulated for 16-24 hours in growth media (RPMI, 10% FBS, 1% Pen/Strep, 5ng/m1 recombinant mouse IL-4, 100uM beta-mercaptoethanol) with 5 pg/ml Lipopolysaccharide. 5X106 B cells were then intratumorally injected into the CT26 mouse model, and anti-tumor responses in the distal (abscopal) tumor where measured. Tumors were implanted at day 0, and at day 6 palpable tumor mass was observed. Treatment was initiated on day 6 intratumorally.

[0031] FIGS. 13A-13C illustrates expression of three CAR-B receptors (also referred to as CAR-B receptors) in mouse B cells 24 hours post transfection.
100321 FIG. 14 illustrates the efficacy of PSMA-specific CAR engineered murine B cells on tumor volume and survival in BALB/c mice with CT26-PSMA tumors.
[0033] FIG. 15 illustrates the efficacy of PSMA-specific CAR engineered allogenic B cells on tumor volume and survival in BALB/c mice with CT26-PSMA tumors.
[0034] FIGS. 16 illustrates the efficacy of PSMA-specific CAR engineered murine B cells on immunocompromised BALB/c mice with CT26-PSMA tumors.
[0035] FIG. 17 illustrates the efficacy of murine B cells on tumor volume and survival in C57B1/6 mice with HEPA 1-6 GPC3 tumors.
100361 FIG. 18 illustrates NFKb signaling in luciferase reporter cells in B
cells engineered with four different CAR constructs that recognized GPC3, using GFP as a control.
[0037] FIG. 19 illustrates basal or tonic NFKb activity in the absence of cognate target antigen in CAR constructs expressed in human B cell reporter line.
[0038] FIG. 20 illustrates the efficacy of murine B cells electroporated with anti-GPC3CAR-CD79a and a CD80 payload mRNAs in syngeneic C57B1/6 mice with HEPA1-6GPC3 tumors.
[0039] FIGS. 21A-21C illustrate the responses of the saline control, anti-GPC3CAR-CD79a, and anti-GPC3CAR-CD79a plus CD80 combo B cell groups.
[0040] FIGS. 22A-22C illustrate the expression of the GPC3 CAR post-electroporation, using FACS plots.
DETAILED DESCRIPTION
[0041] The invention disclosed herein relates to several embodiments of engineered or modified B cells:
1. B cells that have been modified to home to a site/target of interest, using, e.g., a binding domain such as an scFv, antibody, ligand, receptor, or fragments thereof;
2. B cells that have been modified with a homing domain, further comprising an activation, and optionally a costimulatory domain, such that the B cells can home and activate upon interaction with a desired target;
3. B cells engineered to be capable of making a desired protein payload, such as an antibody, therapeutic protein, polypeptide, nucleic acid sequence (such as RNAi) or the like;
4. Engineered B cells comprising a homing/binding domain, an activating domain, an optional costimulatory domain, and further engineered to express a desire protein payload, such as an antibody, therapeutic protein, polypeptide, nucleic acid sequence (such as RNAi) or the like;

11 5. B cells that have been modified to express an integrin, a homing antibody, protein, or a receptor, such that the B cells are attracted to specific ligands, chemokines, or attractants at a specific site/target of interest (e.g., a homing tissue) and can thereby home to the site/target of interest, for example, to deliver a desired payload;
6. B cells that have been modified to express an immune inhibitory molecule, such that the inflammation and autoimmune activity of B cells localized to a site/target of interest is decreased, thereby leading to a positive therapeutic response;
7. B cells that have been treated with a compound or derivatives thereof, such that trafficking of the B cells is altered by expression of specific B cell integrins and/or homing receptors;
8. B cells that have been (i) treated with a Toll-like receptor (TLR) agonist, and/or (ii) engineered to express a constitutively active TLR, for potentiating B cells and/or producing potent effector B cells for increasing immune responses in a subject;
9. B cells that have been electroporated with an mRNA encoding specific antigens of interest fused to a targeting signal of a lysosomal protein, such that the B cells can simultaneously and efficiently present the specific antigens and/or antigen-derived epitopes of interest in both HLA
class I and class II molecules.
10. B cells that have been electroporated with a self-amplifying RNA that encodes any items noted heretofore in 1-9.
[0042] It is understood that the various embodiments of engineered or modified B cells of the present application are not mutually exclusive and can be combined with each other in any way and without any restriction unless explicitly indicated, for achieving of facilitating any of the results and/or therapeutic responses contemplated herein.
[0043] Tumor Antigen. In certain embodiments, the site/target of interest is a tumor antigen.
The selection of the antigen-binding domain (moiety) of the invention will depend on the particular type of cancer to be treated. Some tumor antigens may be membrane bound, whereas other may be secreted. For example, a tumor antigen may be secreted and accumulate in the extracellular matrix, or the tumor antigen may be expressed as part of the MHC complex. Tumor antigens are well known in the art and may include, for example, CD19, KRAS, HGF, CLL, a glioma-associated antigen, carcinoembryonic antigen (CEA); fl-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LACE- 1 a, p53, protein, PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, EGFR, BCMA, KIT and 1L-13.

12 [0044] Infections Disease Antigen. In certain embodiments, the site/target of interest is an infectious disease antigen against which an immune response may be desired.
Infectious disease antigens are well known in the art and may include, but are not limited to, viruses, bacteria, protists, and parasitic antigens, such as parasites, fungi, yeasts, mycoplasma, viral proteins, bacterial proteins and carbohydrates, and fungal proteins and carbohydrates. In addition, the type of infectious disease of the infectious disease antigen is not particularly limited, and may include, but are not limited to, intractable diseases among viral infectious diseases such as AIDS, hepatitis B, Epstein Barr Virus (EBV) infection, HPV infection, HCV infection, SARS, SARS-CoV2, etc.
Parasitic antigens may include, but are not limited to, the malaria parasite sporozoide protein.
[0045] In certain embodiments the modified B cells express an engineered B
cell receptor (CAR-B) comprising an extracellular domain, a transmembrane domain and an intracellular domain. In certain embodiments, the extracellular domain comprises a binding domain and a hinge domain. In certain embodiments, the extracellular domain comprises a binding domain, such as an scFv, ligand, antibody, receptor, or fragment thereof which allows the modified B cell to target specific target cells by binding to proteins expressed on the surface of those cells. In certain embodiments, the modified tumor cells target and bind to proteins/antigens expressed on the surface of tumor cells.
In certain embodiments, the modified B cell further expresses a payload. In certain embodiments, the payload is capable of increasing the number of cross-presenting antigen or antigenic fragments to dendritic cells (DC) in tumors or in lymph nodes. In certain embodiments, the payload is capable of activating and attracting T cells into tumors. In certain embodiments, the payload is capable of fomenting the formation of tertiary lymphoid structures (TLS) in tumors. In certain embodiments of the invention, the modified B cell expresses both a CAR-B and a payload. In certain embodiments, the CAR-B comprises stimulatory domains that activate expression of the payload when bound to an antigen or protein expressed on the surface of a tumor cell.
1. Design and domain orientation of Chimeric Antigen Receptors in B Cells (CAR-Bs) [0046] In various embodiments, the invention provides a chimeric B Cell Receptor (CAR-B). It will be appreciated that chimeric B cell receptors (CAR-Bs) are genetically engineered receptors.
These engineered receptors can be readily inserted into and expressed by B
cells in accordance with techniques known in the art. With a CAR-B, a single receptor can be programmed to both recognize a specific protein or antigen expressed on a tumor cell, and when bound to said protein or antigen elicit an anti-tumor response. In various embodiments, the CAR-Bs serve in part as a homing mechanism to deliver B cells to target tissue.
100471 It will be appreciated that relative to the cell bearing the receptor, the chimeric B cell receptor of the invention will comprise an extracellular domain (which will comprise an antigen-binding domain and may comprise an extracellular signaling domain and/or a hinge domain), a

13 transmembrane domain, and an intracellular domain The intracellular domain comprises at least an activating domain, preferably comprised of CD79a (Immunoglobulin a), CD79b (Immunoglobulin 13), CD40, CD19, CD137, CD3 Fcyr2a and/or MyD88. It will further be appreciated that the antigen-binding domain is engineered such that it is located in the extracellular protion of the molecule/construct, such that it is capable of recognizing and binding to its target or targets.
[0048] Structurally it will be appreciated that these domains correspond to locations relative to the immune cell. Exemplary CAR-B constructs in accordance with the invention are set forth in Table 1:

mmwiiiiiiiiiiiiiiiiimmWwv*gqwgmwRmmmmgOmummngmmmmwsPWAm]q49**
pWF-82 anti-PSMA CD8 CD28 hCD19 pWF-83 anti-PSMA CD8 CD28 hCD40 pWF-84 anti-PSMA CD8 CD28 hCD40 CD79b pWF-85 anti-PSMA CD8 CD28 hCD40 CD137 pWF-86 anti-PSMA CD8 CD28 hCD40 Fcyr2a pWF-87 anti-PSMA CD8 CD28 hMyd88 hCD40 pWF-88 anti-PSMA CDS CD28 CD79a pWF-89 anti-PSMA CD8 CD28 CD79b pWF-391 anti-PSMA 3x strep II tag CD28 CD79b pWF-394 anti-Sarcoglycan 3x strep II tag CD28 CD79b pWF-396 anti-GPC-3 CD8 CD28 CD79a pWF-397 anti-GPC-3 CD8 CD28 CD79b pWF-460 anti-GPC-3 Human IgG1 Fc CD28 CD79a pWF428 anti -GPC-3 Human Lambda Human Lambda Constant region Constant region pWF429 anti-GPC-3 Human IgG1 Fc Human IgG1 Fc pWF-521 Anti-GPC3 vL- Human IgG1 Fc Human IgG1 Endogenous hclambda constant BCR complex region-linker-vH-hcHl-cH2-cH3 pWF-533 Anti-GPC3-vL-hcH1 Human IgG1 Endogenous (complex with BCR complex pWF534) pWF-534 Anti-GPC3-vH- Human IgG1 Fc Human IgG1 Endogenous hcKappa-hcH2-cH3 BCR complex

14 [0049] In various embodiments, chimeric B cell receptors are comprised of an extracellular domain, a transmembrane domain and a cytoplasmic domain. In various embodiments, the cytoplasmic domain comprises an activating domain. In various embodiments, the cytoplasmic domain may also comprise a co-stimulatory domain. In various embodiments, the extracellular domain comprises an antigen-binding domain. In various embodiments, the extracellular domain further comprises a hinge region between the antigen-binding domain and the transmembrane domain. FIG. 1 provides a schematic representation of a chimeric B cell receptor of various embodiments of the present invention.
[0050] Extracellular Domain. A number of extracellular domains may be used with the present invention. In various embodiments, the extracellular domain comprises an antigen-binding domain.
In various embodiments, the extracellular domain may also comprise a hinge region and/or a signaling domain. In various embodiments, the extracellular domains containing IgG1 constant domain may also comprise either IgG1 (hole) or IgG1 (knob) to facilitate directed CAR-B
formation.
[0051] Antigen-Binding Domain and Binding Domain. As used herein, an -antigen binding domain," "antigen-binding domain" or "binding domain" refers to a portion of the CAR-B capable of binding an antigen or protein expressed on the surface of a cell. In some embodiments, the antigen-binding domain binds to an antigen or protein on a cell involved in a hyperproliferative disease. In preferred embodiments, the antigen-binding domain binds to an antigen or protein expressed on the surface of a tumor cell. The antigen-binding molecules will be further understood in view of the definitions and descriptions below.
[0052] An antigen-binding domain is said to "specifically bind" its target antigen or protein when the dissociation constant (Ka) is 1x10' M. The antigen-binding domain specifically binds antigen with "high affinity" when the Ka is 1-5x10-9 M, and with "very high affinity"
when the Ka is 1-5x10-1 M. In one embodiment, the antigen-binding domain has a Ka of 10-9M. In one embodiment, the off-rate is <1x10-5. In other embodiments, the antigen-binding domain will bind to antigen or protein with a Ka of between about 10 M and 10-'3 M, and in yet another embodiment the antigen-binding domain will bind with a Ka 1.0-5.0x1 .
[0053] An antigen-binding domain is said to be -selective" when it binds to one target more tightly than it binds to a second target.
[0054] The term "neutralizing" refers to an antigen-binding domain that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking a binding site on the ligand or by binding to the ligand and altering the ligand's ability to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments, the term can also denote an antigen-binding domain that prevents the protein to which it is bound from performing a biological function.
100551 The term "target" or "antigen" refers to a molecule or a portion of a molecule capable of being bound by an antigen-binding molecule. In certain embodiments, a target can have one or more epitopes.
[0056] The term -antibody" refers to what are known as immunoglobulins. Y-shaped proteins that are produced by the immune system to recognize a particular antigen. The term "antibody fragment- refers to antigen-binding fragments and Fc fragments of antibodies.
Types of antigen-binding fragments include: F(ab')2, Fab, Fab' and scFv molecules. Fc fragments are generated entirely from the heavy chain constant region of an immunoglobulin.
100571 Extracellular Signaling Domains. The extracellular domain is beneficial for signaling and for an efficient response of lymphocytes to an antigen. Extracellular domains of particular use in this invention may be derived from (i.e., comprise) CD28, CD28T (See e.g., U.S. Patent Application US2017/0283500A1), 0X40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, CD79a (Immunoglobulin a), CD79b (immunoglobulin 13), DAP-10, Fc gamma receptor, MHC class 1 molecule, 'TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM
proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF I), NKp44, NKp30, NKp46, CD19, CD4, CD8a1pha, CD8beta, IL-2R beta, IL-2R gamma, IL-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source.
[0058] Hinge Domains. As described herein, extracellular domains often comprise a hinge portion. This is a portion of the extracellular domain proximal to the cell membrane. The extracellular domain may further comprise a spacer region. A variety of hinges can be employed in accordance with the invention, including costimulatory molecules as discussed above, as well as immunoglobulin (Ig) sequences a 3X strep IT spacer or other suitable molecules to achieve the desired special distance from the target cell_ In some embodiments, the hinge region comprises the extracellular domain of CD28, or CD8 or a portion thereof as described herein.
100591 Transmembrane Domains. The CAR-B can be designed to comprise a transmembrane domain that is fused or otherwise linked to the extracellular domain of the CAR-B-B. It can similarly be fused to the intracellular domain of the CAR-B. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in a CAR-B is used. In some instances, the transmembrane domain can be 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. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
Transmembrane regions of particular use in this invention may be derived from (i.e. comprise) CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1 a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, CD79a (Immunoglobulin a), CD79b (Immunoglobulin 13), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof [0060] Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR-B.
[0061] In certain embodiments, the transmembrane domain in the CAR-B of the invention is the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 1. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 2. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ

ID NO: 3. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4.
100621 In one embodiment, the transmembrane domain in the CAR-B of the invention is a CD8 transmembrane domain.
[0063] Intracellular (Cytoplasmic) Domains. The intracellular (IC, or cytoplasmic) domain of the CAR-B receptors of the invention can provide activation of at least one of the normal effector functions of the immune cell.
[0064] It will be appreciated that suitable intracellular molecules, include, but are not limited to CD79a (Immunoglobulin a), CD79b (Immunoglobulin (3), CD40, CD19, CD137, Fcyr2a CD and MyD88. Intraceullar molecules may further include CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-I (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM
proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1). NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD' Id, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDI lc, ITGBI, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/ RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRT AM, Ly9 (CD229), CD160 (BY55), PSGLI, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR-B of the invention may be linked to each other in a random or specified order.
[0065] The term -co-stimulatory" domain or molecule as used herein refers to a heterogenous group of cell surface molecules that act to amplif,7 or counteract initial activating signals of the cell.
[0066] In one preferred embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD19, wherein the hCD19 domain comprises the nucleic acid sequence set forth in SEQ ID NO. 5. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD40, wherein the hCD40 domain comprises the nucleic acid sequence set forth in SEQ ID NO. 7. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD40 and hCD79b, wherein the hCD40 domain comprises the nucleic acid sequence set forth in SEQ ID No. 7 and the hCD79b domain comprises the nucleic acid sequence set forth in SEQ ID NO. 25. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD40 and hCD137, wherein the hCD40 domain comprises the nucleic acid sequence set forth in SEQ ID NO. 7 and the hCD137 domain comprises the nucleic acid sequence set forth in SEQ ID NO. 13. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD40 and hFcyr2a, wherein the hCD40 domain comprises the nucleic acid sequence set forth in SEQ ID NO. 7 and the hFcyr2a domain comprises the nucleic acid sequence set forth in SEQ ID NO. 17. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD40 and hMyd88, wherein the hCD40 domain comprises the nucleic acid sequence set forth in SEQ ID NO. 7 and the hMyd88 domain comprises the nucleic acid sequence set forth in SEQ ID NO. 21. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD79a, wherein the hCD79a domain comprises the nucleic acid sequence set forth in SEQ ID NO. 23. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of hCD79b, wherein the hCD79b domain comprises the nucleic acid sequence set forth in SEQ ID NO. 25.
These embodiments are preferably of human origin but may be derived from other species. In various embodiments the signaling domain comprises both hCD79a in tandem with hCD79b or another hCD79a domain. In various embodiments the signaling domain comprises both hCD79b in tandem with hCD79a or another hCD79b domain.
2. Modified B Cells [0067] Modified B Cells that Express Payloads. In various embodiments of the present invention a modified B cell is provided that is capable of expressing a payload. As used herein the term "payload" refers to an amino acid sequence, a nucleic acid sequence encoding a peptide or protein, or an RNA molecule, for use as a therapeutic agent. In certain embodiments the payload is for delivery to the tumor or tumor microenvironment or the draining lymph node. In certain embodiments, it is desirable that the B cell deliver to the tumor or tumor microenvironment or draining lymph node a payload capable of, for example, increasing the number of cross-presenting dendritic cells (Des) in tumors. Cross-presenting DCs will allow for improved presentation of tumor antigens. In various embodiments, the payload may be capable of activating and attracting T cells into tumors. Activating more T cells in tumors will complement the cross-presenting DCs to remold the tumor environment to have more potent antitumor immune capabilities. Payloads may also foment the formation of tertiary lymphoid structures (TLS) in tumors.
Clinical studies have demonstrated that there is a relationship between B cells, TLS and responses to immune checkpoint blockade.

[0068] Nonexclusive examples of payloads of the present invention include: IL-1, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, IL-21, interferon a, interferon 13, interferon y, TSLP, CCL21, FLT3L, XCL1, LIGHT(TNFSF14), OX4OL, CD137L, CD4OL, ICOSL, anti-CD3 antibody, CD47, TIM4-FC, CXCL13, CCL21, CD80, CD4OL, IFNa A2, LIGHT, 4-1BBL, MDGF (C19orf10), FGF10, PDGF, agrin, TNF-a, GM-CSF, an anti-FAP antibody, an anti-TGF-13 antibody; a TGF-(3 trap, decoy or other inhibitory molecule; an anti-BMP antibody; a BMP trap, decoy or other inhibitory molecule.
[0069] Signaling for Payload Expression. In various embodiments of the present invention, the payload is expressed in the modified B cell as a DNA construct under the control of an activated transcriptional pathway. In certain embodiments, the expression of the payload is controlled of the Nuclear Factor of Activated T cell ("NFAT") pathway. The NFAT pathway is a transcription factor pathway activated during an immune response and is activated by the NFx13. In various embodiments, the modified B cell expresses both a payload and a CAR-B. In various embodiments, where the modified B cell expresses both a payload and a CAR-B, the CAR-B may further encode signaling molecules that induce activation of the NFid3 pathway. Such molecules include but are not limited to: CD79a (Immunoglobulin a), CD79b (Immunoglobulin 13), CD40, CD19, CD137, Fcyr2a, C,D3. and MyD88.
[0070] In various embodiments, the invention relates to isolated B cells that express at least one payload. In various embodiments, the invention relates to isolated B cells that express more than one payload. In various embodiments, the invention relates to isolated B cells that express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 different payloads.
[0071] Modification of B Cells for homing. In various embodiments of the present invention, the engineered B cells can be modified with homing domains (e.g., as illustrated in FIG. 2) such that the B cells can home to a site/target of interest and activate upon interaction with the target.
Additionally, B cell homing receptors expressed on B cell membranes that recognize addressins and ligands on target tissues, compound or derivatives thereof that alter the trafficking of B cells to a particular site, and inhibitory molecules inflammation and autoimmune activity of the B cells, can play a role in B cell homing and development of specialized immune responses.
[0072] Modified B cells that Express Integrin of Interest. The major homing receptors expressed by lymphocytes are the integrins, which are a large class of molecules characterized by a heterodimeric structure of a and 13 chains. In general, the pairing of specific a and 13 chains of the integrin determines the type of the homing receptor. For example, pairing of the a4 chain with 137 chain characterizes the major integrin molecule (a4137) responsible for lymphocyte binding to Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venules (HEVs) in Peyer's patches (PP) and gastrointestinal (GI) tract lamina propria endothelial venules (LPVs). Similarly, pairing of the a4 chain with f31 chain characterizes the homing receptor (a4131) for the skin.
[0073] In various embodiments of the present inventions, a B cell to be modified can be selected for in advance, with specific traits that mediate preferred localizations. For example, memory B
cells expressing CXCR3 may be enriched for and then subjected to engineering.
CXCR3 cells may be attracted to ligands expressed at sites of inflammation. As such, modified B cells can preferentially localize to such sites.
[0074] In various embodiments of the present invention, a modified B cell is provided that expresses the a4 and f37 chains of an integrin. It is desirable that expression of the a4137 integrin will promote homing of the modified B cell to the colon. In various embodiments, a modified B
cell is provided that expresses the a4 and f31 chains of an integrin. It is desirable that expression of the a4131 integrin will promote homing of the modified B cell to the skin. In various embodiments, a modified B cell is provided that expresses a desired pairing of an a and a 13 chain of an integrin, such that the expressed integrin promotes homing of the modified B cell to a desired site/target of interest. Accordingly, in various embodiments, any desired combination of the a and 13 chains of an integrin is contemplated for expression in the B cells, such that the modified B cells expressing the specific integrin is targeted to a desired site/target of interest.
[0075] Modified B cells that Express Homing Receptors of Interest. B cells have an ability to home to inflammatory tissues and altering their homing receptor expression can complement their native homing tendencies. B cell localization is also driven by expression of attractant molecules (e.g., targets such as ligands and chemokines) at inflammatory sites in specific locations or tissues. Such molecules can also include antibodies, such as the MECA79 antibody that targets cells to peripheral node addressin (PNAd). Bahmani et al., J Clin Invest.
2018;128(11):4770-4786;
Azzi et al., Cell Rep. 2016;15(6)1202-13. Accordingly, B cells can be engineered to express certain antibodies, proteins, and receptors that facilitate B cell homing to a site/target of interest and interactions of such B cells with the desired target. In certain instances, expression of such receptors redirects the B cells to the tissue of interest.
[0076] In various embodiments of the present invention, a modified B cell is provided that is capable of expressing a homing antibody, protein, or a receptor, expression of which is capable of directing the B cell to a specific site/target of interest. Exemplary homing of '1 cells to specific homing tissues (target tissues) using specific homing receptor/ligand pairs are set forth in Table 2.
The same specific homing receptor/ligand pairs are also capable of facilitating homing of B cells to a specific homing tissue (target tissue). Accordingly, in various embodiments of the present invention, homing of the modified B cells to an exemplary homing tissue (target tissue) is facilitated using the corresponding homing receptor/ligand pairs as set forth in Table 2.

Tea cell homing receptors and their cognate ligands mediating organotropic targeting Homing Tissue Type Teff Cell Homing Receptor Cognate Ligand Skin CLA (PSGL-1 glycoform) E/P-selectin CD43E E-selectin VLA-4 (a4f3i) VCAM-1 LFA-1 (aLI32) ICAM-1 Gut (intestine, colon, mLN, a437 MAdCAM-1 PP) CCR9a CCL25a Selectin ligandsb E/P-selectinb VLA-4b VCAM- lb LFA-lb ICAM-lb C CR6b CCL20 (MIP-3a)b Liver CD44 Hyaluronate Selectin ligandsb E/P-selectin 114137b MAdCAM- lb Lung LFA-1 ICAM-1 Selectin ligandsb E/P-selectinb VLA-4b VCAM- lb LFA-lb ICAM-lb Bone Marrow CLA (PSGL-1 glycoform) E/P-selecan CD43E E-selectin 014137b MAdC AM- lb Heart CCR5 CCL4, CCL5 c-Met HGF
Brain VLA-4b VCAM-lb LFA-lb ICAM-lb CXCR3b CXCL9/CXCL10b Peripheral L1STe Selectin ligandsb E/P-selectinb LFA-lb ICAM-lb CXCR3 b CXCL9/CXCL10b aInvolved in Teff cell homing to the intestine but not colon.
bInflammatory reactions, tissue injury.
'Under non-inflamed, steady-state conditions, Teff cells typically lose L-selectin and CCR7 expression and are largely restricted from LN access though may enter during inflammatory reactions (b) as shown. In contrast, both naïve T cells and Tcrii cells express L-selectin, CCR7, and CXCR4 and engage PNAd, CCL19/CCL21, and CXCL12, respectively, to undergo T-cell rolling and LFA-1/ICAM-1/2- mediated adhesion and transmigration into LNs.
100771 Exemplary homing tissue (target tissue) type and ligand or chemokine that enables tissue-restricted B cell homing in accordance with the invention are set forth in Table 3.

Homing Tissue Type Ligand/Chemokines CNS VCAM-1, CD62P, ligands for CCR1,2, 5, Liver CD62P, VAP-1, CXCL16 Small Intestine MAdCAM, CD62P, CCL25 Colon MAdCAM, CD62P, CCL20, Skin CD62E, CD62P, CCL17(22), Thymus VCAM, CD62P, CCL25 Peripheral Lymph Node PNAd, CCL21, ICAM-1 Peyer's Patch MAdCAM, CCL21, CXCL13 Bone Marrow VCAM, CD62P, CXCL12, ICAM-1 100781 In various embodiments of the present invention, a modified B cell is provided that expresses one or more of an antibody, a protein, or a receptor that facilitate homing of the modified B cell to the exemplary target/homing tissues using the specific homing receptor/ligand pairs as set forth in Table 2. In various embodiments of the present invention, a modified B cell is provided that expresses one or more of a homing receptor that facilitate homing of the modified B cell to the exemplary target/homing tissue using the ligand or chemokines are set forth in Tables 2 and/or 3.
As used herein, the term "B cell homing" refers to localizing, targeting, trafficking, directing, or redirecting of the B cell of the present application to a site/target of interest, for example, a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment, where delivery of therapeutic payloads is desirable. As used in the context of B cell homing, the term "antibody", "protein" or a "receptor" refers to an amino acid sequence, a nucleic acid sequence encoding a peptide or protein, or an RNA molecule, for use as a therapeutic agent, which when expressed in a modified B cell of the present invention will direct the B cell to a site/target of interest.
[0079] In certain embodiments, the homing antibody, protein, or receptor molecule is for homing/targeting the modified B cell expressing such a molecule to a site/target of interest. In certain embodiments, the homing antibody, protein, or receptor molecule is for homing/targeting the modified B cell expressing such a molecule to inflammatory sites in specific locations or tissues.
In certain embodiments, the homing antibody, protein or receptor is for targeting the B cell to a tumor or tumor microenvironment and to the tumor draining lymph node In certain embodiments, targeting B cells to particular locations is desirable so that the engineered or modified B cells of the present invention can deliver therapeutic payloads to desired locations of interest, for example, a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment. Accordingly, in certain embodiments, it is desirable that the B cells home to a site/target of interest, for example, a tumor or tumor microenvironment and tumor-draining lymph node and deliver to the site/target of interest a payload capable of, for example, increasing the number of cross-presenting dendritic cells (DCs) at the site/target of interest (e.g., in tumors).
[0080] In various embodiments, the homing antibody, protein, or receptor is expressed in the modified or engineered B cell as a DNA construct. In various embodiments, the homing antibody, protein, or receptor is expressed in the modified B cell as a DNA construct under the control of a constitutively activated transcriptional pathway. In various embodiments, the homing antibody, protein, or receptor involved in the B cell homing/targeting is either not naturally expressed in a B
cell or is expressed at higher levels than is naturally expressed in a B cell.
Exemplary homing of the modified B cells to specific homing/target tissues using specific homing receptor/ligand pairs in accordance with the present invention is set forth in Table 4. It should be understood that, notwithstanding the exemplary homing tissues, homing receptor, and ligand pairs set forth in Table 4, a modified B cell of the present invention may be engineered to express any homing antibody, protein, or a receptor (e.g., any homing receptor set for in Table 2), such that the modified B cell can be directed to a specific site/target of interest.

Homing Tissue Type Homing Receptor Ligand/Chemokine Liver CXCR6 CXCL16 Small Intestine CCR9 CCL25 Large Intestine (Colon) CCR6 CCL20 Lymph Node CCR7 CCL21 Bone Marrow CXCR4 CXCL12 Peyer's Patch CCR7 and CXCR5 CCL21 and CXCL13, respectively Skin CCR4 CCL17(22) [0081] Nonexclusive examples of homing (target) tissue types for the specific homing receptor/ligand pairs of the present invention include: skin, gut (intestine, colon, mesenteric lymph nodes (mLN), Peyer's Patch (PP), small intestine), liver, lung, bone marrow, heart, peripheral lymph node (LN), CNS, thymus, and bone marrow.
[0082] Nonexclusive examples of homing receptors that can be paired with specific or corresponding attractants/ligands/chemokines of the present invention include:
CLA (PSGL-1 glycoform), CLA (PSGL-1 glycoform), CCRIO, CCR3, CCR4, CCR5, CCR6, CCR9, CD43E, CD44, c-Met, CXCR3, CXCR4, LFA-1, LFA-1 (aL132), Selectin ligands, VLA-4, VLA-4 (a4f31), and a4137.
[0083] Nonexclusive examples of ligands/chemokines that can be paired with specific or corresponding homing receptors of the present invention include: CXCL16, CCL17, CCL17(22), CCL20 (MIP-3a), CCL21, CCL25, CCL27, CCL28, CCL4, CCL5, CD62E, CD62P, CXCL10, CXCL12, CXCL13, CXCL16, CXCL9/CXCL10, CXCR3, E/P-selectin, E-selectin, GPR15L, HGF, Hyaluronate, ICAM- I, ligands for CCR1,2, 5, MAdCAM, MAdCAM-1, PNAd, VAP-1, VCAM, and VCAM-1.
[0084] In certain embodiments of the present invention, a modified B cell is provided that express or have increased expression of the exemplary B cell homing receptors (e.g., as set forth in Table 2), such that the modified B cell is targeted to the corresponding homing tissue of interest that expresses the corresponding ligand/chemokines (e.g., as set forth in Tables 2 and/or 3)_ In certain embodiments of the present invention, a modified B cell is provided that co-expresses an integrin with a specific a and 13 chain pairing and a specific B cell homing receptor (e.g., as set forth in Tables 2 and/or 3), expression of which integrin and/or homing receptor promote or facilitate homing/targeting of the modified B cell to a site/target of interest. In some embodiments, a modified B cell is provided that co-expresses an a4137 integrin and CCR9. It is desirable that co-expression of a4137 and CCR9 will promote small intestine homing of the modified B cells of the present invention. In some embodiments, a modified B cell is provided that co-expresses an a4131 integrin and CCR4. It is desirable that co-expression of a4131 and CCR4 will promote small intestine homing of the modified B cells of the present invention.
[0085] Modified B cells that Express Immune Inhibitory Molecules. B cells are key contributors to many autoimmune diseases. However, B cells can be used therapeutically to antagonize autoimmunity. Specifically, B cells can be engineered to express at least one or more immune inhibitory molecules, which may decrease the autoimmune activity of the B cells, leading to decrease in an autoimmune disease. Immune inhibitory molecules are well known in the art.
Such inhibitory molecules may include, but are not limited to, 1L-10, PD-L1, PD-L2, LAG-3, and TIM-3. In certain embodiments of the present invention, a modified B
cell is provided that is engineered to express at least one or more of an inhibitory molecule selected from IL-10, TGF-13, PD-L1, PD-L2, LAG-3, and TIM-3, or any combinations thereof, such that the inflammation at the site and autoimmune activity of the B cells localized to the site are decreased, thereby leading to a positive therapeutic response.
[0086] Compounds that alter B cell Trafficking. In certain embodiments of the present invention, a modified B cell is provided that is treated with at least one or more compound or derivatives thereof that alter the trafficking of B cells by inducing expression of a specific B cell integrin and/or a homing receptor. Compounds or derivatives thereof that alter the trafficking of B
cells are well known in the art. In certain embodiments, a modified B cell is provided that is treated with all-trans-retinoic acid (ATRA) or derivatives thereof that promote homing of the B cells to gut (small intestine) due to the increased expression of cc4f37 integrin and CCR9 homing receptor. As used herein, the term -compound" refers to a chemical, drug, a therapeutic agent, or derivatives thereof, that alter the trafficking of B cells in a desired manner.
[0087] In various embodiments of the present invention, a modified B cell engineered to co-express a specific integrin (e.g., with a specific a and 13 chain pairing) and a specific B cell homing receptor of interest is treated with at least one or more compounds or derivatives thereof that alter the trafficking of the modified B cells and promote homing of the cells to a specific site/target of interest due to the increased expression of the specific integrin and/or the homing receptor. In various embodiments, a B cell modified to co-express an integrin with a specific a and 13 chain pairings and a specific B cell homing receptor further expresses at least one or more immune inhibitory molecules, such that the autoimmune activity of the modified B
cells targeted to a specific site of inflammation is decreased, leading to a decrease in the autoimmune disease. In some embodiments, a modified B cell engineered to express one or more immune inhibitory molecules, for example IL-10, TGF-13, PD-L1, PD-L2, LAG-3, and TIM-3, or combinations thereof, is treated with ATRA or derivatives thereof for a specified period of time, such that expression of the a4137 integrin and CCR9 homing receptor is induced to promote B cell homing to a specific site/target of interest (e.g., the gut), but the inflammation at the site and autoimmune activity of B cells localized to the site are decreased, leading to a positive therapeutic response. In one embodiment, a modified B cell engineered to express one or more immune inhibitory molecules, for example IL-10, TGF-13, or combinations thereof, is treated with ATRA or derivatives thereof for a specified period of time, such that expression of the oc4137 integrin and CCR9 homing receptor is induced to promote B cell homing to a specific site/target of interest (e.g., the gut), but the inflammation at the site and autoimmune activity of B cells localized to the site are decreased, leading to a positive therapeutic response.
[0088] It is understood that, any B cell of the present invention modified to co-express a specific B cell integrin and homing receptor that targets the B cell to a particular homing/target tissue of interest, may be further engineered to express one or more immune inhibitory molecules for reducing inflammation and autoimmune activity of the B cells localized to the site, and/or treated with a compound that alter the homing/targeting of the modified B cells by inducing expression of the specific B cell integrin and/or the homing receptor.
[0089] Activation of B cells with TLR agonists and TLRs. B cells have a natural ability to uptake and present antigens recognized by their specific B cell receptors (BCRs). B cells activated by Toll-like receptors (TLRs) result in potent effector B cells in defending the body in an immune response. Expression of or increasing the expression of TLRs in B cells can provide a mechanism for potentiating B cells for innate signals regulating adaptive immune responses.
[0090] Activation of B cells with TLR agonists. In various embodiments of the present invention, a B cell is provided, where the B cell is treated in vitro with at least one TLR agonist. In various embodiments, the TLR can be a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1, TLR12, and/or a TLR13. In various embodiments, the TLR
agonist preleientialiy binds to one or more TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. TLR
agonists are well known in the art and may include, but are not limited to, CpG-rich oligonucleotides and the double-stranded RNA mimic, polyinosinic acidrpolycytidylic acid (poly-IrC)_ In various embodiments, the TLR agonist can be CpG oligonucleotides.
100911 In various embodiments, each B cell may be treated with one TLR
agonist. In various embodiments, each B cell may be treated with more than one TLR agonist. For example, each B
cell may be treated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 different TLR
agonists. Alternatively, the patient may be administered a heterogeneous population of B cells, each B cell treated with a unique TLR agonist or a combination of TLR agonists. In some embodiments, the B cells for use a therapeutic agent is treated with one or more TLR agonists at the same time or in advance of the administration of the B cells to a subject or patient in need thereof In certain embodiments, treatment with one or more TLR agonist is capable of producing more potent effector B cells for defending the body in an immune response. In certain embodiments, treatment with one or more TLR agonist is capable of potentiating B cells for immune responses. In some embodiments, treating a B cell of the present invention with at least one or more TLR
agonists induces expression or activation of one or more TLRs.
[0092] Activation of B cells with TLR Expression. In various embodiments of the present invention, a modified B cell is provided that is capable of expressing a constitutively active TLR.
In various embodiments, the TLR is expressed in the modified or engineered B
cell as a DNA
construct under the control of a constitutively activated transcriptional pathway. In various embodiments, the TLR is either not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell. In various embodiments, the TLR can be a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and/or a TLR13.
[0093] In various embodiments, each B cell may express more than one constitutively active TLR. For example, each B cell may express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 different constitutively active TLRs. Alternatively, the patient may be administered a heterogeneous population of B cells, each B cell capable of expressing and/or secreting a unique TLR or combination of TLRs, which are constitutively active. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 different constitutively active TLRs may be administered to the subject or patient through a heterogeneous population of B cells.
[0094] In certain embodiments of the present invention, the B cell is a modified B cell that expresses at least one constitutively active TLR. In certain embodiments, the modified B cell that expresses at least one constitutively active TLR is treated with one or more TLR agonist. In certain embodiments, the expression of the constitutively active TLR is capable of producing more potent effector B cells for defending the body in an immune response. In certain embodiments, the expression of the constitutively active TLR is capable of potentiating B cells for immune responses.
In certain embodiments, the modified B cell expresses both a TLR that is constitutively active and any CAR-B of the present application. In various embodiments, the modified B
cell expressing a TLR that is constitutively active and/or a CAR-B is further treated with one or more TLR agonist at the same time or in advance of the administration of the modified B cells to a subject or patient in need thereof. In certain embodiments, B cells may be engineered to express payloads and modifiers, such as TLRs, in the absence of CAR-B, for intratumoral administration.
[0095] Modified B Cells that Present Antigens Simultaneously in HLA Class I
and Class II
Molecules. B cells, in addition to their function in antibody production, also express high level of Human Leukocyte Antigen (HLA) class II molecules and can present antigens to CD4+ T
cells(Hong et al., 2018, Immunity 49, 695-708). In various embodiments of the present invention, a modified B cell is provided that is capable of presenting specific antigens and/or antigen-derived epitopes of interest, such as tumor antigens or infectious disease antigens, simultaneously in both HLA class I and class II molecules. Tumor antigens and infectious disease antigens are well known in the art and are described in the foregoing sections. In certain embodiments, a specific antigen of interest, e.g., a tumor antigen or an infectious disease antigen, is fused to a targeting signal of a lysosomal protein that targets the antigen to the lysosomes and presents the antigen simultaneously and efficiently in both HLA class I and class II molecules. In some embodiments, the targeting signal is the targeting signal of lysosome-associated membrane protein-1 (LAMP1). In some embodiments, the targeting signal is capable of entering endosomal recycling compartments The c-terminal sequence of Clec9A is such a targeting moiety. As used herein, a specific tumor antigen or an infectious disease antigen fused to a targeting signal refers to an amino acid sequence, a nucleic acid sequence encoding a peptide or protein, or an RNA molecule (e.g., an mRNA
molecule), for use as a therapeutic agent. In one embodiment, a specific tumor antigen or an infectious disease antigen fused to a targeting signal refers to an mRNA
molecule for use as a therapeutic agent. In certain embodiments, it is desirable that the specific tumor antigens and/or infectious disease antigens fused to a targeting signal, such as the targeting signal of LAMP1 or Clec9A, be targeted to the lysosomes or endosomes and presented simultaneously and efficiently in both HLA class I and class II molecules. In certain embodiments, it is desirable that electroporation of B cells (e.g., human B cells), before or after maturation, with an mRNA encoding specific tumor antigens and/or infectious disease antigens of interest fused to a targeting signal, such as the targeting signal of LAMP1 or Clec9A, be capable of simultaneously and efficiently presenting the specific antigens and/or antigen-derived epitopes in both HLA
class I and class II
molecules. In various embodiments, the specific tumor antigens and/or infectious disease antigens of interest is either not naturally presented by a B cell, is not presented by a B cell simultaneously in both HLA class I and class II molecules naturally, or is not presented by a B cell with high efficiencies in both HLA class I and class II molecules naturally. It is contemplated that, introduction of such electroporated B cells into a subject, e.g., a human host, will promote development of or potentiate antigen-specific immune responses by presenting specific antigens and/or antigen-derived epitopes of interest simultaneously and efficiently in both HLA class I and class II molecules.
[0096] In various embodiments, the invention relates to a nucleic acid sequence, e.g., an mRNA
sequence, encoding at least one specific antigen of interest, e.g., a tumor antigen or an infectious disease antigen, fused to a targeting signal, such as the targeting signal of LAMP1, for use as a therapeutic agent in electroporation of B cells for simultaneously and efficiently presenting the specific antigen and/or antigen-derived epitopes in both HLA class I and class II molecules. In various embodiments, the invention relates to nucleic acid sequence, e.g., an mRNA sequence, encoding more than one (e.g., 1, 2, 3, 4, 5, or more) specific tumor antigen and/or an infectious disease antigen of interest fused to a targeting signal. In various embodiments, the invention relates to pools of different nucleic acid sequences, e.g., pools of different mRNA
sequences, for use as a therapeutic agent in electroporation of B cells as described above, where each pool encodes at least one specific antigen of interest, e.g., a tumor antigen or an infectious disease antigen, fused to a targeting signal that is different from the other pools of the mRNA sequences.
Accordingly, in some embodiments, the subject may be administered a homogeneous population of B cells, where each B cell is electroporated with an mRNA encoding at least one specific antigen of interest fused to a targeting signal. In some embodiments, the subject may be administered a homogeneous a population of B cells, where each B cell is electroporated with an mRNA
encoding more than one specific antigen of interest fused to targeting signal. In some embodiments, the subject may be administered a heterogeneous population of B cells, where each B cell is electroporated with a combination of rnRNAs each encoding at least one specific antigen of interest fused to a different targeting signal.
[0097] In some embodiments, the B cells for use in electroporation as described above may be any of the modified B cells of the present application. In some embodiments, the modified B cell comprises a chimeric antigen receptor for B cells (CAR-B). In various embodiments, the modified B cell can express a CAR-B and simultaneously and efficiently present specific antigen and/or antigen-derived epitopes of interest in both HLA class I and class II
molecules.
[0098] In various embodiments, the invention relates to a method of administering an isolated B
cell to a patient in need thereof In various embodiments, a population of B
cells may be administered to the patient. In various embodiments, each B cell may express more than one payload peptide or protein. For example, each B cell may express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 different payloads. Alternatively, the patient may be administered a heterogeneous population of B cells, each B cell capable of expressing and/or secreting a unique payload or combination of payloads. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 different payloads may be administered to the patient through a heterogeneous population of B cells.
3. Methods of Treatment [0099] In some aspects, the invention therefore comprises a method for treating or preventing a tumor or cancerous tissue, comprising administering to a patient in need thereof an effective amount of at least one CAR-B disclosed herein.
101001 Methods are provided for treating diseases or disorders, including cancer. In some embodiments, the invention relates to creating a B cell-mediated immune response in a subject, comprising administering an effective amount of the engineered immune cells of the present application to the subject. In some embodiments, the B cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor for B cells (CAR-B). In some embodiments, the target cell is a tumor cell. In some aspects, the invention comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen-binding molecule described herein. In some aspects, the invention comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor.
101011 In some aspects, the invention comprises a pharmaceutical composition comprising at least one antigen-binding molecule as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.
[0102] In some embodiments, the subject is diagnosed with a metastatic disease localized to the liver. In other embodiments, the metastatic disease is a cancer. In still other embodiments, the cancer metastasized from a primary tumor in the breast, colon, rectum, esophagus, lung, pancreas and/or stomach. In still other embodiments, the subject is diagnosed with unresectable metastatic liver tumors. In yet other embodiments, the subject is diagnosed with unresectable metastatic liver tumors from primary colorectal cancer. In some embodiments, the subject is diagnosed with hepatocellular carcinoma.
[0103] It will be appreciated that target doses for modified B cells can range from 1 x106-2x10' cells/kg, preferably 2x106 cells/kg, more preferably. It will be appreciated that doses above and below this range may be appropriate for certain subjects, and appropriate dose levels can be determined by the healthcare provider as needed. Additionally, multiple doses of cells can be provided in accordance with the invention.

[0104] Also provided are methods for reducing the size of a tumor in a subject, comprising administering to the subject a modified B cell of the present invention, wherein the cell comprises a CAR-B receptor comprising an antigen-binding domain that binds to an antigen on a tumor, a payload or both a CAR-B and a payload. In some embodiments, the subject has a solid tumor, or a blood malignancy such as lymphoma or leukemia. In some embodiments, the modified B cell is delivered to a tumor bed. In some embodiments, the cancer is present in the bone marrow of the subject.
[0105] Also provided are methods for homing B cells to a site/target of interest in a subject, comprising administering to the subject a modified B cell of the present invention, wherein the cell comprises an integrin, a homing antibody, protein, or a receptor that is attracted to a ligand, chemokine, or an attractant at the site/target of interest. In some embodiments, the site/target of interest is, for example, a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment, where delivery of therapeutic payloads is desirable.
[0106] Also provided are methods for decreasing inflammation and autoimmune activity of B
cells at a site/target of interest in a subject, comprising administering to the subject a modified B
cell of the present invention, wherein the cell comprises an immune inhibitory molecule. In some embodiments, the site/target of interest is, for example, a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment, where delivery of therapeutic payloads is desirable.
101071 Also provided are methods for altering trafficking of B cells to a site/target of interest in a subject, comprising treating a B cell of the present invention with a compound or derivatives thereof suitable for altering B cell trafficking, and administering the treated B cell to the subject in need thereof In some instances, the compound or derivatives thereof alters B
cell trafficking by increasing the expression of an integrin, homing antibody, protein, receptor, or combinations thereof, expressed by the B cells.
[0108] Also provided are methods for potentiating B cells and/or producing potent effector B
cells for increasing immune responses in a subject, comprising treating a B
cell of the present invention with at least one or more TLR agonists, and administering the treated B cell to the subject in need thereof In some embodiments, treating a B cell of the present invention with at least one or more TLR agonists induces expression or activation of one or more TLRs. In some embodiments, the method for potentiating B cells and/or producing potent effector B cells for increasing immune responses in a subject, further comprises administering to the subject a modified B cell of the present invention that expresses at least one or more constitutively active TLRs. Also provided are methods for potentiating B cells and/or producing potent effector B cells for increasing immune responses in a subject, comprising administering to the subject a modified B cell of the present invention, wherein the cell expresses a CAR-B receptor comprising an antigen-binding domain that binds to an antigen on a tumor, a constitutively active TLR or both a CAR-B and a constitutively active TLR, where the cell is treated with at least one or more TLR agonists at the same time or in advance of the administration of the cells to the subject [0109] Also provided are methods for increasing antigen-specific immune responses in a subject, comprising administering to the subject a modified B cell of the present invention, wherein the cell is electroporated with a nucleic acid sequence, e.g., an mRNA, encoding specific tumor antigens and/or infectious disease antigens fused to a targeting signal, such as the targeting signal of LAMP1 or Clec9A, for simultaneously and efficiently presenting the specific antigens and/or antigen-derived epitopes in both HLA class I and class II molecules. In some embodiments, the subject has a solid tumor, or a blood malignancy such as lymphoma or leukemia.
[0110] It is understood that the various embodiments of the methods of treatment using the engineered or modified B cells of the present application are not mutually exclusive and can be combined with each other in any way and without any restriction unless explicitly indicated, for achieving of facilitating any of the results and/or therapeutic responses contemplated herein.
[0111] In some embodiments, the modified B cells are autologous B cells. In some embodiments, the modified B cells are allogeneic B cells. In some embodiments, the modified B cells are heterologous B cells. In some embodiments, the modified B cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo.
[0112] As used herein, the term 'subject or -patient" means an individual. In some aspect, a subject is a mammal such as a human. In some aspect, a subject can be a non-human primate. Non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons, to name a few. The term "subject" also includes domesticated animals, such as cats, dogs, etc., livestock (e.g., llama, horses, cows), wild animals (e.g., deer, elk, moose, etc.,), laboratory animals (e.g., mouse, rabbit, rat, gerbil, guinea pig, etc.) and avian species (e.g., chickens, turkeys, ducks, etc.). Preferably, the subject is a human subject. More preferably, the subject is a human patient.
[0113] The methods can further comprise administering one or more chemotherapeutic agents.
In certain embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic. Beneficial preconditioning treatment regimens, along with correlative beneficial biomarkers are described in U.S. Provisional Patent Applications 62/262,143 and 62/167,750, which are hereby incorporated by reference in their entirety herein. These describe, e.g., methods of conditioning a patient in need of a T cell therapy comprising administering to the patient specified beneficial doses of cyclophosphamide (between 200 mg/m2/ day and 2000 mg/m2/day) and specified doses of fludarabine (between 20 mg/m2/day and 900 mg/m2/day). A

preferred dose regimen involves treating a patient comprising administering daily to the patient about 500 mg/m2/day of cyclophosphamide and about 60 mg/m2/day of fludarabine for three days prior to administration of a therapeutically effective amount of engineered B
cells to the patient.
[0114] In other embodiments, the antigen-binding molecule, transduced (or otherwise engineered) cells (such as CARs), and the chemotherapeutic agent are administered each in an amount effective to treat the disease or condition in the subject.
[0115] In certain embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents.
Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTm); alkyl sulfonates such as busulfan, improsulfan and piposulfan;
aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-di azo-5 -oxo-L-norl eucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, que-lamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;
bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;
nitracrine;
pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSI(*);
razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',27-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine;
arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (Taxol Cf-k, Bristol-Myers Squibb) and doxetaxel (Taxoterek, Rhone-Poulenc Rorer); chlorambucil;
gemcitabine;
thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C;
mitoxantrone; vincristine;
vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;
xeloda; ibandronate;
CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMF0);
retinoic acid derivatives such as TargretinTm (bexarotene), PanretinTm, (alitretinoin);
OntakTM (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including, but not limited to CHOP, i.e., Cyclophosphamide (Cytoxank) Doxorubicin (hydroxydoxorubicin), Fludarabine, Vincristine (Oncoving), and Prednisone.
101161 In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the cell or nucleic acid. In some embodiments, the methods further comprise administering two or more chemotherapeutic agents.
[0117] A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 (or PD-L1) inhibitors such as nivolumab (Opdivok), pembrolizumab (Keytrudak), pembrolizumab, pidilizumab, and atezolizumab (Tecentrie). Other additional therapeutics include anti-CTLA-4 antibodies (e.g., Ipilimumabg), anti-LAG-3 antibodies (e.g., Relatlimab, BMS), alone or in combination with PD-1 and/or PD-Li inhibitors.
[0118] Additional therapeutic agents suitable for use in combination with the invention include, but are not limited to, ibrutinib (Imbruvicak), ofatumumab (Arzerrak), rituximab (Rituxank), bevacizumab (Avastink), trastuzumab (Hercepting), trastuzumab emtansine (KADCYLAg), imatinib (Gleevecg), cetuximab (Erbitux0), panitumumab (Vectibixk), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacri tinib, cobi me tinib, s el ume tinib, trametinib, binimetinib, al ec tinib, ceri tinib, crizo tinib, aflibercept, adipoti de, den il euki n di fti tox , mTOR inhibitors such as Everoli mus and Temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitor (palbociclib).
[0119] In additional embodiments, the composition comprising CAR-containing B
cells can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF
medications, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium. Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone.
Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL ,), adalimumab (HUMIRAO) and infliximab (REMICADEO)), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
[0120] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. "Cytokine" as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators.
Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin;
relaxin; prorelaxin;
glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF);
prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin;
thrombopoietin (TP0); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II;
erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF
(GM-CSF); and granulocy te-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, 1L-4, 1L-5, IL-6, 1L-7, 1L-8, 1L-9, IL-10, IL-11, 1L-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including L1F and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.
4. Methods of Making [0121] A variety of known techniques can be utilized in making the polynucleotides, polypeptides, vectors, antigen-binding molecules, immune cells, compositions, and the like according to the invention.
[0122] Prior to the in vitro manipulation or genetic modification of the immune cells described herein, the cells may be obtained from a subject. In some embodiments, the immune cells comprise B cells. B cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, B cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLLTM separation. Cells may preferably be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing. The cells may be washed with PBS. As will be appreciated, a washing step may be used, such as by using a semiautomated flowthrough centrifuge for example, the CobeTM 2991 cell processor, the Baxter Cyto-MateTm, or the like. After washing, the cells may be resuspended in a variety of biocompatible buffers, or other saline solution with or without buffer. In certain embodiments, the undesired components of the apheresis sample may be removed.
[0123] The immune cells, such as B cells, can be genetically modified following isolation using known methods, or the immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In another embodiment, the immune cells, such as B cells, are genetically modified with the chimeric B cell receptors described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR-B) and then are activated and/or expanded in vitro. Methods for activating and expanding B cells are known in the art and are described, for example, in U.S. Pat_ Nos.
6,905,874; 6,867,041;
6,797,514; and PCT WO 2012/079000, the contents of which are hereby incorporated by reference in their entirety. Generally, such methods include contacting PBMC or isolated B cells with a stimulatory agent and cos timulatory agent generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2.
[0124] In other embodiments, the B cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S.
Pat. Nos. 6,040,177; 5,827,642; and WO/2012129514, the contents of which are hereby incorporated by reference in their entirety.
[0125] Certain methods for making the constructs and engineered immune cells of the invention are described in PCT application PCT/US2015/14520, the contents of which are hereby incorporated by reference in their entirety. Additional methods of making the constructs and cells can be found in U.S. provisional patent application No. 62/244,036 the contents of which are hereby incorporated by reference in their entirety.
[0126] For cloning of polynucleotides, the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the origin of replication may be selected to promote autonomous replication of the vector in the host cell.
[0127] In certain embodiments, the present disclosure provides isolated host cells containing the vector provided herein. The host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector. Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E.
Enterobacter, Erwima, Klebsiella. Proteus, Salmonella, e.g., Salmonella typhimurnan, Serratia, e.g., Serratia marc-escans, and Shigella, as well as Bacilli such as B. subtilis and B.
hcheniformis, Pseuclomonas such as P. aeruginosa, and Streplomyces.
[0128] The vector can be introduced to the host cell using any suitable methods known in the art, including, without limitation, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used in genetically modifying a donor population of immune effector cells wherein each vector encodes a different CAR-Bs as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a proportion of the engineered cells expressing more than one different CAR-Bs.
[0129] In one embodiment, the invention provides a method of storing genetically engineered cells expressing CAR-Bs that target a protein. This involves cryopreserving the immune cells such that the cells remain viable upon thawing. A fraction of the immune cells expressing the CAR-Bs can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with a malignancy. When needed, the cryopreserved transformed immune cells can be thawed, grown and expanded for more such cells.
[0130] As used herein, "cryopreserve- refers to the preservation of cells by cooling to sub-zero temperatures, such as (typically) 77 Kelvin or 196 C. (the boiling point of liquid nitrogen).
Cryoprotective agents are often used at sub-zero temperatures to prevent the cells being preserved from damage due to freezing at low temperatures or warming to room temperature.
Cryopreservative agents and optimal cooling rates can protect against cell injury. Cryoprotective agents which can be used in accordance with the invention include but are not limited to: dimethyl sulfoxide (DMSO) (Lovelock & Bishop, Nature, 1959, 183, 1394-1395; Ashwood-Smith, Nature, 1961, 190, 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y.
Acad. Sci., 1960, 85, 576), and polyethylene glycol (Sloviter 8z Ravdin, Nature, 1962, 196, 48). The preferred cooling rate is 1 -3 C/minute.
[0131] The term, "substantially pure," is used to indicate that a given component is present at a high level. The component is desirably the predominant component present in a composition.
Preferably it is present at a level of more than 30%, of more than 50%, of more than 75%, of more than 90%, or even of more than 95%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. At very high levels (e.g. at levels of more than 90%, of more than 95% or of more than 99%) the component can be regarded as being in "pure form." Biologically active substances of the present invention (including polypeptides, nucleic acid molecules, antigen-binding molecules, moieties) can be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. When a composition is substantially free of a given contaminant, the contaminant will be at a low level (e.g., at a level of less than 10%, less than 5%, or less than 1% on the dry weight/dry weight basis set out above).
[0132] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount Suitable infusion media can be any isotonic medium formulation, typically normal saline, NormosolTM R (Abbott) or Plasma-LyteTm A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin.
[0133] Desired treatment amounts of cells in the composition is generally at least 2 cells or is more typically greater than 102 cells, and up to 106, up to and including 108 or 109 cells and can be more than 1010 cells. The number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein. The density of the desired cells is typically greater than 106 cells/ml and generally is greater than 107 cells/ml, generally 108 cells/ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. In some aspects of the present invention, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of 106/kilogram (106-1011 per patient) may be administered. CAR-B treatments may be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy. In some aspects, different CAR-B cells are found in a single product.
The composition can be as few as 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 different CAR-B cells. These can consist of cells expressing a chimeric CAR protein and B cells expressing other CARs and/or payloads.
[0134] The B cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions of the present invention may comprise a CAR-B expressing cell population, such as B cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;
proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA
or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
Compositions of the present invention are preferably formulated for intravenous administration.
Treatment may also include one or more corticosteroid treatment, such as dexamethasone and/or methylprednisolone.
[0135] The compositions of the present application can comprise, consist essentially of, or consist of, the components disclosed.
[0136] The pharmaceutical compositions of the invention (solutions, suspensions or the like), may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents;
antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylene-diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.
101371 It will be appreciated that adverse events may be minimized by transducing the immune cells (containing one or more CAR-B) with a suicide gene. It may also be desired to incorporate an inducible "on" or "accelerator" switch into the immune cells. These techniques may employ the use of dimerization domains and optional activators of such domain dimerization. These techniques include, e.g., those described by Wu et al., Science 2014, 350(6258) utilizing FKBP/Rapalog dimerization systems in certain cells, the contents of which are incorporated by reference herein in their entirety. Additional dimerization technology is described in, e.g., Fegan et al. Chem. Rev.
2010, 110, 3315-3336 as well as U.S. Patent Nos. 5,830,462; 5,834,266;
5,869,337; and 6,165,787, the contents of which are also incorporated by reference herein in their entirety. Additional dimerization pairs may include cyclosporine-A/cyclophilin, receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoids/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. Further examples of dimerization technology can be found in e.g., WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US
2016/0046700, U.S. Patent No. 8,486,693, US 2014/0171649, and US 2012/0130076, the contents of which are further incorporated by reference herein in their entirety.
[0138] Suitable techniques include use of inducible caspase-9 (U.S.
Appl. Pub. No.
2011/0286980) or a thymidine kinase, before, after or at the same time, as the cells are transduced with the CAR-B construct of the present invention. Additional methods for introducing suicide genes and/or "on- switches include CRISPR, TALENS, MEGATALEN, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.
[0139] Anti-CD20 or anti-CD19 represent additional means to reduce or eliminate engineered B
cells if such cells are responsible for adverse events or pathologies.
[0140] It will be understood that descriptions herein are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise.
[0141] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
101421 In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes"
and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
[0143] The term "polynucleotide-, "nucleotide-, or -nucleic acid- includes both single-stranded and double-stranded nucleotide polymers. The nucleotides 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, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
[0144] The term -oligonucleotide" refers to a polynucleotide comprising 200 or fewer nucleotides. Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can 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 can be used, for example, as PCR primers, cloning primers or hybridization probes.
[0145] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend upon the host organism. In particular embodiments, control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence. "Control sequences" can include leader sequences (signal peptides) and/or fusion partner sequences.
[0146] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
[0147] 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 "expression vector- or "expression construct- refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
[0148] The term "host cell" refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence 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.
[0149] 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 can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can 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.
101501 The term -transfection" refers to the uptake of foreign or exogenous DNA by a cell. A
number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., VIROLOGY, 1973, 52:456; Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2001, supra; Davis et al., Basic Methods in Molecular Biology, 1986, Elsevier; Chu et al., Gene, 1981, 13:197.
[0151] The term "transduction" refers to the process whereby foreign DNA is introduced into a cell via viral vector. See, e.g., Jones et al., Genetics: principles and analysis, 1998, Boston: Jones & Bartlett Publ.
[0152] The terms -polypeptide" or "protein" refer to a macromolecule having the amino acid sequence of a protein, including deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein"
specifically encompass antigen-binding molecules, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigen-binding protein. The term -polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein.
Such fragments can also contain modified amino acids as compared with the native protein. Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules.
101531 The term "isolated- means (i) free of at least some other proteins with which it would normally be found, (ii) is essentially free of other proteins from the same source, e.g., from the same species, (iii) separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (iv) operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (v) does not occur in nature.
[0154] A "variant" of a polypeptide (e.g., an antigen-binding molecule) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
Variants include fusion proteins.
[0155] 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) are preferably addressed by a particular mathematical model or computer program (i.e., an "algorithm").
[0156] To calculate percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., Nucl. Acid Res., 1984, 12, 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or 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). In certain embodiments, a standard comparison matrix (see, e.g., Dayhoff etal., 1978, Atlas of Protein Sequence and Structure, 1978, 5:345-352 for the PAM 250 comparison matrix; Henikoff et at., Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 10915-10919 for the BLO-SUM 62 comparison matrix) is also used by the algorithm.
[0157] As used herein, the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunology A Synthesis (2nd Edition, Golub and Green, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, .gamma. -carboxy-glutamate, epsilon-N,N,N-trimethylly sine, e-N-acetylly sine, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3 -methy lhi sti dine, 5 -hy droxy ly sine, . sigma. -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hy droxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
[0158] Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, 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. Naturally occurring residues can be divided into classes based on common side chain properties:
a) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
c) acidic: Asp, Glu;
d) basic: His, Lys, Arg;
e) residues that influence chain orientation: Gly, Pro; and f) aromatic: Trp, Tyr, Phe.
[0159] For example, non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
[0160] In making changes to the antigen-binding molecule, the costimulatory or activating domains of the engineered T cell, according to certain embodiments, the hydropathic index of amino acids can 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/cystine (+2.5); methionine (+1.9); al anine (+1.8);
glycine (-0.4); threonine (-0.7); serine (-0.8); try ptophan (-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). See, e.g., Kyte et al., J. Mol. Biol., 1982, 157, 105-131. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution 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 in the present case. Exemplary amino acid substitutions are set forth in Table 5.

Original Residues Exemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys, Gin, Asn Lys Asn Gln Gln Asp Glu Glu Cy s Ser, Ala Ser Gin Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gin, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Leu Norleucine Leu Norleucine, Ile, Va, Met, Ala, Tie Phe Lys Arg, 1, 4 Diamino-butyric Arg Acid, Gin, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine [0161] The term "derivative- refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids). In certain embodiments, derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties. In certain embodiments, a chemically modified antigen-binding molecule can have a greater circulating half-life than an antigen-binding molecule that is not chemically modified. In some embodiments, a derivative antigen-binding molecule is covalently modified to include one or more water soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
[0162] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed -peptide mimetics" or "peptidomimetics." Fauchere, J. L., Adv. Drug Res., 1986, 15, 29; Veber, D. F. & Freidinger, R. M., Trends in Neuroscience, 1985, 8, 392-396; and Evans, B. E., et al., J. Med. Chem., 1987, 30, 1229-1239, which are incorporated herein by reference for any purpose.
[0163] The term "therapeutically effective amount" refers to the amount of CAR-B cells determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
[0164] The terms -patient" and -subject" are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
101651 The term -treat- and -treatment- includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. The term -prevent"
does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
[0166] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
5. Sequences [0167] The following sequences will further exemplify the invention:
CD28 transmembrane domain - mouse (SEQ ID NO: 1) TTCTGGGCCCTTGTGGTGGTTGCC GGAGTGCTGTTTTGCTATGGGCTC CTGGTTAC C GT
TGCCCTTTGTGTGATTTGGACC
CD28 transmembrane domain - mouse (SEQ ID NO: 2) F W AL V V VAGVLECYGLLVTVALCVIWT
CD28 transmembrane domain - human (SEQ ID NO: 3) TTTTGGGTATTGGTAGTGGTGGGCGGAGTTTTAGCCTGCTACAGCCTCCTGGTAACAGT
GGCTTTTATCATCTTTTGGGTG
CD28 transmembrane domain - human (SEQ ID NO: 4) FWVLVVVGGVLACYSLLVTVAFIIFWV
CD19 cytoplasmic domain - human (SEQ ID NO: 5) CAGCGGGCTTTAGTCTTGCGGCGTAAACGTAAAAGAATGACAGATCCAACTCGCAGGT
TCTTCAAAGTGACC C CC CCACCTGGGTCCGGACCGCAGAACCAATATGGGAATGTCCT
GTCTCTGC C TAC GC C TAC AAGTGGACTGGGTAGGGCTCAGAGGTGGGCTGC C GGTCTC

GGCGGAACTGCGCCATCTTACGGAAATCCCTCCTCCGACGTTCAGGCAGACGGGGCCC
TGGGGTCTCGATCCCCGCCTGGTGTTGGACCAGAAGAGGAAGAGGGCGAGGGCTACG
AAGAGCC CGACTC CGAAGAGGACAGTGAGTTTTACGAGAACGACAGCAACCTGGGGC
AGGATCAGCTGTCACAGGATGGCTCAGGATATGAAAACCCTGAGGACGAGCCTTTGGG
GCCTGAAGATGAGGACTCCTTTTCTAATGCAGAGTCATATGAGAATGAGGACGAAGAA
TTGACTCAACCCGTGGCAAGAACAATGGATTTCCTCAGTCCACACGGGAGTGCATGGG
ACCCCTCCAGAGAGGCTACTAGCCTCGGTTCTCAAAGCTATGAGGACATGAGGGGTAT
TC TGTAC GC AGC GC C TC AGTTGAGGTC C ATC C GC GGC C AGC C AGGC C C AAA C C
ATGAG
GAAGATGC CGATTCTTAC GAAAACATGGACAACC C C GAT GGTC CTGACC C C GC ATGGG
GGGGCGGCGGGAGGATGGGCACCTGGTCTACTCGC
CD19 cytoplasmic domain ¨ human (SEQ ID NO: 6) QRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGG
TAP SYGNP S S DV QAD GAL GS RS P P GV GP EEEEGEGYEEPD S EED S EFYEND SNL
GQDQLSQ
DGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSL
GS Q S YEDMRGILY AAPQLRSIRGQP GPNHEEDAD SYEN MDN PDGPDPAW GGGGRWIGTWS
TR
CD40 cy toplasmic domain ¨ human (SEQ ID NO: 7) AAGAAGGTTGCAAAAAAACCTACTAATAAGGCTCCCCATCCTAAGCAAGAGCCCCAA
GAAATTAACTTTCCCGATGATCTTCCGGGTTCTAACACGGCAGCCCCGGTGCAGGAGA
CCCTGCATGGTTGTCAACCCGTCACTCAGGAGGACGGGAAAGAGTCTCGTATCTCCGT
CCAGGAGAGACAG
CD40 cytoplasmic domain ¨ human (SEQ ID NO: 8) KKVAKKP TNKAPHPKQEP QEINFPDDLP GSNTAAPV QETLH GC QPVTQED GKES RI SV QER
CD40 + CD79b cytoplasmic domain ¨ human (SEQ ID NO: 9) AAGAAGGTTGCAAAAAAACCTACTAATAAGGCTCCCCATCCTAAGCAAGAGCCCCAA
GA A ATT A ACTTTCC CGATGATC TTC C GGGTTCT A AC ACGGCAGCCCCGGTGC A GGA GA
CCCTGCATGGTTGTCAACCCGTCACTCAGGAGGACGGGAAAGAGTCTCGTATCTCCGT
C CAGGAGAGACAGGACAAGGAC GATAGTAAAGCAGGGATGGAGGAGGAC C ATAC AT
ACGAGGGACTGGATATCGATCAGACAGCCACGTACGAAGACATTGTGACACTGAGAA
CTGGCGAGGTGAAGTGGTCAGTGGGAGAACATCCGGGGCAGGAA
CD40 + CD79h cytoplasmic domain ¨ human (SEQ ID NO: 10) KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED
GKESRISVQE RQDKDDSKAG MEEDHTYEGL DIDQTATYED IVTLRTGEVK
W S V GEHP GQE
CD40 + CD137 cytoplasmic domain ¨ human (SEQ ID NO: 11) AAGAAGGTTGCAAAAAAACCTACTAATAAGGCTCCCCATCCTAAGCAAGAGCCCCAA
GAAATTAACTTTCCCGATGATCTTCCGGGTTCTAACACGGCAGCCCCGGTGCAGGAGA
CCCTGCATGGTTGTCAACCCGTCACTCAGGAGGACGGGAAAGAGTCTCGTATCTCCGT
CCAGGAGAGACAGAAAAGAGGCCGAAAAAAGCTGCTGTACATCTTCAAACAACCCTT

CATGCGACCTGTTCAGACGACACAGGAGGAGGACGGCTGCAGCTGTAGGTTTCCCGAA
GAAGAGGAGGGAGGATGCGAACTT
CD40 + CD137 cytoplasmic domain ¨ human (SEQ ID NO: 12) KKVAKKP TNKAPHPKQEP QEINFPDDLP GSNTAAPV QETLH GC QPVTQED GKES RI SV QER
QKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD137 cytoplasmic domain ¨ human (SEQ ID NO: 13) AAAAGAGGCCGAAAAAAGCTGCTGTACATCTTCAAACAACCCTTCATGCGAC CTGTTC
AGACGACACAGGAGGAGGACGGCTGCAGCTGTAGGTTTCCCGAAGAAGAGGAGGGAG
GATGCGAACTT
CD137 cytoplasmic domain ¨ human (SEQ ID NO: 14) KRGRKKLLY IFKQP FMRP V QTTQEED GC SCRFPEEEEGGCEL
CD40 and Fc gamma receptor 2a cytoplasmic domain ¨ human (SEQ ID NO: 15) AAGAAGGTTGCAAAAAAACCTAC TAATAAGGC TCCC CATC CTAAGCAAGAGCC C C AA
GA A ATT A A CTTTCC CGATGATC TTC C GGGTTCT A AC A CGGC AGC C CCGGTGC A GGA GA
CCCTGCATGGTTGTCAACCCGTCACTCAGGAGGACGGGAAAGAGTCTCGTATCTCCGT
CCAGGAGAGACAGCGCAAAAAACGTATAAGCGCAAACTCTACAGATCCAGTAAAAGC
CGCGCAATTCGAGCCTCCCGGCCGCCAGATGATTGCAATACGGAAACGTCAACTGGAG
GAAACTAATAATGACTATGAGACGGCCGACGGTGGATACATGACCCTTAATCCCCGCG
C GC CAAC CGAC GATGATAAGAAC ATATATCTGAC GC TC CCC CC TAAC GATC AC GTTAA
CAGTAATAAT
CD40 and Fc gamma receptor 2a cytoplasmic domain ¨ human (SEQ ID NO: 16) KKVAKKP TNKAPHPKQEP QEINFPDDLP GSNTAAPV QETLH GC QPVTQED GKES RI SV QER
QRKKRI S AN S TDPVKAAQF EPP GRQMIAIRKRQLEETNNDYETAD GGYMTLNP RAP TDDD
KNIYLTLPPNDHVNSNN
Fc gamma receptor 2a cytoplasmic domain ¨ human (SEQ ID NO: 17) C GC AAAAAAC GTATAAGC GC AAAC TC TAC AGATC C AGTAAAAGC C GC GC AATTC GAG
CCTCCCGGCCGCCAGATGATTGCAATACGGAAACGTCAACTGGAGGAAACTAATAATG
ACTATGAGACGGCCGACGGTGGATACATGACCCITAATCCCCGCGCGCCAACCGACGA
TGATAAGAACATATATCTGACGCTCCCCC CTAAC GATC AC GTTAAC AGTAATAAT
Fc gamma receptor 2a cytoplasmic domain ¨ human (SEQ ID NO: 18) RKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDK
NIYLTLPPNDHVN SNN
Myd88 + CD40 cytoplasmic domain ¨ human (SEQ ID NO: 19) ATGGCGGCGGGCGGGCCCGGCGCCGGAAGCGCCGCGCCAGTCTCATCTACGTCCAGTC
TGCCACTGGCTGCCCTGAACATGAGAGTGAGACGCCGTTTATCCCTCTTCCTGAATGTG
CGGACCCAGGTCGCCGCTGATTGGACCGCCCTGGCCGAAGAGATGGACTTTGAATACT
TGGA A ATC AGA C A GCTGGA A A C A C A GGC A GA C CC A A CCGGGAGA CTGC TTGA C
GC CT
GGCAGGGACGCCCAGGGGCAAGTGTTGGTCGGTTACTGGAGCTTTTAACTAAGTTGGG

CCGCGATGACGTGCTGTTGGAGTTAGGACCCAGTATCGAGGAGGATTGTCAGAAATAC
ATCTTGAAACAGCAGCAGGAGGAGGCGGAAAAGCCCCTGCAGGTGGCGGC CGTTGAC
AGC AGTGTACC CAGAAC AGCTGAGCTGGCCGGCATCACAAC C CTGGATGATC CCCTGG
GC CACATGC CTGAGAGGTTC GAC GCTTTC ATAAAGAAGGTTGCAAAAAAACCTACTAA
TAAGGCTCCCCATCCTAAGCAAGAGCCCCAAGAAATTAACTTTCC CGATGATCTTCCG
GGTTCTAAC AC GGC AGC C C C GGTGCAGGAGAC C CTGC ATGGTTGTCAAC C C GTCACTC
A GGA GGA CGGGA A A GA GTCTCGT ATC TC CGTCC A GGA GA GA C AG
Myd88 + CD40 cytoplasmic domain ¨ human (SEQ ID NO: 20) MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYL
EIRQLETQ ADPTGRLL DAWQ GRP GAS V GRLLEL LTKL GRDDVLL ELGP S TEED C QKYILKQ
QQEEAEKPLQVAAVDS SVP RTAELAGITTLDDPL GHMPERFDAFIKKVAKKPTNKAPHPK
QEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ
Myd88 cytoplasmic domain ¨ human (SEQ ID NO: 21) ATGGCGGCGGGCGGGCCCGGCGCCGGAAGCGCCGCGCCAGTCTCATCTACGTCCAGTC
TGCCACTGGCTGCCCTGAACATGAGAGTGAGACGCCGTTTATCCCTCTTCCTGAATGTG
C GGACC CAGGTC GC C GC TGATTGGAC C GC C C TGGC CGAAGAGATGGACTTTGAATACT
TGGA A ATC AGA C A GCTGGA A AC AC A GGC A GA C CC A A CCGGGAGA CTGC TTGA C GC
CT
GGCAGGGACGCCCAGGGGCAAGTGTTGGTCGGTTACTGGAGCTTTTAACTAAGTTGGG
CCGCGATGACGTGCTGTTGGAGTTAGGACCCAGTATCGAGGAGGATTGTCAGAAATAC
ATCTTGAAACAGCAGCAGGAGGAGGCGGAAAAGCCCCTGCAGGTGGCGGC CGTTGAC
AGCAGTGTACCCAGAACAGCTGAGCTGGCCGGCATCACAACCCTGGATGATCCCCTGG
GCCACATGCCTGAGAGGTTCGACGCTTTCATA
Myd88 cytoplasmic domain ¨ human (SEQ ID NO: 22) MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAFEMDFEYL
EIRQLETQ ADPTGRLL DAWQ GRP GASV GRLLEL LTKL GRDDVLLEL GP S IEED C QKYILKQ
QQEEAEKPLQVAAVDS SVPRTAELAGITTLDDPLGHMPERFDAFI
CD79a cytoplasmic domain ¨ human (SEQ ID NO: 23) AGGAAACGATGGCAGAACGAGAAGCTCGGGTTGGATGCCGGGGATGAATATGAAGAT
GAAAACCTTTATGAAGGC CTGAACCTGGAC GACTGCTCCATGTATGAGGAC ATCTCCC
GGGGC CTCCAGGGCACCTACCAGGATGTGGGCAGCCTCAACATAGGAGATGTCCAGCT
GGAGAAGCCG
CD79a cytoplasmic domain ¨ human (SEQ ID NO: 24) RKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLE
KP
CD79b cytoplasmic domain ¨ human (SEQ ID NO: 25) CTGGACAAGGATGACAGCAAGGCTGGCATGGAGGAAGATCACACCTACGAGGGCCTG
GACATTGACCAGACAGCCACCTATGAGGACATAGTGACGCTGCGGACAGGGGAAGTG
AAGTGGTCTGTAGGTGAGCACCCAGGCCAGGAG
CD79b cytoplasmic domain ¨ human (SEQ ID NO: 26) LDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE
CD8 hinge domain - human (SEQ ID NO: 27) TTCGTGCCTGTGTTCCTCCCAGCTAAGCCCACTACCACCCCCGCTCCAAGGCCGCCCAC
GCCCGCTCCTACTATTGCTAGTCAGCCTTTAAGTTTACGACCCGAAGCTTGCAGGCCCG
C C GC C GGCGGC GC TGTGC AC AC C AGGGGGC TTGATTTTGC C TGC GAC
CD8 hinge domain - human (SEQ ID NO: 28) FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
Spacer with 3X strep II tag (SEQ ID NO: 29) GGCGCTGGTAGTGGCGGTAACTGGAGCCACCCTCAATTTGAGAAGGGCGGGTCAGGC
GGATCAGGTGGTAGTGGTGGGTCCAACTGGAGCCATCCGCAATTTGAAAAGGGCGGA
AGCGGCGGTTCCGGCGGTTCAGGCGGTAGCAACTGGTCACATCCGCAATTTGAGAAAG
GCGGGTCAGGCGGCGGG
Spacer with 3X strep II tag (SEQ ID NO: 30) GAGS GGNW SHP QFEKGGS GGS GGS GGSNWSHPQFEKGGS GGS GGS GGSNWSHPQFEKGG
S GGG
human IgG1 Fc (transmembrane form) (SEQ ID NO: 31) C C CAAGAGC TGC GACAAGAC C CAC AC CTGC C CCC CC TGC C CAGC C C CAGAGCTGCTGG
GC GGACCCTC CGTGTTC CTGTTC C CCCC CAAGC CCAAGGAC AC CC TGATGATC AGCAG
GAC CCC C GAGGTGAC CTGC GT GGTGGTGGAC GTGAGC CAC GAGGACCCAGAGGTGAA
GTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGA
GGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC
TGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCC
ATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACC
CTGCC CC CCTC C CGGGAGGAGATGAC C A AGA ACC AGGTGTCCCTGACCTGTCTGGTGA
AGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGA
AC AACTAC AAGAC CAC C CCC C CAGTGC TGGAC AGC GAC GGC AGC TTC TTC C TGTAC AG
CAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT
GATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGAG
CTGCAACTGGAGGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGACGGGCTGTGG
ACGACCATCACCATCTTCATCACACTCTTCCTGTTAAGCGTGTGCTACAGTGCCACCGT
CACCTTCTTCAAGGTGAAGTGGATCTTCTCCTCGGTGGTGGACCTGAAGCAGACCATC
ATCCCCGACTACAGGAACATGATC GGACAGGGGGCCTGA
human IgG1 Fc (transmembrane form) (SEQ ID NO: 32) PKS CDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQ
DGELDGLWTTITIFITLFLLSVCYSATVTFFKVKWIFSSVVDLKQTIIPDYRNMIGQGA
anti-huPSMA scFv (SEQ ID NO: 33) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGC CCGGGGCATCTGTGAAAG
TATCATGCAAAACATCC GGCTATACGTTTACC GAATAC AC C ATTC AC TGGGTC AGAC A
GGCTC CCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGAAC C
ACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACGTCCA
CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GC TGGA TGGA ACTTCGAC T A TTGGGGAC A AGGGACTC TTGTT ACGGTGTC C A GTGGC A
AAC CAGGTAGTGGTAAACC CGGAAGCGGCAAGCC CGGGAGCGGTAAACCTGGTAGCG
AC ATC GTCATGACTCAAAGCC C TGAC TC AC TC GC C GTGAGC C TGGGAGAGC GTGCAAC
GCTATCTTGTCGGGCCTCTCAGGATGTCGGAACTGCTGTAGACTGGTATCAACAGAAA
C C TGAC C AATC AC CAAAAC TC CTGATTTATTGGGC CTCAAC ACGTCACACAGGAGTGC
CAGATAGGTTCACAGGTAGTGGCAGTGGAACTGATTTTACTTTGACAATTAGCAGCCT
GCAAGC C GAAGATGTAGC C GTTTACTTCTGTC AAC AATATAACTCATAC C C ACTAAC G
TTCGGTGCCGGGACGAAGGTAGAGATTAAA
anti-huPSMA scFv (SEQ ID NO: 34) EVQLVQSGAE VKKPGASVKV SCKTSGYTFT EYTIHWVRQA PGQSLEWMGN
INPNNGGTTY NQKFQGRVTI TVDKSTSTAY MELSSLRSED TAVYYCAAGW
NFDYWGQGTL VTVSSGKPGS GKPGSGKPGS GKPGSDIVMT QSPDSLAVSL
GERATLSCRA SQDVGTAVDW YQQKPDQSPK LLIYWASTRH TGVPDRFTGS
GSGTDFTLTI SSLQAEDVAV YFCQQYNSYP LTFGAGTKVE IK
anti-S arcogly can scFv (SEQ ID NO: 35) GAAGTCCAATTGGTTGAAAGCGGTGGTGGACTCGTCAAACCTGGCGGTAGCCTTAAAC
TTTCATGTGCCGCAAGCGGCTTCACGTTTAGTAACTATGCTATGAGTTGGGTCCGCCAA
AGTC C AGAAAAGC GC C TC GAATGGGTGGC GGAGATC TC TGGAGGAGGAAC ATATAC A
TATTATCCAGACACCATGACCGGTAGGTTTACAATCTCAAGAGACAACGCTAAGAACA
CCCTGTACCTGGAAATGTCAAGCCTGAGATCAGAAGATACGGCCATGTATTATTGTAC
GCGCCTACTCGACTATTGGGGTCAAGGAACTTCCGTGACGGTGTCAAGCGGAGGAGGT
GGGAGCGGAGGAGGCGGAAGTGGCGGTGGTGGCTCTGGTGGCGGTGGAAGTGATATA
GTGATGACGCAAGCTGCCTTTTCAAACCCTGTTACTTTGGGGACTAGCGCATCAATCTC
CTGTAGGTCCAGCAAATCTTTGCTGCACAGTAATGGAATCACCTATCTTTTCTGGTATT
TGC A A A A GC CTGGGC AGA GCCC GC A ACTGC TGATCT ATC A A ATGTC A A ATC TTGC
TTC
CGGAGTTCCAGACCGCTTCTCAAGTTCCGGGTCCGGCACTGATTTTACCTTGAGAATTT
CTAGGGTCGAAGCTGAAGACGTCGGTGTCTATTATTGC GC GC AAAAC C TTGAGC TTC C
ATACACCTTCGGGGGGGGCACAAAACTTGAGATCAAG
anti-S arcogly can scFv (SEQ ID NO: 36) EV QLVES GGGLVKPGGSLKL SCAAS GFTFSNYAMSWVRQSPEKRLEWVAEISGGGTYTYY
PDTMTGRFTISRDNAKNTLYLEMS SLRS EDTAMYYC TRLLDYWGQ GTS V TV S SGGGGSG
GGGS GGGGS GGGGS DIVMTQAAF SNPVTLGT S ASI S CRS S KS LLHSNGITYLFWYL QKP GQ
SP QLLIY QMSN LAS GVPDRFS S S GS GTDFTLRI SRVEAED V GV Y Y CAQN LELPY TF GGGTK

LEIK
anti-hu GPC 3 scFv (SEQ ID NO: 37) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCACCA
TCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAACAC
CTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGGGA
TTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGA

CTCCAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAATG
GTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTTCTAGAGGTGGTGGTGG
TAGCGGC GGCGGCGGC TC TGGTGGTGGTGGATC CCTCGAGATGGCC CAGGTGC AGCTG
GTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG
CCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCACATACTATGCAGAC
TCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCA AGAACACGCTGTATCTGC
AAATGAACAGCC TGAGAGCCGAGGAC AC GGC C GTATATTACTGTGC GC GC AC TTC TTA
C CTGAACC ATGGTGATTAC TGGGGTCAAGGTACTCTGGTGACCGTGTC TAGC GC C GC T
GCA
anti-hu GPC3 scEv (SEQ ID NO: 38) QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDR
ESASKSGTSATLGITGLQTGDEADYYCGTVVDYTLNGVVEGGGTKLTVLGSRGGGGSGGG
GS GGGGS LEMAQV QLVE S GGGLV QP GGSLRL SCAASGFTF S SYAMSWVRQAP GKGLEWV
S VIY SGGS S TY Y AD S VKGRETISRDN SKNTLYLQMN S LRAEDTAV Y Y CART S Y LNHGDY W

GQGTLVTVSSAAA
pWF-82 (SEQ ID NO: 39) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGCCCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGCTCCCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGAACC
ACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACGTCCA
CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GCTGGATGGAACTTCGACTATTGGGGACAAGGGACTCTTGTTACGGTGTCCAGTGGCA
AAC CAGGTAGTGGTAAACC CGGAAGCGGCAAGC CCGGGAGCGGTAAACCTGGTAGCG
ACATCGTCATGACTCAAAGCCCTGACTCACTCGCCGTGAGCCTGGGAGAGCGTGCAAC
GC TATC TTGTCGGGCCTCTCAGGATGTC GGAACTGC TGTAGACTGGTATCAACAGAAA
CCTGACCAATCACCAAAACTCCTGATTTATTGGGCCTCAACACGTCACACAGGAGTGC
C AGATAGGTTCAC AGGTAGTGGCAGTGGAACTGATTTTAC TTTGACAATTAGC AGC CT
GCAAGCCGAAGATGTAGCCGTTTACTTCTGTCAACAATATAACTCATACCCACTAAC G
TTCGGTGCCGGGACGAAGGTAGAGATTAAATTCGTGCCTGTGTTCCTCCCAGCTAAGC
CC ACTACC ACCCCCGCTCC AAGGCCGCCCACGCCCGCTCCTACTATTGCTAGTCAGCCT
TTAAGTTTAC GAC C C GAAGC TTGC AGGC C C GC C GC C GGC GGC GCTGTGC AC AC CAGGG
GGCTTGATTTTGCCTGC GACTTTTGGGTATTGGTAGTGGTGGGC GGAGTTTTAGC CTGC
TACAGC CTC C TGGTAAC AGTGGCTTTTATCATCTTTTGGGTGCAGC GGGC TTTAGTC TT
GC GGC GTAAAC GTAAAAGAATGAC AGATC C AACTC GCAGGTTCTTCAAAGTGAC CC CC
C C AC CTGGGTC C GGAC C GCAGAAC CAATATGGGAATGTC CTGTCTCTGC C TAC GC CTA
C A A GTGGA C TGGGT A GGGCTC A GA GGTGGGCTGCC GGTC T CGGCGGA A CTGCGC C AT C
TTACGGAAATCCCTCCTCCGACGTTCAGGCAGACGGGGCCCTGGGGTCTCGATCCCCG
C CTGGTGTTGGACCAGAAGAGGAAGAGGGC GAGGGC TACGAAGAGC CCGAC TC CGAA
GAGGACAGTGAGTTTTACGAGAACGACAGCAACCTGGGGCAGGATCAGCTGTCACAG
GATGGCTCAGGATATGAAAACCCTGAGGACGAGCCTTTGGGGCCTGAAGATGAGGAC
TCCTTTTCTAATGCAGAGTCATATGAGAATGAGGACGAAGAATTGACTCAACCCGTGG
CAAGAACAATGGATTTCCTCAGTCCACACGGGAGTGCATGGGACCCCTCCAGAGAGGC
TACTAGCCTC GGTTC TC AAAGC TATGAGGAC ATGAGGGGTATTC TGTAC GC AGC GC C T
CAGTTGAGGTCCATCCGCGGCCAGCCAGGCCCAAACCATGAGGAAGATGCCGATTCTT
ACGAAAACATGGACAACCCCGATGGTCCTGACCCCGCATGGGGGGGCGGCGGGAGGA
TGGGCACCTGGTCTACTCGCTAG
pWF-82 (SEQ ID NO. 40) EVQLVQSGAE VKKPGASVKV SCKTSGYTFT EYTIHWVRQA PGQSLEWMGN
INPNNGGTTY NQKFQGRVTI TVDKSTSTAY MELSSLRSED TAVYYCAAGW
NFDYWGQGTL VTVSSGKPGS GKPGSGKPGS GKPGSDIVMT QSPDSLAVSL
GERATLSCRA SQDVGTAVDW YQQKPDQSPK LLIYWASTRH TGVPDRFTGS
GSGTDFTLTI SSLQAEDVAV YFCQQYNSYP LTFGAGTKVE IKFVPVFLPA KPTTTPAPRP
PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDFWV LVVVGGVLAC
YSLLVTVAFI IFWVQRALVL RRKRKRMTDP TRRFFKVTPP PGSGPQNQYG NVLSLPTPTS
GLGRAQRWAA GLGGTAPSYG NPSSDVQADG ALGSRSPPGV GPEEEEGEGY
EEPDSEEDSE FYENDSNLGQ DQLSQDGSGY ENPEDEPLGP EDEDSFSNAE SYENEDEELT
QPVARTMDFL SPHGSAWDPS REATSLGSQS YEDMRGILYA APQLRSIRGQ
PGPNHEEDAD SYENMDNPDG PDPAWGGGGR MGTWSTR-pWF-83 (SEQ ID NO: 41) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGCCCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGCTCCCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGAACC
ACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACGTCCA
CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GCTGGATGGAACTTCGACTATTGGGGACAAGGGACTCTTGTTACGGTGTCCAGTGGCA
AACCAGGTAGTGGTAAACCCGGAAGCGGCAAGCCCGGGAGCGGTAAACCTGGTAGCG
ACATCGTCATGACTCAAAGCCCTGACTCACTCGCCGTGAGCCTGGGAGAGCGTGCAAC
GCTATCTTGTCGGGCCTCTCAGGATGTCGGAACTGCTGTAGACTGGTATCAACAGAAA
CCTGACCAATCACCAAAACTCCTGATTTATTGGGCCTCAACACGTCACACAGGAGTGC
CAGATAGGTTCACAGGTAGTGGCAGTGGAACTGATTTTACTTTGACAATTAGCAGCCT
GCAAGCCGAAGATGTAGCCGTTTACTTCTGTCAACAATATAACTCATACCCACTAACG
TTCGGTGCCGGGACGAAGGTAGAGATTAAATTCGTGCCTGTGTTCCTCCCAGCTAAGC
CCACTACCACCCCCGCTCCAAGGCCGCCCACGCCCGCTCCTACTATTGCTAGTCAGCCT
TTAAGTTTAC GAC C C GAAGC TTGC AGGC C C GC C GC C GGC GGC GC TGTGC AC AC CAGGG
GGCTTGATTTTGCCTGCGACTTTTGGGTATTGGTAGTGGTGGGCGGAGTTTTAGCCTGC
TACAGCCTCCTGGTAACAGTGGCTTTTATCATCTTTTGGGTGCTGGACAAGGATGACAG
CAAGGCTGGCATGGAGGAAGATCACACCTACGAGGGCCTGGACATTGACCAGACAGC
CACCTATGAGGACATAGTGACGCTGCGGACAGGGGAAGTGAAGTGGTCTGTAGGTGA
GCACCCAGGCCAGGAGTGA
pWF-83 (SEQ ID NO: 42) EVQLVQSGAE VKKPGASVKV SCKTSGYTFT EYTIHWVRQA PGQSLEWMGN
INPNNGGTTY NQKFQGRVTI TVDKSTSTAY MELSSLRSED TAVYYCAAGW
NFDYWGQGTI, VTVSSGKPGS GKPGSGKPGS GKPGSDIVMT QSPDSLAVSI, GERATLSCRA SQDVGTAVDW YQQKPDQSPK LLIYWASTRH TGVPDRFTGS
GSGTDFTLTI SSLQAEDVAV YFCQQYNSYP LTFGAGTKVE IKFVPVFLPA KPTTTPAPRP
PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDFWV LVVVGGVLAC

EVKWSVGEHP GQE-pWF-84:
(SEQ ID NO: 43) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGCCCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGCTCCCGGTCAAAGCCTCGA ATGGATGGGAAATATTA ACCCTAACAATGGCGGAACC
ACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACGTCCA

C AG CTTAC ATG GAAC TCAGC TCTTTG AGATCC GAAGACACTG CAGTTTATTATTGTG CA
GC TGGATGGAACTTC GAC TATTGGGGACAAGGGACTC TTGTTAC GGTGTC CAGTGGCA
AAC CAGGTAGTGGTAAACC CGGAAGCGGCAAGCC CGGGAGCGGTAAACCTGGTAGCG
ACATC GTCATGACTCAAAGCCCTGACTCAC TC GC C GTGAGC C TGGGAGAGC GTGCAAC
GC TATC TTGTC GGGC C TC TC AGGATGTC GGAACTGC TGTAGACTGGTATCAACAGAAA
C C TGAC C AATC AC CAAAAC TCCTGATTTATTGGGCCTCAAC ACGTCACACAGGAGTGC
C AGATA GGTTC AC AGGTAGTGGC, AGTGGA AC TGA TTTT ACTTTGAC A A TTAGC AGC CT
GC AAGC C GAAGATGTAGC CGTTTACTTCTGTC AAC AATATAACTCATACCC AC TAAC G
TTCGGTGCCGGGAC GAAGGTAGAGATTAAATTCGTGC CTGTGTTCCTCCC AGCTAAGC
C C ACTACC ACC CCC GCTC CAAGGCCGC CCAC GCC C GCTCCTAC TATTGCTAGTCAGC CT
TTAAGTTTAC GAC C C GAAGC TTGC AGGC C C GC C GC C GGC GGC GCTGTGC AC AC CAGGG
GGCTTGATTTTGCCTGCGACTTTTGGGTATTGGTAGTGGTGGGCGGAGTTTTAGCCTGC
TACAGCCTCCTGGTAACAGTGGC,TTTTATCATCTTTTGGGTGAAGAAGGTTGCAAAAA
AAC CTACTAATAAGGCTC C C CATC C TAAGC AAGAGC C C C AAGAAATTAACTTTC C C GA
TGATCTTC C GGGTTCTAACAC GGC AGC C C C GGTGCAGGAGAC C CTGCATGGTTGTC AA
CCCGTCACTCAGGAGGACGGGAAAGAGTCTCGTATCTCCGTCCAGGAGAGACAGGAC
AAGGACGATAGTAAAGCAGGGATGGAGGAGGACCATACATACGAGGGACTGGATATC
GATCAGACAGCCACGTACGAAGACATTGTGACACTGAGAACTGGCGAGGTGAAGTGG
TCAGTGGGAGAACATCCGGGGCAGGAATAA
pWF-84:
(SEQ ID NO: 44) EVQLVQSGAE VKKPGASVKV SCKTSGYTFT EYTIHWVRQA PGQSLEWMGN
INPNNGGTTY NQKFQGRVTI TVDKSTSTAY MELSSLRSED TAVYYCAAGW
NFDYWGQGTL VTVSSGKPGS GKPGSGKPGS GKPGSDIVMT QSPDSLAVSL

GSGTDFTLTI SSLQAEDVAV YFCQQYNSYP LTFGAGTKVE IKFVPVFLPA KPTTTPAPRP
PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDFWV LVVVGGVLAC
YSLLVTVAFI IFWVKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV
QETLHGCQPV TQEDGKESRI SVQERQDKDD SKAGMEEDHT YEGLDIDQTA
TYEDIVTLRT GEVKWSVGEH PGQE-pWF-85:
(SEQ ID NO: 45) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGCCCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGCTCCCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGAACC
ACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACGTCCA
CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GCTGGATGGAACTTCGACTATTGGGGACAAGGGACTCTTGTTACGGIGTCCAGTGGCA
AACCAGGTAGTGGTAAACCCGGAAGCGGCAAGCCCGGGAGCGGTAAACCTGGTAGCG
AC A TC GTC ATGACTC A A AGCCCTGACTC A C TC GCCGTGA GCCTGGGAGA GC GTGC A AC
GCTATCTTGTCGGGCCTCTCAGGATGTCGGAACTGCTGTAGACTGGTATCAACAGAAA
CCTGACCAATCACCAAAACTCCTGATTTATTGGGCCTCAACACGTCACACAGGAGTGC
CAGATAGGTTCACAGGTAGTGGCAGTGGAACTGATTTTACTTTGACAATTAGCAGCCT
GCAAGCCGAAGATGTAGCCGTTTACTTCTGTCAACAATATAACTCATACCCACTAACG
TTCGGTGCCGGGACGAAGGTAGAGATTAAATTCGTGCCTGTGTTCCTCCCAGCTAAGC
CCACTACCACCCCCGCTCCAAGGCCGCCCACGCCCGCTCCTACTATTGCTAGTCAGCCT
TTAAGTTTAC GAC C C GAAGC TTGC AGGC C C GC C GC C GGC GGC GC TGTGC AC AC C AGGG

GGCTTGATTTTGCCTGCGACTTTTGGGTATTGGTAGTGGTGGGCGGAGTTTTAGCCTGC
TACAGCCTCCTGGTAACAGTGGCTTTTATCATCTTTTGGGTGAAGAAGGTTGCAAAAA
AACCTACTAATAAGGCTCCCCATCCTAAGC AAGAGC C C C AAGAAATTAACTTTC C C GA
TGATCTTC CGGGTTCT A AC AC GGC AGCCCCGGTGC A GGAGACCCTGCATGGTTGTC A A
CCCGTCACTCAGGAGGACGGGAAAGAGTCTCGTATCTCCGTCCAGGAGAGACAGAAA

AGAGGCCGAAAAAAGCTGCTGTACATCTTCAAACAACCCTTCATGCGACCTGTTCAGA
CGACACAGGAGGAGGACGGCTGCAGCTGTAGGTTTCCCGAAGAAGAGGAGGGAGGAT
GCGAACTTTAA
pWF-85:
(SEQ ID NO: 46) EVQLVQSGAE VKKPGASVKV SCKTSGYTFT EYTIHWVRQA PGQSLEWMGN
INPNNGGTTY NQKFQGRVTI TVDKSTSTAY MELSSLRSED TAVYYCAAGW
NFDYWGQGTL VTVSSGKPGS GKPGSGKPGS GKPGSDIVMT QSPDSLAVSL
GERATLSCRA SQDVGTAVDW YQQKPDQSPK LLIYWASTRH TGVPDRFTGS
GSGTDFTLTI SSLQAEDVAV YFCQQYNSYP LTFGAGTKVE IKFVPVFLPA KPTTTPAPRP
PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDFWV
LVVVGGVLAC YSLLVTVAFI IFWVKKVAKK PTNKAPHPKQ EPQEINFPDD
LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQKRGR KKLLYIFKQP
FMRPVQTTQE EDGCSCRFPE EEEGGCEL-pWF-86 (SEQ ID NO: 150) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGCCCGGGGCATCTGTGAAA
GTATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGA
CAGGCTCCCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGA
ACCACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACG
TCCACAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATT
GTGCAGCTGGATGGAACTTCGACTATTGGGGACAAGGGACTCTTGTTACGGTGTCCA
GTGGCAAACCAGGTAGTGGTAAACCCGGAAGCGGCAAGCCCGGGAGCGGTAAACCT
GGTAGCGACATCGTCATGACTCAAAGCCCTGACTCACTCGCCGTGAGCCTGGGAGAG
CGTGCAACGCTATCTTGTCGGGCCTCTCAGGATGTCGGAACTGCTGTAGACTGGTATC
AACAGAAACCTGACCAATCACCAAAACTCCTGATTTATTGGGCCTCAACACGTCACA
CAGGAGTGCCAGATAGGTTCACAGGTAGTGGCAGTGGAACTGATTTTACTTTGACAA
TTAGCAGCCTGCAAGCCGAAGATGTAGCCGTTTACTTCTGTCAACAATATAACTCATA
C C CAC TAAC GTTC GGTGC CGGGACGAAGGTAGAGATTAAATTCGTGCCTGTGTTCCT
CCCAGCTAAGCCCACTACCACCCCCGCTCCAAGGCCGCCCACGCCCGCTCCTACTATT
GCTAGTCAGCCTTTAAGTTTACGACCCGAAGCTTGCAGGCCCGCCGCCGGCGGCGCT
GTGCACACCAGGGGGCTTGATTTTGCCTGCGACTTTTGGGTATTGGTAGTGGTGGGCG
GAGTTTTAGCCTGCTACAGCCTCCTGGTAACAGTGGCTTTTATCATCTTTTGGGTGAA
GAAGGTTGCAAAAAAACCTACTAATAAGGCTCCCCATCCTAAGCAAGAGCCCCAAG
AAATTAACTTTCCCGATGATCTTCCGGGTTCTAACACGGCAGCCCCGGTGCAGGAGA
CCCTGCATGGTTGTCAACCCGTCACTCAGGAGGACGGGAAAGAGTCTCGTATCTCCG
TCCAGGAGAGACAGCGCAAAAAACGTATAAGCGCAAACTCTACAGATCCAGTAAAA
GCCGCGCAATTCGAGCCTCCCGGCCGCCAGATGATTGCAATACGGAAACGTCAACTG
GAGGAAACTAATAATGACTATGAGACGGCCGACGGTGGATACATGACCCTTAATCCC
CGC,GCGCCAACCGACGATGATAAGAACATATATCTGACGCTCCCCCCTAACGATCAC
GTTAACAGTAATAATTAA
pWF-86:
(SEQ ID NO: 47) EVQLVQSGAE VKKPGASVKV SCKTSGYTFT EYTIHWVRQA PGQSLEWMGN
INPNNGGTTY NQKFQGRVTI TVDKSTSTAY MELSSLRSED TAVYYCAAGW
NFDYWGQGTL VTVSSGKPGS GKPGSGKPGS GKPGSDIVMT QSPDSLAVSL
GERATLSCRA SQDVGTAVDW YQQKPDQSPK LLIYWASTRH TGVPDRFTGS
GSGTDFTLTI SSLQAEDVAV YFCQQYNSYP LTFGAGTKVE IKFVPVFLPA KPTTTPAPRP
PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDFWV LVVVGGVLAC
YSLLVTVAFI IFWVKKVAKK P'TNKAPHPKQ EPQETNFPDD LPGSNTAAPV

QETLHGCQPV TQEDGKESRI SVQERQRKKR ISANSTDPVK AAQFEPPGRQ
MIAIRKRQLE ETNNDYETAD GrGYMTLNPRA PTDDDKNIYL TLPPNDHVNS NN-pWF-87:
(SEQ ID NO: 48) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGC CCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGC TC CC GGTC AAAGCCTC GAATGGATGGGAAATATTAACCC TAAC AATGGCGGAAC C
AC ATATAATCAGAAATTC C AAGGC C GAGTGAC GATAAC TGTC GATAAGAGTAC GTC C A
CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GCTGGATGGAACTTCGACTATTGGGGACAAGGGACTCTTGTTACGGTGTCCAGTGGCA
AACCAGGTAGTGGTAAACCCGGAAGCGGCAAGCCCGGGAGCGGTAAACCTGGTAGCG
ACATC GTCATGACTCAAAGCCCTGACTCAC TC GC C GTGAGC C TGGGAGAGC GTGCAAC
GCTATCTTGTCGGGCCTCTCAGGATGTCGGAACTGCTGTAGACTGGTATCAACAGAAA
C C TGAC C AATC AC CAAAAC TCCTGATTTATTGGGCCTCAAC ACGTCACACAGGAGTGC
CAGATAGrGTTCACAGGTAGTGGrCAGTGGAACTGATTTTACTTTGACAATTAGCAGCCT
GCAAGCCGAAGATGTAGCCGTTTACTTCTGTCAACAATATAACTCATACCCACTAAC G
TTCGGTGCCGGGACGAAGGTAGAGATTAAATTCGTGCCTGTGTTCCTCCCAGCTAAGC
CCACTACCACCCCCGCTCCAAGGCCGCCCACGCCCGCTCCTACTATTGCTAGTCAGCCT
TTAAGTTTACGAC CCGAAGC TTGC AGGCC C GC C GC C GGC GGC GC TGTGC AC AC C AGGG
GGCTTGATTTTGCCTGC GA CTTTTGGGT ATTGGT A GTGGTGGGC GGAGTTTT A GC CTGC
TACAGCCTCCTGGTAACAGTGGCTTTTATCATCTTTTGGGTGATGGCGGC GGGCGGGCC
CGGCGCCGGAAGCGCCGCGCCAGTCTCATCTACGTCCAGTCTGCCACTGGCTGCCCTG
AACATGAGAGTGAGACGCCGTTTATCCCTCTTCCTGAATGTGCGGACCCAGGTCGCCG
CTGATTGGACCGCCCTGGCCGAAGAGATGGACTTTGAATACTTGGAAATCAGACAGrCT
GGAAACACAGGCAGACCCAACCGGGAGACTGC TTGAC GC CTGGCAGGGAC GC C C AGG
GGCAAGTGTTGGTCGGTTACTGGAGCTTTTAACTAAGTTGGGCCGCGATGACGTGCTG
TTGGAGTTAGGACC CAGTATC GAGGAGGATTGTC AGWTACATC TTGAAAC AGC AGC
AGGAGGAGGCGGAAAAGCCCCTGCAGGTGGCGGC CGTTGACAGCAGTGTACCCAGAA
CAGCTGAGC TGGCCGGC ATC AC AAC C CTGGATGATCC C C TGGGC C AC ATGC C TGAGAG
GTTCGACGCTTTCATAAAGAAGGTTGCAAAAAAACCTACTAATAAGGCTCCCCATCCT
AAGCAAGAGCCCCAAGAAATTAACTTTCCCGATGATCTTCCGGGTTCTAACACGGCAG
CCCCGGTGCAGGAGACCCTGCATGGTTGTCAACCCGTCACTCAGGAGGACGGGAAAG
AGTCTCGTATCTCCGTCCAGGAGAGACAGTGA
pWF-87:
(SEQ ID NO: 49) EV QLV QS GAEVKKP GAS VKV SCKTSGYTFTEYTIHWVRQAPGQ SLEWMGNINPNNGGTT
YNQKFQ GRVTITVD KS T S TAYMEL S SLRSEDTAVYYCAAGWNFDYWGQGTLVTVS SGKP
GS GKP GS GKP GS GKP GS DIVMTQ SP D SLAV S LGERATL SCRAS QDVGTAVDWYQQKPDQ S
PK I I,TYW A STRHTGVPDRF TGS GS GTDFTI ,TIS SI ,Q A-F-1)V AVYFCQQYNSYPI ,TF GA
GTK V
EIKFVPVFLPAKPTTTPAPRPPTPAPTIAS QPL SLRPEACRPAAGGAVHTRGLDFACDFWVL
VVV GGVLACY S LLVTVAFIIFWVMAAGGP GAGS AAPV S STS SLPLAALNMRVRRRL SLFL
NVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKL
GRDD V LLELGP SIEEDC QKY ILKQ Q QEEAEKPL Q V AAVD S S V PRTAELAGITTLDDPL GHM
PERFDAFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGC QPVTQ ED GKE
SRISVQERQ-pWF-88:
(SEQ ID NO: 50) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGC CCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGCTC CCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGAAC C

ACATATAATCAGAAATTCCAAG GC CGAGTGACGATAACTG TCGATAAGAGTAC GTCCA
CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GC TGGATGGAAC TTC GAC TATTGGGGAC AAGGGAC TC TTGTTAC GGTGTC C AGTGGC A
AACCAGGTAGTGGTAAACCCGGAAGCGGCAAGCCCGGGAGCGGTAAACCTGGTAGCG
ACATC GTC ATGACTC AAAGC C CTGACTCAC TC GC C GTGAGC C TGGGAGAGC GTGCAAC
GC TATC TTGTC GGGC C TC TC AGGATGTC GGAACTGC TGTAGACTGGTATCAACAGAAA
CCTGACC AATC ACCA AA ACTCCTGATTTATTGGGCCTCAAC ACGTCACACAGGAGTGC
C AGATAGGTTC AC AGGTAGTGGC AGTGGAAC TGATTTTAC TTTGAC AATTAGC AGC CT
GC AAGC C GAAGATGTAGC C GTTTAC TTC TGTC AAC AATATAAC TC ATAC C C AC TAAC G
TTCGGTGCCGGGACGAAGGTAGAGATTAAATTCGTGCCTGTGTTCCTCCCAGCTAAGC
C CACTACCACCC CC GCTCCAAGGC CGCC CAC GC C C GCTCCTACTATTGCTAGTCAGC CT
TTAAGTTTAC GAC C C GAAGCTTGC AGGC C C GC C GC C GGC GGC GC TGTGC ACAC CAGGG
GGCTTGATTTTGCCTGCGACTTTTGGGTATTGGTAGTGGTGGGC,GGAGTTTTAGCCTGC
TACAGC CTC CTGGTAACAGTGGC TTTTATCATCTTTTGGGTGAGGAAAC GATGGCAGA
AC GAGAAGC TC GGGTTGGATGC C GGCTG ATGAATATGAAGATGAAAAC CTTTATGAAG
GC CTGAAC CTGGAC GACTGCTC CATGTATGAGGAC ATC TC C C GGGGC CTCCAGGGCAC
CTACCAGGATGTGGGCAGCCTCAACATAGGAGATGTCCAGCTGGAGAAGCCGTGA
pWF - 88:
(SEQ ID NO: 51) EVQLVQSGAEVKKPGASVKV SCKTSGYTFTEYTIHWVRQAPGQSLEWMGNINPNNGGTT
YNQKFQGRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLVTVSSGKP
GS GKP GS GKP GS GKP GS DIVMTQ SPD SLAV S LGERATL S CRAS QDVGTAVDWYQQKPDQS
PKLLIYWAS TRHTGVPDRF TGS GS GTDFTLTI S SLQAEDVAVYFCQQYNSYPLTFGAGTKV
EIKF VP VFLPAKPTTTP APRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDFWVL
VVVGGVLACYSLLVTVAFIIFWVRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYED
I SRGLQGTYQDVGSLNIGDVQLEKP -pWF-89:
(SEQ ID NO: 52) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGC CCGGGGCATCTGTGAAAG
TATCATGCAAAACATC C GGC TATAC GTTTAC C GAATAC AC CATTCACTGGGTCAGAC A
GGCTC CCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGAAC C
AC ATATAATCAGAAATTC C AAGGC C GAGTGAC GATAAC TGTC GATAAGAGTAC GTC CA
C AGCTTAC ATGGA ACTC AGCTCTTTGAGATCC GA AGAC ACTGC A GTTT ATT ATTGTGC A
GC TGGATGGAACTTC GAC TATTGGGGACAAGGGACTC TTGTTAC GGTGTC CAGTGGCA
AAC CAGGTAGTGGTAAACC CGGAAGCGGCAAGCC CGGGAGCGGTAAACCTGGTAGCG
ACATC GTC ATGACTC AAAGC C CTGACTCAC TC GC C GTGAGC C TGGGAGAGC GTGCAAC
GC TATC TTGTC GGGC C TC TC AGGATGTC GGAACTGC TGTAGACTGGTATCAACAGAAA
C C TGAC C AATC AC CAAAAC TC CTGATTTATTGGGC CTCAAC ACGTCAC ACAGGAGTGC
C A GA TA CTGTTC AC AGGT A GTGCTC, A GTGGA A CTGA TTTT A CTTTGA C A A TT A GC
AGC CT
GCAAGCCGAAGATGTAGCCGTTTACTTCTGTCAACAATATAACTCATACCCACTAACG
TTCGGTGCCGGGAC GAAGGTAGAGATTAAATTCGTGC CTGTGTTCCTCCCAGCTAAGC
C CACTACCACCC CC GCTCCAAGGC CGCC CAC GC C C GCTCCTACTATTGCTAGTCAGC CT
TTAAGTTTAC GAC C C GAAGC TTGC AGGC C C GC C GC C GGC GGC GCTGTGC AC AC CAGGG
GGCTTGATTTTGCCTGCGACTTTTGGGTATTGGTAGTGGTGGGCGGAGTTTTAGCCTGC
TACAGCCTCCTGGTAACAGTGGCTTTTATCATCTTTTGGGTGCTGGACAAGGATGACAG
C AAGGC TGGC ATGGAGGAAGATC AC AC C TAC GAGGGC C TGGAC ATTGAC C AGAC AGC
C AC CTATGAGGACATAGTGAC GC TGC GGACAGGGGAAGTGAAGTGGTCTGTAGGTGA
GCACCCAGGCCAGGAGTGA
pWF-89:
(SEQ ID NO: 53) EV QLV QS GAEVKKP GAS VKV SCKTSGYTFTEYTIHWVRQAPGQSLEWMGNINPNNGGTT
YNQKFQ GRVTITVD KS T S TAYMEL S SLRSEDTAVYYCAAGWNFDYWGQGTLVTVS SGKP
GSGKPGS GKPGSGKPGSDIVMTQSPDSLAVSLGERATLSCRAS QDVGTAVDWYQQKPDQS
PKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIS SLQAEDVAVYF CQQYNSYPLTFGAGTKV
EIKFVPVFLPAKPTTTPAPRPPTPAPTIAS QPL SLRPEACRPAAGGAVHTRGLDFACDFWVL
VVVGGVLACYSLLVTVAFIIFWVLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGE
VKWSVGEHPGQE
pWF-391:
(SEQ ID NO: 54) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGCCCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGCTC CCGGTCAAAGCCTCGAATGGATGGGAAATATTAACCCTAACAATGGCGGAAC C
ACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACGTCCA
CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GCTGGATGGAACTTCGACTATTGGGGACAAGGGACTCTTGTTACGGTGTCCAGTGGCA
AACCAGGTAGTGGTAAACCCGGAAGCGGCAAGCCCGGGAGCGGTAAACCTGGTAGCG
ACATCGTCATGACTCAAAGCCCTGACTCACTCGCCGTGAGCCTGGGAGAGCGTGCAAC
GCTATCTTGTCGGGCCTCTCAGGATGTCGGAACTGCTGTAGACTGGTATCAACAGAAA
C CTGACC AATC AC C AAAAC TC C TGATTTATTGGGC C TC AAC AC GTC AC AC AGGAGTGC
C A GATA GGTTC AC AGGTAGTGGC, A GTGGA A C TGA TTTT A CTTTGA C A A TTA GC AGC
CT
GCAAGCCGAAGATGTAGCCGTTTACTTCTGTCAACAATATAACTCATACCCACTAACG
TTCGGTGCCGGGACGAAGGTAGAGATTAAAGGCGCTGGTAGTGGCGGTAACTGGAGC
CACCCTCAATTTGAGAAGGGCGGGTCAGGCGGATCAGGTGGTAGTGGTGGGTCCAACT
GGAGC CATC C GC AATTTGAAAAGGGC GGAAGC GGC GGTTC CGGC GGTTCAGGCGGTA
GCAACTGGTCACATCCGCAATTTGAGAAAGGCGGGTCAGGCGGCGGGTTTTGGGCTCT
CGTGGTGGTGGCTGGAGTGCTTTTCTGCTATGGCCTGC TGGTAACCGTGGCCCTTTGTG
TAATCTGGACC GATAAAGAC GATGGAAAAGCC GGGATGGAAGAAGACCATACC TAC G
AGGGGCTCAATATTGATCAAAC C GC CAC GTATGAAGACATTGTAACAC TGC GCAC AGG
TGAGGTCAAGTGGTCC GTC GGTGAAC AC C C AGGAC AAGAATAA
pWF-391:
(SEQ ID NO: 55) YNQKFQGRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLVTVSSGKP
GS GKP GS GKP GS GKP GSDIVMTQ SPD S LAV S LGERATL S CRASQDVGTAVDWYQQKPDQS
PKLLIYWAS TRHTGVPDRF TGS GS GTDFTLTI S SLQAEDVAVYF CQQYNSYPLTFGAGTKV
EIKGAGSGGNWSHPQFEKGGS GGSGGSGGSNVVSHPQFEKGGSGGSGGSGGSNWSHPQFE
KGGSGGGFWALVVVAGVLECYGLLVTVALCVIWTDKDDGKAGMEEDHTYEGLNIDQTA
TYEDIVTLRTGEVKWSVGEHPGQE
pWF-394:
(SEQ ID NO: 56) GAAGTCCAATTGGTTGAAAGCGGTGGTGGACTCGTCAAACCTGGCGGTAGCCTTAAAC
TTTC ATGTGC C GCAAGC GGCTTCAC GTTTAGTAACTATGCTATGAGTTGGGTC C GC CAA
AGTCCAGAAAAGCGCCTCGAATGGGTGGCGGAGATCTCTGGAGGAGGAACATATACA
TATTATCCAGACACCATGACCGGTAGGTTTACAATCTCAAGAGACAACGCTAAGAACA
C CCTGTACC TGGAAATGTCAAGCCTGAGATCAGAAGATACGGC CATGTATTATTGTAC
GC GC CTACTC GACTATTGGGGTCAAGGAACTTC C GTGAC GGTGTCAAGC GGA GGAGGT
GGGAGCGGAGGAGGCGGAAGTGGCGGTGGTGGCTCTGGTGGCGGTGGAAGTGATATA
GTGATGAC GCAAGC TGC CTTTTCAAAC C C TGTTAC TTTGGGGACTAGC GC ATC AATCTC
CTGTAGGTCCAGCAAATCTTTGCTGCACAGTAATGGAATCACCTATCTTTTCTGGTATT
TGCAAAAGCCTGGGCAGAGCCC GCAACTGCTGATCTATCAAATGTCAAATCTTGCTTC

CGGAGTTCCAGACCGCTTCTCAAGTTCCGGGTCCGGCACTGATTTTACCTTGAGAATTT
C TAGGGTC GAAGCTGAAGAC GTC GG TGTCTATTATTGC GC GC AAAAC C TTGAGCTTC C
ATAC AC CTTCGGGGGGGGC ACAAAACTTGAGATCAAGGGC GCTGGGAGC GGCGGGAA
TTGGAGTCATCCACAATTCGAAAAGGGTGGGTC CGGCGGCAGTGGTGGAAGCGGCGG
GAGTAACTGGTCACATCCCCAGTTTGAGAAAGGCGGTAGTGGTGGCAGCGGCGGTAGT
GGTGGCAGTAATTGGAGCCATC CCCAATTCGAAAAGGGCGGTTCCGGCGGCGGATTTT
GGGCTC TTGTTGTGGTGGC C GGAGTATTGTTTTGC TAT GGC C TGCTC GTTA C A GTGGC A
TTGTGC GTAATTTGGACTGATAAAGACGAC GGCAAAGC C GGGATGGAAGAAGATC AC
AC CTATGAGGGGCTTAATATAGATCAAACAGC CAC ATATGAAGATATTGTGACTCTAA
GGACTGGAGAGGTTAAATGGAGTGTGGGTGAGCATCCAGGACAAGAATAA
pWF -394 :
(SEQ ID NO: 57) EV QLVES GGGLVKPGGSLKL SCAAS GFTF SNYAM SWVRQ S PEKRLEWVAEI S GGGTYTYY
PDTMTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCTRLLDYWGQGTSVTVSSGGGGSG
GGGS GGGGS GGGGSDIVMTQAAF SNPVTLGT S ASI S CRS SKSLLHSNGITYLFWYLQKPGQ
SP QLLIY QMSN LAS GV PDRF S S SGSGTDFTLRISRVEAEDVGVYYCAQNLELPYTFGGGTK
LEIKGAG S GGNW SHP QFEKGG S GG S GG S GGSNWSHPQFEKGGSGGSGGSGGSNW SHP QF
EKGGSGGGFWALVVVAGVLFCYGLLVTVALCVIWTDKDDGK AGMEEDHTYEGLNIDQT
ATYEDIVTLRTGEVKWSVGEHPGQE
pWF -396 :
(SEQ ID NO: 58) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCACCA
TCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAACAC
CTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGGGA
TTCCTGACC GATTCTCTGCCTC C AAGTC TGGC AC GTC AGC C AC C C TGGGC ATC AC C GGA
CTCCAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAATG
GTGTGGTGTTCGGCGGAGGGACCAAGCTGACC GTC CTAGGTTCTAGAGGTGGTGGTGG
TAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGCTG
GTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG
CCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGC TGGAGTGGGTC TC AGTTATTTATAGC GGTGGTAGTAGC AC ATAC TATGCAGAC
TCC GTGA A GGGC CGGTTC ACC A TC TC C A GA GATA ATTCC A A GA A C A CGC TGT
ATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCACTTCTTA
C CTGAACC ATGGTGATTAC TGGGGTC AAGGTACT C TGGTGAC C GTGTC TAGC GC C GC T
GCATTCGTGCCTGTGTTCCTCC CAGCTAAGCCCACTACCACCCCCGCTCCAAGGCCGCC
CACGCCCGCTCCTACTATTGCTAGTCAGCCTTTAAGTTTACGACCCGAAGCTTGCAGGC
CCGCCGCCGGCGGCGCTGTGCACACCAGGGGGCTTGATTTTGCCTGCGACTTTTGGGT
A TTGGT A GTGGTGGGCGGA GTTTT A GC CTGCT AC A GCC TC CTGGT A AC A GTGGCTTTT A
TCATCTTTTGGGTGAGGAAACGATGGCAGAACGAGAAGCTCGGGTTGGATGCCGGGG
ATGAATATGAAGATGAAAAC CTTTATGAAGGCCTGAAC CTGGACGACTGCTC CATGTA
TGAGGACATCTCCCGGGGC CTC CAGGGC AC C TAC CAGG ATGTGGGC AGC CTCAAC ATA
GGAGATGTCCAGCTGGAGAAGCC GTCiA
pWF -396 :
(SEQ ID NO: 59) Q SVLTQP P SV S AAP GQRVTI S C S GTRSNIGSDYV S WYQHLP GTAPKLLVYGDNLRP SGIPDR
FSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVEGGGTKLTVLGSRGGGGSGGG
GS GGGGS LEMAQV QLVE S GGGLV QP GGSLRL S CAAS GF TF S SYAMSWVRQAP GKGLEWV
SVIYSGGS S TYY AD SVK GRFTI SRDNSKNTLYLQMNS LR AEDT AVYYC ART S YLNHGDYW
GQGTLVTVS SAAAFVPVFLPAKPTTTPAPRPPTPAPTIAS QPLS LRPEACRPAAGGAVHTRG

LDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRKRWQNEKLGLDAGDEYEDENLYEGL
NLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP
pWF-397:
(SEQ ID NO: 60) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCACCA
TCTCC TGC TC TGGA A CC A GGTCC A AC ATT GGGAGTGATT ATGTTTCC TGGTACC A AC AC
CTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGGGA
TTCCTGACC GATTCTCTGCCTC C AAGTC TGGC AC GTCAGC C AC C C TGGGC ATC AC C GGA
CTCCAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAATG
GTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTTCTAGAGGTGGTGGTGG
TAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGCTG
GTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG
CCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCACATACTATGCAGAC
TCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCACTTCTTA
CCTGAACCATGGTGATTACTGGGGTCAAGGTACTCTGGTGACCGTGTCTAGCGCCGCT
GCATTCGTGCCTGTGTTCCTCCCAGCTAAGCCCACTACCACCCCCGCTCCAAGGCCGCC
C AC GC C C GC TC CTACTATTGCTAGTCAGCCTTTAAGTTTACGAC CCGAAGCTTGCAGGC
CCGCCGCCGGCGGCGCTGTGCACACCAGGGGGCTTGATTTTGCCTGCGACTTTTGGGT
ATTGGTAGTGGTGGGCGGAGTITTAGCCTGCTACAGCCTCCTGGTAACAGTGGCTTTTA
TCATCTTTTGGGTGCTGGACAAGGATGACAGCAAGGCTGGCATGGAGGAAGATCACAC
CTAC GAGGGCCTGGACATTGAC C AGAC AGC C AC C TATGAGGAC ATAGTGAC GCTGC GG
ACAGGGGAAGTGAAGTGGTCTGTAGGTGAGCACCCAGGCCAGGAGTGA
pWF-397:
(SEQ ID NO: 61) QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDR
FSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGSRGGGGSGGG
GSGGGGSLEMAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
SVIYSGGSSTYYADSVKGRETISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYW
GQGTLVTVS SAAAFVPVFLPAKPTTTPAPRPPTPAPTIAS QPLS LRPEACRPAAGGAVHTRG
LDFACDFWVLVVVGGVLACY SLLVTVAFIIFWVLDKDDSKAGMEEDHTYEGLDIDQTAT
YEDIVTLRTGEVKWSVGEHPGQE
pWF-460:
(SEQ ID NO: 62) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCACCA
TCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAACAC
CTCCC AGGA AC AGCCCCC A A ACTCCTCGTTTATGGCGATA ATCTGCGACCCTC AGGGA
TTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGA
CTC CAGACTGGGGAC GAGGC C GATTATTAC TGC GGC AC ATGGGATTAC AC C CTGAATG
GTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTTCTAGAGGTGGTGGTGG
TAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGCTG
GTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG
CCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCACATACTATGCAGAC
TCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAACACGCTGTATCTGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCACTTCTTA
CCTGAACCATGGTGATTACTGGGGTCAAGGTACTCTGGTGACCGTGTCTAGCCCCAAG
AGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGAC
CCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCC

GAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAG
TAC AAC AGC AC CTACAGGGTGGTGTCC GTGCTGAC C GTGC TGC AC C AGGAC TGGC TGA
ACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAA
AGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCC
CCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTT
CTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA
C AAGAC C AC CCCC CC AGTGC TGGAC AGC GAC GGCAGCTTCTTCCTGTACAGCAAGCTG
AC C GT GGAC AAGTC C AGGTGGCAGC AGGGCAACGTGTTCAGCTGCAGC GTGATGC AC
GAGGC CC TGCAC AAC CAC TACACC CAGAAGAGCC TGAGC CTGTCC CCC GAGC TGC AAC
TGGAGGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGACGGGCTGTGGACGACCA
TCACCATCTTCATCACACTCTTCCTGTTAAGCGTGTGCTACAGTGCCACCGTCACCTTCT
TCAAGGTGAAGTGGATCTTCTCCTCGGTGGTGGACCTGAAGCAGACCATCATCCCCGA
CTACAGGAACATGATCGGACAGGGGGCCTGA
pWF-460:
(SEQ ID NO: 63) QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRP SGIPDR
FSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVEGGGTKLTVLGSRGGGGSGGG
GS GGGGS LEMAQV QLVE S GGGLV QP GGS LRL S C AAS GF TF S SYAMSWVRQAPGKGLEWV
SVIYSGGS STYY AD SVK GRFTI SRDNSKNTLYLQMNS LR AEDT AVYYC ART SYLNHGDYW
GQGTLVTVS SPKS CDKTHTCPP CPAPELLGGP SVFLEPPKPKDTLMIS RTPEV TCVVVDV SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPELQ
LEES CAEAQDGELDGLWTTITIFITLELLSVCYSATVTFEKVKWIFS SVVDLKQTIIPDYRNM
IGQGA
pWF-428:
(SEQ ID NO: 64) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCACCA
TCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAACAC
CTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGGGA
TTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGA
C TC C A GAC TGGGGAC GA GGC CGATT ATT ACTGCGGC AC ATGGGATT AC ACC CTGA ATG
GTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTC CTAGGTCAGCCCAAGGCCAAC CC
C AC TGTC AC TC TGTTC C C GC CCTCCTCTGAGGAGCTCCAAGC C AAC AAGGC C AC AC TA
GTGTGTCTGATCAGTGACTTCTACC CGGGAGCTGTGACAGTGGCCTGGAAGGCAGATG
GCAGCCCCGTCAAGGCGGGAGTGGAGACCACCAAACCCTCCAAACAGAGCAACAACA
AGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAA
GCT A C A GCTGC,C A GGTC A CGC A TGA A GGGA GC A CC GTGGA GA A GAC A GTGGCC
CCT A
CAGAATGTTCA
pWF-428:
(SEQ ID NO: 65) QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRP SGIPDR
FSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVEGGGTKLTVLGQPKANPTVTLF
PP S S EELQANKATLVC LI S DFYP GAVTVAWKADGS PVKAGVETTKP S KQ SNNKYAAS SYL
SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC S
pWF-429:
(SEQ ID NO: 66) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC
TCTC C TGTGCAGC CTCTGGATTC ACC TTTAGCAGC TATGC C ATGAGCTGGGTC C GC CAG
GC TC CAGGGAAGGGGCTGGAGTGGGTC TC AGTTATTTATAGC GGTGGTAGTAGC AC AT
AC TATGCAGAC TC C GTGAAGGGC C GGTTCAC CATCTC CAGAGATAATTC CAAGAACAC
GC TGTATCTGCAAATGAAC AGC C TGAGAGC C GAGGACAC GGC C GTATATTACTGTGC G
CGCACTTCTTACCTGAACCATGGTGATTACTGGGGTCAAGGTACTCTGGTGACCGTGTC
TAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCT
C TGGGGGC AC AGC GGC C C TGGGC TGC C TGGTC AAGGAC TA C TTC CCC GAACC GGTGAC
GGTGT C GTGGAAC TC AGGC GC C CTGAC CAGC GGC GTGC AC AC CTTC CCGGCTGTC C TA
CAGTC CTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGC CC TCCAGCAGCTTGG
GCAC C C AGAC C TACATC TGC AAC GTGAAC CACAAGC C C AGCAACAC C AAGGTGGAC A
AGAGAGTGGAGCCCAAGAGCTGCGACAAGACC CACAC CTGCC C CC C CTGC CCAGC CCC
AGAGC TGC, TGGGCGGACC CTCC GTGTTCCTGTTC CCC CC CAAGCC CAAGGACAC CCTG
ATGATC AGCAGGACCC CC GAGGTGAC CTGC GTGGTGGT GGAC GTGAGCCAC GAGGAC
C C AGAGGTGAAGTTCAAC TGGTAC GTGGAC GGC GTGGAGGTGCACAAC GC CAAGAC C
AAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTG
C TGC AC CAGGAC TGGC TGAAC GGCAAGGAATACAAGTGCAAGGTC TC CAAC AAGGC C
CTGC CAGCC CC CATC GAAAAGACCATCAGCAAGGCCAAGGGCCAG C CAC GGGAGC CC
CAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGA
C CTGTCTGGTGAAGGGCTTCTACC C C AGC GAC ATC GC C GTGGAGTGGGAGAGC AAC GG
CC AGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGAC AGCGACGGC AGCTTC
TTC CTGTACAGCAAGC TGAC C GTGGACAAGTC C AGG TGGCAGC AGGGCAAC GTGTTC A
GCTGC AGC GTGATGC AC GAGGC C C TGC AC AAC CAC TAC AC C C AGAAGAGC CTGAGC C T
GTCCCCCGAGCTGCAACTGGAGGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGA
C GGGCTGTGGAC GAC CATC AC CATCTTCATC ACACTCTTC C TGTTAAGrC GTGTGCTAC A
GTGC CAC C GTCAC C TTCTTCAAGGTGAAGTGG ATCTTCTC CTC GGTGGTGGAC CTGAAG
CAGACCATCATCCCCGACTACAGGAACATGATC GGACAGGGGGCCTGA
pWF-429:
(SEQ ID NO: 67) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIVSGGSSTYY
ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSSA
STKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSA
TVTFFKVKWIFSSVVDLKQUIPDYRNMIGQGA-mu eXCI.13 (SEQ ID NO: 68) ATGAGAC TTTC AAC AGC AAC AC TC C TC C TGTTGC TGGC TTC ATGTC TGAGC C C TGGTC A
TGGTATTTTGGAGGC C CACTATAC AAATCTCAAATGTC GGTGTTCAGGC GTAATATC CA
C C GTAGTC GGCCTGAAC ATTATC GATAGGATTC AGGTTAC ACCC CC C GGGAAC GGATG
TCCTAAGAC CGAGGTGGTGATTTGGACCAAGATGAAGAAGGTCATTTGTGTGAACC CA
CGGGCTAAATGGCTGCAGCGTCTTTTGCGACACGTGCAGTCCAAGAGCTTGTCCAGCA
CAC CTCAGGCCC CAGTTAGCAAGCGACGTGCAGCC
mu CXCL 1 3 (SEQ ID NO: 69) MRLSTATLLLLLASCLSPGHGILEAHYTNLKCRCSGVISTVVGLNIIDRIQVTPPGNGCPKTE
VVIWTKIVIKKVICVNPRAKWLQRLLRHVQSKSLSSTPQ APVSKRRAA

mu FLT3LG
(SEQ ID NO: 70) ATGACAGTGCTGGCCCC CGCGTGGTCTCCCAATAGCTCACTC CTCCTCTTGCTGCTACT
GCTCAGCCCATGCCTCAGGGGCACCCCCGATTGTTACTTCAGCCACAGCCCAATCTCCT
CCAACTTCAAAGTGAAATTTAGGGAACTGACCGACCACCTGCTGAAAGATTATCCTGT
GA C TGTGGC A GTGA A CC TGC A A GA C GA A A A GC ATTGTA A GGCGC T ATGGA GC CTC
TTT
C TTGCC CAAC GATGGATTGAGCAACTCAAAAC TGTAGCC GGAAGCAAAATGC AGACG
C TAC TGGAGGAC GTGAATAC TGAGATTC AC TTC GTTACC AGTTGTACTTTC CAGC C ACT
GCCAGAGTGTCTCAGGTTTGTGCAGACTAATATCAGCCACCTGCTGAAGGATACTTGC
ACCCAGCTCCTGGCTCTCAAGCCTTGTATAGGCAAGGCTTGTCAAAATTTTAGCAGGTG
TCTCGAAGTCCAGTGCCAGCCAGATTCATCC AC ACTGC TGCC GCC CC GAAGCCC TATC
GC ACT C GAAGC GACAGAGTTGC C AGAGC, C TC GTC C CAGAC AGCTTC TGC TGCTGCTAC
TTCTGCTGCTGCCGCTAACTCTGGTGCTACTTGCTGCCGCCTGGGGCCTCAGATGGCAA
CGCGCCAGACGCCGAGGCGAACTCCACCCTGGGGTGCCACTGCCATCCCACCCA
mu FLT3LG
(SEQ ID NO: 71) MTVLAPAWSPNS SLLLLLLLL SP CLRGTPDCYFSHSPIS SNFKVKFRELTDHLLKDYPVTVA
VNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRF
V QTNI SHLLKDTCTQLL ALK P CIGK A CQNF SR CLEV QCQPDS STLLPP RSPI ALE ATEL PEPR

PRQLLLLLLLLLPLTLVLLAAAWGLRWQRARRRGELHPGVPLPS HP
mu XCL1 (SEQ ID NO: 72) ATGC GAC TC TTGTTGTTGAC TTTTCTC GGAGTGTGC TGC C TGACAC C CT GGGTC GTAGA
GGGAGTTGGCACTGAAGTACTAGAAGAGTCCTCCTGCGTTAACCTGCAGACACAGCGG
C TC CCAGTC CAGAAAATTAAGAC CTACATTATATGGGAAGGAGC AATGC GAGCGGTGA
TTTTTGTGACCAAGAGGGGTCTCAAGATTTGCGCGGACCCTGAGGCCAAGTGGGTCAA
AGC AGCTATTAAGACAGTAGACGGAAGAGCC TC CAC C AGGAAGAATATGGC AGAAAC
TGTACCGACCGGTGCGCAGCGGTCAACATCTACCGCAATCACACTCACCGGC
mu XCL1 (SEQ ID NO: 73) MRLLLLTFL GV CC LTPWVVEGV GTEVLEE S SCVNLQTQRLPVQKIKTYITWEGAMRAVIFV
TKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAI TLTG
mu Tim4(ECD)-muIgG2a Fc (SEQ ID NO: 74) ATGAGCAAGGGCCTTCTCCTGCTGTGGCTAGTAACTGAATTGTGGTGGTTGTACCTGAC
A C C TGCCGC T A GTGAGGAC ACC A TC A TTGGTTTCCTTGGGC A GCC C GTC A C CCTCC CTT

GCCATTACCTAAGCTGGAGCCAGTCACGGAACTCTATGTGCTGGGGAAAGGGGTCATG
C C C TAATTC C AAGTGC AAC GC C GAGC TGTTGC GC AC GGAC GGC AC C AGAATAATC TC A
AGAAAGTCCACCAAGTATACGCTGCTCGGCAAGGTGCAATTCGGTGAAGTGAGCTTGA
CCATAAGTAACACCAACCGCGGTGACTCCGGAGTTTATTGTTGCAGGATCGAAGTGCC
AGGCTGGTTTAACGACGTGAAGAAAAACGTGCGGCTGGAACTGAGGAGGGCAACTAC
GAC CAAGAAAC C AAC AAC CAC GAC GAGAC CTAC CAC CACTC CTTAC GTGAC AAC C AC
GAC AC C GGAGC TGTTGC CAAC TAC CGTCATGACAAC ATCTGTGTTGC CAACTACC ACC
CCCCCCCAAACGCTCGCGACAACTGCCTTTTCCACAGCCGTTACCACATGTCCTTCCAC
CACCCCAGGCTCTTTTTCTCAAGAAACTACCAAGGGATCAGCTTTTACCACCGAGTCTG
AAACTCTCCCAGCAAGTAATCACTCACAGCGGTCAATGATGACCATCAGCACAGACAT
C GCTGTC TTGA GA CCT A C TGGC A GC A ATC CAGGC ATTCTGCC CTCC A CTTC A C AGC
TGA
C TAC C CAAAAGAC TACACTAAC CAC CAGC GAAAGTC TGC AGAAAACTACAAAGAGCC

ATCAAATAAACTCCCGGCAGACTCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATG
CAAATGCCCAGCACCTAAC CTCTTGGGTGGAC CATCCGTCTTCATCTTCCCTCCAAAGA
TCAAGGATGTACTCATGATC TC CCTGAGC CCC ATAGTC AC ATGTGTGGTGGTGGATGTG
AGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACA
CAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGC
CCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAAC
AACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTA
AGAGC TC CAC AGGTATATGTCTTGCC TC C AC C AGAAGAAGAGATGAC TAAGAAAC AG
GTC AC TC TGAC C TGC ATGGTC AC AGAC TTC ATGC C TGAAGAC ATTTACGTGGAGTGGA
CCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTG
ATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAA
GAAATAGCTACTCCTGTTCAGTGGTCC ACGAGGGTCTGCACAATCACCACACGACTAA
GAGCTTCTCCCGGACTCCGGGTAAA
mu Tim4(ECD)-muIgG2a Fc (SEQ ID NO: 75) MSKGLLLLWLVTELWWLYLTPAASEDTIIGFLGQPVTLP CHYL SWSQ SRNS MCWGKGS CP
N SKCN AELLRTDGTRII S RKS TKY TLL GKV QF GEV SLTI SN TN RGDSGVYCCRIEVP GWEN D
VKKNVRLELRRATTTKKPTTTTRPTTTPYVTTTTPELLPTTVMTTSVLPTTTPPQTLATTAF
S TAVTTC P S TTP GS F S QETTKGS AFTTE S ETLP ASNH S QRS MMTI S TDIAVLRPTGSNP
GILP S
TSQLTTQKTTLTTSESLQKTTKSHQINSRQTPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIK
DVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQ
HQDWMS GKEF KCKVNNKDLPAP IERTI S KPKGSV RAP QVYVLPPPEEEMTKKQVTLTCMV
TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVV
HEGLHNHHTTKSFSRTP GK
mu 4-1BB-L
(SEQ ID NO: 76) ATGGATC AGC ATAC AC TGGAC GTGGAAGATACAGCC GATGC C AGAC AC C C TGC TGGA
AC GTC CTGTC C CAGC GAC GC TGC C CTGCTC AGAGAC AC C GGGC TGC TC GCAGATGCTG
CTC TGC TGAGTGATAC C GTTC GGC C AAC TAAC GC GGC C C TAC C C AC AGATGC C GC ATA

TCCCGCGGTAAATGTCAGGGACCGGGAAGCTGCCTGGCCACCGGCCCTCAATTTCTGC
TCTAGACATCCGAAACTGTACGGTCTGGTCGCACTGGTACTGCTGCTACTTATAGCAGC
TTGTGTTCCCATATTTACCCGCACTGAACCCAGACCCGCTCTCACTATTACAACTTCAC
CAAACTTGGGCACACGTGAAAACAATGCAGATCAGGTTACCCCTGTAAGTCATATTGG
ATGCCCCAACACCACACAAC AGGGAAGTCCGGTGTTTGCAAAACTCCTTGCTAAGAAT
CAGGCTTCACTGTGTAACACTACTCTTAATTGGCACTCACAAGACGGGGCCGGGAGTA
GC TATC TCAGCCAAGGTCTCC GC TATGAAGAAGATAAGAAAGAGTTGGTGGTGGAC AG
CCCAGGACTCTACTACGTCTTCCTGGAGCTAAAACTAAGCCCCACTTTTACTAACACTG
GACATAAGGTCCAAGGTTGGGTGTCCCTCGTACTTCAAGCTAAACCCCAGGTGGACGA
CTTCGATAACCTGGCGTTGACAGTTGAGCTCTTTCCTTGCTCTATGGAAAATAAGCTCG
TGCTATCCTCTAGCTGCTTCTCAACTCTTTCTCTCTCTTAAAGCCCTCTTCATCCTTCTCTTCTCTTTGGA
CTACGCGCATACTTGCATGGAGCCCAGGACGCATATCGTGATTGGGAACTGAGCTACC
C GAATAC C AC TAGC TTTGGACTATTTCTTGTTAAACC AGATAATCC TTGGGAG
mu 4-1BB-L
(SEQ ID NO: 77) MD QHTLDVEDTADARHPAGT S CP SDAALLRDTGLLADAALLSDTVRPTNAALPTDAAYP
AVNVRDREAAWPPALNFC S RHPKLYGLVALV LLLLIAACVPIF TRTEPRPALTITTS PNL GT
RENNAD QVTPV SHI GCPNTTQQ GS PVFAKLLAKNQAS L CNTTLNWH S QD GAGS SYL S QGL
RYEEDKKELVVDSP GLYYVFLELKL SPTFTNTGHKVQGWVSLVLQAKP QVDDFDNLALT
VELFPC S MENKLVD RSW S QLLLLKAGHRL S V GLRAYLH GAQDAYRDWEL SYPNTTSFGL
FLVKPDNPWE

mu LIGHT (cleavage-deficient mutant) (SEQ ID NO: 78) ATGGAGAGC GTAGTGC AACC CAGCGTATTTGTGGTGGATGGACAGACC GACATCC CAT
TCAGACGCTTGGAACAGAACCACCGAAGAAGGCGGTGCGGCACCGTCCAGGTGTCCCT
CGCTCTCGTGCTGCTGCTTGGTGCTGGCCTCGCAACACAAGGGTGGTTTCTTTTGAGAC
TCCATCAACGCTTGGGAGACATAGTGGCCCACCTGCCTGATGGTGGGAAGGGCTCTTG
GCAGGACCAGCGATCACACCAGGCTAACCCCGCCGCTCACCTGACAGGGGCGAATGC
C AGCTTGATCGGAATAGGTGGGC C GC TGC TGTGGGAAAC TAGGC TTGGAC TTGC C TTT
CTGAGAGGGCTTACATACC ATGACGGAGC CCTC GTAACAATGGAGC CTGGTTATTACT
AC GTGTAC AGTAAGGTGCAGCTTTC TGGAGTC GGGTGTC C C C AGGGGC TGGCTAAC GG
AC TGC C C ATC ACTC ATGGACTATACAAAC GC AC ATC CAGATATC CTAAAGAGCTGGAA
CTGTTGGTGTCCCGTAGGAGCCCGTGTGGCAGGGCCAACTCTTCCCGTGTGTGGTGGG
ACTCCTCTTTTCTGGGCGGCGTGGTCCATCTGGAAGCTGGTGAGGAAGTCGTCGTAAG
AGTACCTGGAAACCGTCTGGTTCGCCCCC GCGATGGCACCAGGTCCTACTTCGGAGCT
TTCATGGTA
mu LIGHT (cleavage-deficient mutant) (SEQ ID NO: 79) ME SVV QP S VFVVD GQTDIPFRRLEQNHRRRRC GTV QV S LALVLLL GAGLATQ GWFLLRLH
QRLGDIVAHLPDGGKGSWQDQRSHQANPAAHLTGANASLIGIGGPLLWETRLGLAFLRGL
TYHD GALVTMEP GYYYVYS KV QL SGVGCPQGLANGLPITHGLYKRTSRYPKELELLV S RR
S P C GRAN S SRVWWDS SFLGGVVHLEAGEEVVVRVPGNRLVRPRDGTRSYFGAFMV
mu IL12 (transmembrane form) (SEQ ID NO: 80) ATGTGCCCACAGAAACTCACAATTTCTTGGTTCGCAATCGTCCTGCTGGTGTCACCCCT
GATGGCAATGTGGGAGTTGGAAAAGGATGTATACGTCGTCGAGGTCGACTGGACACCT
GAC GCTCCGGGTGAAACTGTCAAC C TC AC TTGC GATACTC CTGAAGAGGAC GA C ATC A
CGTGGACGAGCGACCAGCGACATGGAGTGATAGGGTCTGGCAAGACGCTTACTATCAC
GGTTAAGGAATTTC TC GAC GC AGGGC AGTAC AC ATGTC AC AAGGGC GGC GAGAC TC TG
AGCCACTCCCATTTGCTGCTGCACAAGAAGGAGAATGGTATCTGGTCTACCGAAATCC
TGAAGAATTTTAAGAACAAGACTTTTCTGAAATGCGAGGCCCCAAATTATTCCGGACG
TTTCACTTGCAGTTGGCTCGTTCAAAGAAATATGGACTTGAAATTTAACATTAAATCCA
GCTCTTCATCTCCTGACAGCAGGGCCGTAACTTGTGGAATGGCTTCATTGTCAGCTGAG
AAAGTTACGCTTGACCAAAGGGATTATGAGAAATACAGCGTGAGTTGCCAGGAAGAT
GTGACATGTCCAACGGCAGAGGAAACGTTGCCAATTGAGCTCGCTTTGGAAGCTCGTC
AAC AAAACAAGTATGAAAACTATAGTACTAGCTTCTTCATACGGGAC ATCATCAAACC
AGATCCACCTAAGAATTTGCAGATGAAGCCTCTGAAGAATTCACAAGTCGAGGTATCC
TGGGAATACCCAGATTCATGGTCCACTCCTCATAGTTACTTTAGCCTGAAATTCTTTGT
ACGCATACAGCGGAAGAAGGAGAAAATGAAGGAGACGGAAGAAGGCTGCAATCAGA
A A GGC GCTTTTCTTGTTGA A A A GA CGA GC A C TGAGGTTC A A TGC A A A GGCGGGA A
TGT
ATGTGTTCAAGCCCAAGATAGGTATTATAATAGCTCCTGCTCTAAGTGGGCTTGCGTAC
C ATGCAGAGTTAGAAGTGGCTC AACCTC AGGCTCC GGAAAACCTGGTTCCGGTGAAGG
TTCCACAAAAGGGCGTGTGATTCCTGTGTCCGGCCCAGCTAGGTGTCTCTCCCAGTCAC
GGAATCTCCTGAAAACCACGGATGACATGGTAAAGACAGCTAGGGAGAAACTCAAGC
ACTAC TC CTGCAC AG CT GAG GATATC GATC ATGAGGACATCACC AGGGAC CAGAC ATC
CACTCTGAAAACTTGCCTGCCTTTGGAACTCCACAAGAACGAATCTTGTCTGGCAACG
C GTGAAAC GAGTTC TAC TAC AAGAGGGTC C TGTC TTC CC CC TC AAAAGACAAGCCTTA
TGATGACCTTGTGTCTCGGTAGCATTTATGAGGACCTAAAGATGTATCAAACCGAGTTT
CAGGCTATCAATGCAGCGCTCCAGAATCATAACCATCAGCAGATCATTCTTGACAAAG
GAATGCTCGTGGCCATTGATGAACTAATGCAGAGCCTAAACCACAATGGCGAGACTCT
TCGAC AGA A ACCGCCTGTGGGCGAGGCCGATCC AT ATAGAGTC A A A ATGA A ACTGTGT
ATTCTCCTGCATGCATTTAGTACTCGTGTAGTGACTATTAACAGAGTGATGGGTTACCT

TTCCTCAGCTAATACACTTGTCCTCTTTGGCGCTGGGTTCGGCGCCGTCATAACGGTTG
TTGTCATCGTGGTAATAATCAAGTGCTTTTGCAAGCACAGGTC TTGTTTTCGCAGGAAT
GAAGC C TC TAGAGAAAC AAATAATTC AC TGAC C TTTGGC CC CGAAGAAGC TCTTGC AG
AGCAAACGGTGTTTCTC
mu IL12 (transmembrane form) (SEQ ID NO: 81) MC P QKLTI S WFAIVLLV S P LMAMWELEKDVYVV EVDWTP DAP GETVNLTC DTPEEDDI T
WTSDQRHGVIGS GKTLTITVKEFLDAGQYTCHKGGETL SHSHLLLHKKENGIWSTEILKNF
KNKTFLKCEAPNYSGRFTC SWLVQRNMDLKFNIKS S S S SP D SRAVTC GMASL SAEKVTLD
QRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMK
PLKNSQVEVSWEYPDSWSTPHSYF S LKFFVRI QRKKEKMKETEEGCNQKGAFLV EKT S TE
V Q CKGGNVCV QAQDRYYNS SC SKWACVP CRVRS GS TS GS GKP GS GEGS TKGRVIPVS GP A
RCL SQSRNLLKTTDDMVKTAREKLKHYS C TAEDI DHEDITRD QT S TLKTC LP LELHKNES C
LATRETS S TTRGS C LPP QKTS LMMTL CL GS IYEDLKMYQTEF QAINAAL QNHNHQ QIILDK
GMLVAIDELMQ S LNHNGETLRQKP PVGEADPYRVKMKLC ILLHAF S TRVVTINRVMGYL S
SAN TL VLF GAGF GAVITV V VIV VIIKCFCKHRSCFRRN EASRETNN SLTFGPEEALAEQTVF
mu IL12 (secreted form) (SEQ ID NO: 82) ATGTGTCAGTCACGCTATCTTCTCTTCCTTGCTACTCTGGCCTTGCTCAATCACTTGTCC
CTTGCTCGTGTGATTCCTGTGTCCGGCCCAGCTAGGTGTCTCTCCCAGTCACGGAATCT
C C TGAAAAC CAC GGATGACATGGTAAAGACAGC TAGGGAGAAACTC AAGC,ACTACTC
CTGCACAGCTGAGGATATCGATCATGAGGACATCACCAGGGACCAGACATCCACTCTG
AAAAC TTGC C TGC CTTTGGAAC TC CACAAGAACGAATC TTGTC TGGCAAC GC GTGAAA
C GAGTTCTACTAC AAGAGGGT CC TGTCTTCC CCC TCAAAAGAC AAGC CTTATGATGAC
C TTGTGTCTCGGTAGCATTTATGAGGACCTAAAGATGTATCAAAC CGAGTTTC AGGC TA
TCAATGCAGCGCTCCAGAATCATAACCATCAGCAGATCATTCTTGACAAAGGAATGCT
C GTGGC CATTGATGAAC TAATGCAGAGC C TAAAC CAC AATGGCGAGACTCTTCGACAG
AAAC C GC CTGTGGGC GAGGC C GATC C ATATAGAGTC AAAATGAAACTGTGTATTCTCC
TGCATGCATTTAGTACTCGTGTAGTGACTATTAACAGAGTGATGGGTTACCTTTCCTCA
GC TGGAAGC GGC GC CAC CAAC TTCTC C CTGC TGAAGCAGGCCGGCGACGTGGAGGAG
AAC C C C GGC C C CATGTGC C C ACAGAAACTCACAATTTCTTGGTTC GC AATC GTC C TGCT
GGTGTCACCCCTGATGGCAATGTGGGAGTTGGAAAAGGATGTATACGTCGTCGAGGTC
GAC TGGACAC CTGAC GC TC C GGGTGAAACTGTCAACC TCACTTGCGATACTCCTGAAG
AGGAC GAC ATC AC GTGGAC GAGC GAC C AGC GACATGGAGTGATAGGGTCTGGCAAGA
CGCTTACTATCACGGTTAAGGAATTTCTCGACGCAGGGCAGTACACATGTCACAAGGG
CGGCGAGACTCTGAGCCACTCCCATTTGCTGCTGCACAAGAAGGAGAATGGTATCTGG
TCTAC C GAAATC CTGAAGAATTTTAAGAACAAGACTTTTCTGAAAT GC GAGGC C C C AA
A TT A TTC CGGA CGTTTC A CTTGC A GTTGGCTC GTTC A A A GA A AT A TGGA CTTGA A A
TTT
AACATTAAATCCAGCTCTTCATCTC CTGACAGCAGGGCCGTAACTTGTGGAATGGCTTC
ATTGTC AGC TGAGAAAGTTAC GC TTGAC C AAAGGGATTATGAGAAATAC AGCGTGAGT
TGC C AGGAAGATGTGACATGTC C AAC GGC AGAGGAAAC GTTGC C AATTGAGC TC GC TT
TGGAAGCTCGTCAACAAAACAAGTATGAAAACTATAGTACTAGCTTCTTCATACGGGA
CATCATCAAACCAGATC CAC C TAAGAATTTG CAGATGAAG C C TC TG AAGAATTCACAA
GTC GAGGTATC CTGGGAATAC C C AGATTCATGGT C CAC TC CTCATAGTTACTTTAGC CT
GAAATTC TTTGTAC GC ATAC AGCGGAAGAAGGAGAAAATGAAGGAGAC GGAAGAAGG
CTGCAATCAGAAAGGCGCTTTTCTTGTTGAAAAGACGAGCACTGAGGTTCAATGCAAA
GGCGGGAATGTATGTGTTCAAGCCCAAGATAGGTATTATAATAGCTC CTGCTCTAAGT
GGGCTTGCGTACCATGCAGAGTTAGAAGT
mu IL12 (secreted form) (SEQ ID NO: 83) MCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSC
TAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETS STTRGSCLPPQKTSLMMTLCLGSI
YEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEAD
PYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGSGATNFSLLKQAGDVEENPGPMCPQKL
TISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRH
GVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLK
CEAPNYSGRFTC SWLVQRNMDLKFNIKS S S S SPDSRAVTCGMASLSAEKVTLDQRDYEKY
SVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVE
VSWEYPDSWSTPHSYFSLKFFVRIQRKKEKIVIKETEEGCNQKGAFLVEKTSTEVQCKGGNV
CVQ AQDRYYNSSCSKWACVPCRV RS
mu IFN alpha A2 (SEQ ID NO: 84) ATGGCCAGGCTTTGCGCTTTTCTCGTCATGCTGATCGTCATGAGTTACTGGTCCATTTG
CAGCCTCGGATGTGATCTGCCCCACACCTACAACCTGCGCAACAAACGAGCTCTCAAA
GTGTTGGCCCAAATGAGGCGGTTGCCCTTCCTTTCCTGTCTCAAAGACAGGCAAGATTT
TGGATTTCCACTAGAGAAAGTAGACAATCAACAGATACAGAAAGCTCAAGCTATCCCC
GTGTTGAGGGACTTGACTCAACAGACGTTGAATCTATTTACTAGCAAGGCCAGCTCTG
CTGCTTGGAATGCCACCCTTCTTGACTCATTTTGCAATGACCTACATCAACAACTGAAT
GATCTCCAAACATGTTTGATGCAGCAGGTAGGTGTCCAAGAACCCCCGCTTACTCAGG
AAGACGCCCTTCTGGCTGTCCGCAAGTACTTTCACAGAATCACAGTGTACCTGCGCGA
AAAGAAACACTCCCCCTGCGCTTGGGAAGTGGTCAGGGCCGAGGTTTGGCGAGCCCTG
AGTAGCTCCGTCAATCTCCTTCCTCGGTTGTCCGAGGAGAAAGAG
mu IFN alpha A2 (SEQ ID NO: 85) MARLC AF LVMLIVM S YVV S IC S L GC DLPHTYNLRNKRALKVLAQMRRLPFL S C LKDRQDF
GFP LEKVDNQ QI QKAQAIPV LRDLTQ QTLNLF TS KAS SAAWNATLLDSFCNDLHQQLNDL
QTCLMQQVGVQEPPLTQEDALLAVRKYFHRITVYLREKKHSPCAWEVVRAEVWRALSSS
VNLLPRLSEEKE
mu CD80 (SEQ ID NO: 86) ATGGCTTGC A A CTGTC A GCTC ATGC A A GAT A CTCC CCTGC TT A A GTTTC CCT GCC CT A
G
ACTCATTCTCCTCTTCGTCCTTCTCATTCGCCTAAGCCAGGTGAGTTCCGATGTGGATG
AACAACTGAGTAAATCTGTCAAGGATAAAGTTCTGCTCCCATGCCGCTACAATAGCCC
CCATGAGGACGAGTCCGAAGATAGGATTTACTGGCAGAAACATGATAAGGTGGTGCT
ATCCGTCATTGCCGGTAAATTGAAGGTGTGGCCCGAATATAAGAATAGAACCCTGTAT
GACAACACAACTTATAGCCTAATCATCCTCGGTCTCGTACTGAGCGACCGAGGTACTT
A CTC A TGC GTTGTGC AGA AGA A GGAGCGCGGA AC AT A CGA A GTC A A GC A CCTTGC A
TT
GGTGAAATTGTCAATAAAAGCTGACTTTTCAACTCCTAATATTACTGAATCAGGTAACC
CTTCCGCAGACACTAAAAGAATTACATGCTTCGCCTCTGGCGGGTTTCCCAAACCACG
GTTCTCTTGGCTAGAGAATGGGAGAGAACTTCCAGGTATCAATACAACCATCTCTCAA
GACCCAGAATCAGAACTGTACACCATCTCCAGCCAACTCGATTTCAATACCACAAGAA
ATCATACAATAAAATGTCTGATAAAGTACGGAGATGCACATGTCTCTGAAGATTTCAC
ATGGGAGAAACCACCAGAGGACCCGCCAGACAGCAAGAATACACTTGTCCTCTTTGGC
GCTGGGTTCGGCGCCGTCATAACGGTTGTTGTCATCGTGGTAATAATCAAGTGCTTTTG
CAAGCACAGGTCTTGTITTCGCAGGAATGAAGCCTCTAGAGAAACAAATAATTCACTG
ACCTTTGGCCCCGAAGAAGCTCTTGCAGAGCAAACGGTGTTTCTC
mu CD80 (SEQ ID NO: 87) MACNCQLMQDTP LLKFP CP RLILLFVLLIRL S QV S SDVDEQL SKSVKDKVLLPCRYNSPHE
DE S EDRIYWQKHDKVVL S VIAGKLKVWP EYKNRTLYDNTTY S LIILGLVL S DRGTY S CVV
QKKERGTYEVKHLALVKL S IKADF S TPNITES GNP S ADTKRITC FA S GGFPKPRF SWLENGR
ELPGINTTISQDPESELYTISSQLDFNTTRNHTIKCLIKYGDAHVSEDFTWEKPPEDPPDSKN
TLVLF GAGF GAVITVVVIVVIIKCFCKHRSCFRR NEASRETNNS LTF GP EEALAEQTVFL
mu CD4O-L
(SEQ ID NO: 88) ATGATC GAAACTTATTCC CAACCC TC AC C GC GC TC AGTAGC AAC T GGC CTAC CAGC CA
GCATGAAGATATTCATGTACCTCTTGACTGTATTCTTGATCACGCAAATGATTGGTAGT
GTTTTGTTCGCCGTTTATCTCCACAGGCGCCTGGATAAAGTTGAAGAAGAGGTTAATCT
CCATGAAGACTTCGTGTTCATTAAGAAACTC AAAAGATGTAACAAAGGTGAGGGAT CT
CTGTCTCTTCTGAACTGTGAGGAGATGCGACGGCAATTCGAGGACC TCGTAAAAGACA
TAACTCTCAACAAAGAAGAGAAGAAAGAAAACTCTTTCGAGATGCAACGGGGCGACG
AGGAC CCTCAAATTGCC GCACATGTC GTTTCTGAAGC GAATTC CAATGC C GC GTC C GT
GC TC CAGTGGGC GAAGAAGGGATAC TACAC GATGAAGAGCAAC C TTGTGATGCTTGA
AAATGGCAAGCAGCTCACAGTTAAACGCGAGGGACTCTACTATGTATACACCCAAGTG
ACCTTTTGTTCCAACCGGGAGCCAAGTAGCCAACGCCCGTTCATCGTTGGGCTGTGGCT
C AAGC C TTCTTCAGGGAGTGAAC GAATC CTTC TCAAGGCAGC CAAC AC GCATTCCAGC
AGC CAAC TGTGTGAGC AACAATC C GTGC ATC TT GGC GGGGTC TTTGAGC TGC AAGC GG
GCGCCTCTGTGTTCGTGA ATGTT A C CGA A GCC A GCC A GGTTATC C A CCGCGTGGGTTTC
AGTAGTTTTGGCCTGCTCAAGCTG
mu CD4O-L
(SEQ ID NO: 89) MI ETY S QP S P RSV ATGLPAS MKIF MYLLTVFLITQ MI GSVLFAVYLHRRLDKVEEEVNLHED
FVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAA
HVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNRE
PS SQRPFIVGLWLKP S S GSERILLKAANTHS S SQLCEQQSVHLGGVFELQAGASVFVNVTEA
SQVIHRVGFS SF GLLKL
mu IL21 (SEQ ID NO: 90) ATGGAGCGTACTCTGGTCTGCCTTGTTGTGATATTCTTGGGGACAGTTGC ACACAAATC
ATC A C CC C A A GGA CC GGATA GA CTC CTC AT A CGC CTGCGCC ATCTGATTGAC ATTGTC
GAGCAGTTGAAGATTTATGAGAAC GAC CTGGAC C CTGAACTATTGAGC GC GC C TCAAG
AC GTCAAAGGGCATTGC GAGCATGCTGCATTTGC ATGTTTTC AGAAAGCTAAGC TC AA
AC CAAGTAATC C C GGTAAC AATAAAACATTCATCATC GAC CTGGTGGC CC AACTAAGA
CGCCGGTTGCCGGCGCGCCGGGGTGGTAAGAAACAGAAACATATTGCTAAATGCCCCT
CTTGCGACTCTTACGAGAAAAGGACACCTAAGGAATTCCTCGAACGATTGAAATGGTT
GTTGCAGA AGATGATCC ATC A ACATCTGAGC
mu IL21 (SEQ ID NO: 91) MERTL V CL V VIFLGTVAHKS S P Q GP DRLLIRLRHLIDI VEQLKIY EN DLDPELL S AP Q DVKG

HCEHAAFACFQKAKLKP SNP GNNKTF IIDLVAQLRRRLPARRG GKKQK

mu CCL21 (SEQ ID NO: 92) ATGGCACAAATGATGACACTGTCCCTACTTAGTCTAGTTCTAGCTTTGTGTATTCCCTG
GACTCAAGGCAGTGACGGAGGAGGACAAGACTGCTGCCTCAAATATTCTCAAAAGAA
AATCCCTTATTCTATAGTCCGAGGTTACCGTAAGCAAGAACCGAGTCTAGGTTGTCCTA
TCCCCGCAATCCTCTTTCTACCACGGAAACATAGCAAACCAGAATTGTGCGCCAACCC

AGAAGAGGGTTGGGTCCAAAATTTGATGAGGCGCCTTGAC CAACC ACC GGCC CCGGGT
AAACAATCACCGGGGTGTCGGAAGAATAGGGGTACATCCAAATCCGGGAAGAAAGGG
AAGGGGAGTAAGGGC TGTAAGAGAACGGAACAAACTC AACCTAGC AGAGGT
mu CCL21 (SEQ ID NO: 93) MA QMMTL SLL SLVL AL CIPWTQ GSDGGGQDCCLKYS QKKIPYSIVRGYRK QEP SLGCPIP A
ILFLPRKH S KPEL C ANPEEGWV QNLMRRLD QP P AP GKQ S P GC RKNRGT S KS GKKGKGS KG
CKRTEQTQPSRG
anti-mu CD3 scFv-transmembrane (SEQ ID NO: 94) ATGGAAACCGACACATTGCTC CTCTGGGTTCTCCTTC TATGGGTC C C C GGTT C C AC C GG
AGATATCCAAATGACACAATCACCCAGCAGCCTGCCTGCCTCTCTGGGCGACCGCGTT
ACCATCAATTGTCAAGCTTCCCAAGATATAAGTAATTATCTCAACTGGTACCAGCAAA
AGCCCGGTAAAGCGCCTAAATTGCTGATTTATTATACTAATAAACTCGCAGATGGAGT
TCCTAGTAGATITTCTGGTTCAGGGAGTGGACGGGACTCCAGTTTTACCATATCAAGTC
TGGAATCCGAGGATATCGGCAGCTACTATTGCCAGCAATATTATAATTACCCTTGGACT
TTTGGACCCGGGACTAAACTTGAGATCAAAAGAGGCGGAGGAGGCAGTGGTGGTGGT
GGATCAGGC GGCGGTGGTAGTGAGGTAC AACTCGTGGAATC AGGCGGC GGAC TGGTC
C A A CCC GGC A A GA GCCTT A A A CTCTCTTGTGA GGCC A GTGGATTTAC A TTC A GCGGTT
ATGGAATGCACTGGGTGAGAC AAGCTCCCGGCAGGGGCCTAGAATCAGTGGCGTAC A
TCACCAGCTCATCAATAAACATTAAATACGCTGATGCAGTCAAGGGCCGGTTTACTGT
ATCCCGCGACAACGCTAAGAATCTTCTCTTTCTGCAAATGAACATACTTAAGAGCGAG
GATACTGCCATGTATTATTGTGCCC GCTTCGATTGGGATAAGAATTATTGGGGACAAG
GCACCATGGTTACCGTTAGTAGTCCAAACATC AC ATCAAATAATAGCAAC C C C GTGGA
AGGGGACGACTCTGTTTCACTCACCTGTGATTCCTATACCGATCCTGATAATATCAACT
ATCTATGGTC TC GTAACGGTGAAAGTC TC AGCGAAGGCGAC CGGTTGAAACTC TC C GA
AGGTAACAGAACCCTTACGCTTCTGAACGTCACCCGGAACGATACCGGGCCCTATGTT
TGCGAAAC TAGGAACC CTGTTAGC GTGAATCGTAGCGACCC TTTCTC CCTAAATAATA
CTCTAGTGCTATTCGGAGCGGGATTCGGTGCCGTCATCACAGTAGTCGTTATTGTAGTC
ATTATTAAATGCTTTTGTAAACATAGGTCTTGCTTCAGAAGAAATGAGGCCAGCCGTG
AAACTAATAATTCCCTGACCTTTGGGCCCGAAGAAGCTTTGGCTGAACAGACTGTGTTT
CTC
anti-mu CD3 scFv -transmembrane (SEQ ID NO: 95) METDTLLLWVLLLWVP GS TGDIQMTQ SP S SLP AS LGDRVTINCQAS QDI SNYLNWYQQKP
GKAPKLLIYYTNKLADGVP SRFS GS GS GRD S SFTIS S LE S EDI GSYYC Q QYYNYPWTF GP GT
KLEIKRGGGGS GGGGS GGGGS EV QLVE S GGGLV QP GKSLKL SCEASGFTFSGYGMHWVR
Q AP GR GLESV AYITS SSINIKY AD AVK GRFTV SR DNAKNI .I.FI ,QMNII.K SFDT A MYYC
AR F
DWDKNYVVGQGTMVTVS SPNITSNNSNPVEGDDSVSLTCDSYTDPDNINYLWSRNGESL SE
GDRLKLSEGNRTLTLLNVTRNDTGPYVCETRNPVSVNRSDPFSLNNTLVLFGAGFGAVITV
VVIVVIIKCF CKHRS C FRRNEAS RETNN S LTF GPEEALAEQTVF L
mu TSLP
(SEQ ID NO: 96) ATGGTTC TTCTC AGGAGCCTC TTCATCC TGC AAGTAC TAGTACGGATGGGGCTAACTTA
CAACTTTTCTAACTGCAACTTCACGTCAATTACGAAAATATATTGTAACATAATTTTTC
ATGACCTGACTGGAGATTTGAAAGGGGCTAAGTTCGAGCAAATCGAGGACTGTGAGA
GC AAGC C AGCTTGTCTC C TGAAAATC GAGTAC TATAC TC TC AATC CTATC C C TGGCTGC
CCTTCACTCCCCGACAAAACATTTGCCCGGAGAACAAGAGAAGCCCTCAATGACCACT
GC C CAGGC TAC C CTGAAAC TGAGAGAAATGACGGTACTCAGGAAATGGCACAAGAAG

TCCAAAACATCTGCCTGAATCAAACCTCACAAATTCTAAGATTGTGGTATTCCTTCATG
CAATCTCCAGAA
mu TSLP
(SEQ ID NO: 97) MVLLRSLFILQVLVRMGLTYNFSNCNFTSITKIYCNIIFHDLTGDLKGAKFEQIEDCESKPAC
LLKIEYYTLNPIPGCPSLPDKTFARRTREALNDHCPGYPETERNDGTQEMAQEVQNICLNQ
TSQILRLWYSFMQ SPE
mu GM-CSF
(SEQ ID NO: 98) ATGTGGCTGCAGAATTTACTTTTC CTGGGCATTGTGGTCTACAGCCTCTC AGCAC C C AC
CCGCTCACCCATC AC TGTC AC C C GGC C TTGGAAGCATGTAGAGGC CAT CAAAGAAGC C
CTGAACCTCCTGGATGACATGCCTGTCACGTTGAATGAAGAGGTAGAAGTC GTC TC TA
ACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCA
GGGTCTAC GGGGCAATTTCAC CAAACTCAAGGGC GC C TTGAACATGACAGC CAGC TAC
TACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCT
ATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAA
AAACCAGGCCAAAAA
mu GM-CSF
(SEQ ID NO: 99) MWLQNLLFLGIVVYSL SAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEF
SFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFID
SLKTFLTDIPFECKKPGQK
mu IFN gamma (SEQ ID NO: 100) ATGAACGCTACACACTGCATCTTGGCTTTGCAGCTCTTCCTCATGGCTGTTTCTGGCTG
TTAC TGC C AC GGC AC AGTC ATTGAAAGCCTAGAAAGTCTGAATAACTATTTTAAC TCA
AGTGGCATAGATGTGGAAGAAAAGAGTCTCTTCTTGGATATCTGGAGGAAC TGGC AAA
AGGATGGTGACATGAAAATCC TGCAGAGCCAGATTATCTCTTTCTAC CTCAGACTCTTT
GAAGTCTTGAAAGACAATCAGGCCATCAGCAACAACATAAGCGTCATTGAATC ACACC
TGATTACTACCTTCTTCAGCAACAGCAAGGCGAAAAAGGATGCATTCATGAGTATTGC
C A AGTTTGAGGTC A AC A AC CC A C A GGTCC AGCGCC A AGC ATTC A ATGAGCTC ATCCGA
GTGGTCCACCAGCTGTTGCCGGAATCCAGCCTCAGGAAGCGGAAAAGGAGTCGCTGC
mu IFN gamma (SEQ ID NO: 101) MNATHCILALQLFLMAV S GCYCHGTVIES LE S LNNYFN S SGIDVEEKSLFLDIWRNWQKDG
DMK II ,QSQIISFYI ,RI ,FEVI ,KDNQ AI SNNI SVIESHI ITTFFSNSK A KK AF MS I AK
FEVNNP Q
V QRQAFNELIRVVHQLLPE S SLRKRKRS RC
mu IL7 (SEQ ID NO: 102) ATGTTCCATGTTTCTTTTAGATATATCTTTGGAATTCCTCCACTGATCCTTGTTCTGCTG
CCTGTCACATCATCTGAGTGCCACATTAAAGACAAAGAAGGTAAAGCATATGAGAGTG
TACTGATGATC AGCATCGATGAATTGGACAAAATGAC AGGAACTGATAGTAATTGCCC
GAATAATGAACCAAACTTTTTTAGAAAACATGTATGTGATGATACAAAGGAAGCTGCT
TTTCTAAATCGTGCTGCTCGCAAGTTGAAGCAATTTCTTAAAATGAATATCAGTGAAGA
ATTCAATGTCCACTTACTAACAGTATCACAAGGCACACAAACACTGGTGAACTGCACA
AGTAAGGAAGAAAAAAACGTAAAGGAACAGAAAAAGAATGATGCATGTTTCCTAAAG
AGACTACTGAGAGAAATAAAAACTTGTTGGAATAAAATTTTGAAGGGCAGTATA

mu IL7 (SEQ ID NO: 103) MFFIVSFRYIEGIPPLILVLLPVTS SECHIKDKEGKAYESVLMISIDELDKMTGTDSNCPNNEP
NFERKHVCDDTKEAAFLNRAARKLKQELKMNISEEFNVHLLTVSQGTQTLVNCTSKEEKN
VKEQKKNDACFLKRLLREIKTCWNKILKGSI
mu ICOS-L
(SEQ ID NO: 104) ATGCAGCTAAAGTGTC C CTGTTTTGTGTC CTTGGGAACCAGGCAGCCTGTTTGGAAGA
AGC TC CATGTTTCTAGCGGGTTCTTTTC TGGTC TTGGTC TGTTCTTGCTGCTGTTGAGC A
GC CTC TGTGC TGC CTCTGCAGAGACTGAAGTCGGTGCAATGGTGGGCAGCAATGTGGT
GC TC A GC TGC ATTGAC C C C C AC AGAC GC CATTTCAAC TTGAGTGGTCTGTATGTC TATT
GGCAAATC GAAAACC CAGAAGTTTCGGTGACTTACTAC CTGC CTTAC AAGTCTCCAGG
GATCAATGTGGACAGTTCCTACAAGAACAGGGGC CATCTGTC CCTGGACTC CATGAAG
CAGGGTAACTICTCTCTGTACCTGAAGAATGTCACCCCTCAGGATACCCAGGAGTICA
CATGCCGGGTATTTATGAATACAGCCACAGAGTTAGTCAAGATCTTGGAAGAGGTGGT
CAGGCTGCGTGTGGCAGCAAACTTCAGTACACCTGTCATCAGCACCTCTGATAGCTCC
AAC C C GGGC CAGGAAC GTAC C TAC AC C TGC ATGTCC AAGAATGGCTAC C C AGAGC C CA
AC C TGTATTGGATCAAC AC AAC GGAC AATAGC CTAATAGAC AC GGCTCTGC AGAATAA
C ACTGTCTACTTGA AC A A GTTGGGCCTGT ATGATGT A ATC AGCAC A TT A AGGCTCCCTT
GGACATCTCGTGGGGATGTTCTGTGCTGCGTAGAGAATGTGGCTCTCCACCAGAACAT
C ACTA GCATTAGCCAGGC AGAAAGTTTC ACTGGAAATAAC ACAAAGAACCC ACAGGA
AAC C C AC AATAATGAGTTAAAAGTC C TTGTCCC CGTCC TTGCTGTACTGGCGGCAGCG
GC ATTC GTTTC CTTCATCATATACAGAC GCAC GC GTC C C CAC CGAAGCTATACAGGACC
CAAGACTGTACAGCTTGAACTTACAGACCACGCC
mu ICOS-L
(SEQ ID NO: 105) MQLKCPC FVSLGTRQ PVWKKLHVS S GEE SGLGLFLLLL S SLCAASAETEV GAMV GSNVVL
SC IDPHRRHFNLS GLYVYWQIENPEVS V TYYLPYKSP GINVD S SYKNRGHL SLDSMKQGNF
SLYLKNVTP QDTQEFTCRVFMNTATELVKILEEVVRLRVAANF S TPVI S T SD S SNP GQERTY
TCMSKNGYPEPNLYWINTTDNSLIDTALQNNTVYLNKLGLYDVISTLRLPWTSRGDVLCC
VEN ALHQN1TS1S QAESFTGNNTKNPQETHNN ELKVLVP VLAVLAAAAF SEllY RRTRPH
RSYTGPKTVQLELTD HA
mu CD47 (SEQ ID NO: 106) ATGTGGCCCTTGGCGGCGGCGCTGTTGCTGGGCTCCTGCTGCTGCGGTTCAGCTCAACT
AC TGTTTAGTAAC GTCAACTCCATAGAGTTCACTTCATGC AATGAAACTGTGGTCATC C
CTTGC A TC GTC C GT A ATGTGGA GGC GC A A AGC ACC GA A GA A ATGTTTGTGA A GTGGA
A
GTTGAACAAATCGTATATTTTCATC TATGATGGAAATAAAAATAGCACTACTACAGAT
C AAAACTTTAC CAGTGC AAAAATC TC AGTC TC AGACTTAATC AATGGC ATTGCC TCTTT
GAAAATGGATAAGCGCGATGCCATGGTGGGAAACTACACTTGCGAAGTGACAGAGTT
ATCCAGAGAAGGCAAAACAGTTATAGAGCTGAAAAACCGCACGGTTTCGTGGTTTTCT
C CAAATGAAAAGATCCTCATTGTTATTTTC CCAATTTTGGCTATACTCC TGTTCTGGGG
AAAGTTTGGTATTTTAACACTCAAATATAAATCCAGCCATAC GAATAAGAGAATCATT
C TGC TGCTC GTTGC C GGGC TGGTGC TC AC AGTC ATC GTGGTTGTTGGAGC CATCCTTCT
CATCCCAGGAGAAAAGCCCGTGAAGAATGCTTCTGGACTTGGCCTCATTGTAATCTCT
AC GGGGATATTAATAC TAC TTC AGTACAATGTGTTTATGAC AGCTTTTGGAATGACC TC
TTTC ACCATTGCC ATATTGATC AC TCAAGTGCTGGGCTAC GTCC TTGC TTTGGTC GGGC
TGTGTCTCTGC A TC ATGGC ATGTGAGCC A GTGC A CGGC CCC C TTTTGATTT C A GGTTTG

GGGATCATAGCTCTAGCAGAACTACTTGGATTAGTTTATATGAAGTTTGTCGCTTCCAA
CCAGAGGACTATCCAACCTCCTAGGAATAGG
mu CD47 (SEQ ID NO: 107) MWPLAAALLLGS CCCGSAQLLFSNVNS IEFTSCNETVVIPCIVRNVEAQSTEEMFVKWKLN
KSYIFIYDGNKNSTTTDQNFTS AKISVSDLINGI A SLKMDKRD AMVGNYTCEVTEL SREGK
TVIELKNRTV SWF S PNEKILIVIFPILAILLFWGKF GILTLKYKS SHTNKRIILLLVAGLVLTVI
VVVGAILLIPGEKPVKNASGLGLIVISTGILILLQYNVFMTAFGMTSFTIAILITQVLGYVLAL
VGLCLCIMACEPVHGPLLISGLGIIALAELLGLVYMKFVASNQRTIQPPRNR
Mu Sarcoglycan alpha:
(SEQ ID NO: 108) ATGGCAGCAGCAGTAACTTGGATACCTCTCCTGGCAGGTCTCCTGGCAGGACTGAGGG
ACACCAAGGCCCAGCAGACAACTTTACACCTACTTGTGGGTCGTGTGTTTGTGCATCCT
TTGGAACATGCCACCTTCCTGCGCCTTCCAGAACACGTTGCGGTGCCACCCACTGTCCG
ACTCACCTACCACGCTCACCTCCAGGGACATCCAGACCTGCCCAGGTGGCTGCACTAC
ACACAGCGCAGTCCCTATAAC CCTGGCTTCCTCTACGGCTCCCCCACTCCAGAAGATC G
TGGGTAC CAAGTCATC GAGGT CACAGC CTAC AATC GAGAC AGTTTTGAC AC CAC TAGA
CAGAGGCTGCTGCTGCTGATTGGGGAC CCC GAAGGTC C CCGGTTGC CATACCAAGCTG
A GTTCCTGGTGCGC A GCC A TGATGTGGA GGA GGTGC TGC CC A CC A C A CCT GCC A A CCG
CTTCCTCACCGCCTTGGGGGGACTGTGGGAGCCAGGAGAGCTTCAGCTGCTCAACATC
ACTTCCGCCTTGGACCGGGGAGGCCGAGTCCCTCTTCCTATTGAGGGACGGAAGGAAG
GGGTATAC ATTAAGGTAGGC TC TGC CAC AC C C TTCTC C AC C TGC CTGAAGATGGTGGC
GTCGC CC GACAGCTATGCCC GTTGTGCCCAGGGACAGC CTCCACTACTGTCC TGCTAC G
ACACTTTGGCACCCCACTTCCGCGTTGACTGGTGCAATGTGTCTCTGGTAGACAAGTCA
GTACC CGAGCCCCTGGATGAGGTACCTACTC CAGGCGATGGGATCTTGGAGCACGACC
C GTTC TTC TGCC C AC C C AC TGAAGC C AC AGAC CGAGACTTCCTGACAGATGCCTTGGTG
ACCCTCTTGGTGCCTTTGTTGGTGGCTCTGCTGCTTACTCTGTTGCTGGCTTACATCATG
TGC TTTC GGC GTGAAGGAC GGC TGAAGAGAGAC ATGGC C AC C TC TGAC ATCC AGATGT
TTCACCACTGTTCCATCCATGGGAATACAGAAGAGCTTCGGCAGATGGCAGCCAGCCG
AGAGGTGC C C C GGC C TC TTTC C AC C TTGC CCATGTTTAATGTTCGTACAGGAGAGCGGT
TACCTCCCCGAGTAGACAGCGCACAGATGCCTCTTATCCTGGACCAGCAC
Mu Sarcoglycan alpha:
(SEQ ID NO: 109) MAAAVTWIPLLAGLLAGLRDTKAQ QTTLHLLV GRVFVHPLEHATFLRLPEHVAVPP TVRL
TYHAHLQGHPDLPRWLHYTQRSPYNPGFLYGSPTPEDRGYQVIEVTAYNRDSFDTTRQRL
LLLIGDPEGPRLPYQAEFLVRSHDVEEVLPTTPANRFLTALGGLWEPGELQLLNITSALDRG
GRVPLP IEGRKEGVYIKV GS ATPF S TC LKMVAS PD SYARCAQ GQPPLL SCYDTLAPHFRVD
WCNV SI NDK SVPEPI ,DEVPTPGDGII THDPFFCPPTE A TDR DFI ,TD AI ,VTLI NMI ,V ALI
,T
LLLAYIMCFRREGRLKRD MATS DI QMFHHC S IHGNTEELRQMAAS REVPRP L STLPMFNVR
TGERLPPRVDSAQM PLILDQH
Mu FGF10 (SEQ ID NO: 110) ATGTGGAAATGGATACTGACACATTGTGCCTCAGC CTTTC C CC AC CTGC C GGGC TGCTG
TTGC TGC TTC TTGTTGC TC TTTTTGGTGTC TTC GTTC C C TGTC AC C TGC C AAGC TC TTGGT

CAGGACATGGTGTCACAGGAGGC CAC C AACTGC TCTTCTTCCTCC TC GTCCTTCTCC TC
TCCTTCCAGTGC GGGAAGGC ATGTGC GGAGC TACAATC AC CTC CAAGGAGATGTC C GC
TGGAGAAGGCTGTTCTCCTTCACCAAGTACTTTCTCACGATTGAGAAGAACGGCAAGG
TC A GC GGGA CC A A GA ATGA A GA CTGTCCGTA C A GTGTCC TGGA GAT A A C ATC A
GTGGA
AATCGGAGTTGTTGCCGTCAAAGCCATCAACAGCAACTATTACTTAGC CATGAACAAG

AAGGGGAAACTCTATGGCTCAAAAGAGTTTAACAACGACTGTAAGCTGAAAGAGAGA
ATAGAGGAAAATGGATACAACACCTATGCATCTTTTAACTGGCAGCACAATGGCAGGC
AAATGTATGTGGCATTGAATGGAAAAGGAGCTCCCAGGAGAGGACAAAAAACAAGAA
GGAAAAACACCTCTGCTCACTTCCTCCCCATGACGATCCAAACA
Mu FGF10 (SEQ ID NO: 111) MWKWILTHCASAFPHLPGCCCCFLLLFLVSSFPVTCQALGQDMVSQEATNCSSSSSSFSSPS
SAGRHVRSYNHLQGDVRWRRLFSFTKYFLTIEKNGKVSGTKNEDCPYSVLEITSVEIGVVA
VKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVAL
NGKGAPRRGQKTRRKNTSAHFLPMTIQT
Mu Agrin (SEQ ID NO: 112) ATGCCTCCTCTGCCACTGGAACACAGACCCAGGCAGCAGCCTGGTGCCTCCGTGCTGG
TTCGGTACTTCATGATCCCCTGCAACATCTGCTTGATCCTCTTGGCTACTTCTACGTTGG
GCTTTGCGGTGCTGCTTTTCCTCAGCAACTACAAACCTGGGATCCACTTCACAGCAGCG
CCTTCTATGCCTCCTGATGTGTGCAGGGGAATGTTATGTGGCTTTGGTGCTGTGTGTGA
ACCTAGTGTTGAGGATCCAGGCCGGGCCTCCTGTGTGTGCAAGAAGAATGTCTGCCCT
GCTATGGTAGCTCCTGTGTGTGGCTCAGATGCTTCCACCTATAGCAACGAGTGTGAGCT
ACAGCGTGCACAGTGCAACCAGCAACGGCGCATCCGCCTACTCCGCCAAGGGCCATGT
GGGTCCCGGGACCCCTGTGCCAATGTGACCTGCAGTTTCGGTAGTACCTGTGTACCTTC
AGCCGATGGACAGACCGCCTCGTGTCTGTGTCCTACAACCTGCTTTGGGGCCCCTGATG
GCACAGTGTGTGGCAGTGATGGTGTTGACTACCCTAGTGAGTGCCAGCTGCTCCGTCA
TGCCTGTGCCAACCAGGAGCACATCTTTAAGAAGTTCGATGGTCCTTGTGACCCCTGCC
AGGGCAGCATGTCAGACCTGAATCATATTTGCCGGGTGAACCCACGTACACGGCACCC
AGAAATGCTTCTGCGGCCTGAGAACTGCCCCGCCCAACACACACCTATCTGTGGAGAT
GATGGGGTCACCTATGAAAACGACTGTGTCATGAGCCGTATAGGTGCAGCCCGTGGCC
TGCTTCTCCAGAAAGTGCGCTCTGGTCAATGCCAGACTCGAGACCAGTGCCCGGAGAC
CTGCCAGTTTAACTCTGTATGCCTGTCCCGCCGCGGCCGTCCCCACTGTTCCTGCGATC
GCGTCACCTGTGATGGGGCTTACAGGCCAGTGTGTGCCCAGGATGGGCACACGTATGA
CAATGACTGTTGGCGCCAACAGGCCGAGTGTCGACAACAGCAGACCATTCCCCCCAAG
CACCAGGGCCCGTGTGACCAGACCCCATCCCCGTGCCGTGGAGCGCAGTGTGCATTTG
GGGCAACATGCACAGTGAAGAATGGGAAAGCTGTGTGCGAGTGCCAGCGGGTGTGCT
CGGGCGGCTACGATCCTGTGTGCGGCAGTGATGGTGTCACTTACGGCAGTGTGTGCGA
GCTGGAATCCATGGCCTGTACCCTTGGGCGGGAAATCCGAGTGGCCCGCAGAGGACCG
TGTGACCGATGTGGGCAGTGC CGGTTTGGATCC TTGTGCGAGGTGGAGAC TGGAC GC T
GTGTGTGCCCCTCTGAGTGTGTGGAGTCAGCCCAGCCCGTATGTGGCTCTGACGGACA
CACATATGCTAGTGAATGTGAGCTGCATGTCCACGCCTGTACACACCAGATCAGCCTA
TACGTGGCCTCAGCCGGACACTGCCAGACCTGTGGAGAAACAGTTTGTACCTTTGGGG
CTGTGTGCTC A GCTGGA C A GTGTGT ATGTC CCC GTTGTGAGC A CCCCCC A C CTGGC C CT
GTGTGCGGCAGTGATGGCGTCACCTACCTCAGTGCCTGTGAGCTCCGAGAGGCTGCCT
GTCAGCAGCAGGTACAAATTGAGGAGGCCCGTGCAGGGCCGTGTGAGCCGGCTGAGT
GTGGCTCAGGGGGCTCTGGGTCTGGGGAAGACAATGCGTGTGAGCAGGAGCTGTGTCG
GCAGCATGGTGGTGTCTGGGATGAGGACTCAGAAGACGGGCCGTGTGTCTGTGACTTT
AGTTGCCAGAGTGTCCTTAAAAGCCCAGTGTGTGGCTCAGATGGAGTCACCTATAGCA
CGGAGTGCCATCTGAAGAAGGCCAGATGTGAAGCGCGGCAAGAGCTGTACGTCGCTG
CTCAGGGAGCCTGCCGGGGCCCTACCTTGGCTCCACTGCTACCTATGGCCTCCCCACAC
TGTGCCCAAACCCCCTATGGCTGCTGCCAGGACAATGTCACTGCTGCCCAGGGTGTGG
GCTTGGCTGGCTGTCCCAGCACCTGCCATTGCAACCCACACGGCTCCTATAGCGGCACT
TGTGACCCAGTCACAGGGCAGTGCTCCTGCCGACCAGGTGTAGGAGGCCTCAGGTGTG
ATCGCTGTGAGCCTGGCTTCTGGAACTTCCGTGGCATTGTCACCGATGGACATAGTGGT
TGCACTCCCTGCAGCTGTGACCCTCGGGGTGCTGTAAGAGATGACTGTGAGCAGATGA

CTGGATTGTGTTCCTGTAGACCTGGTGTGGCTGGTCCCAAGTGTGGGCAGTGTCCAGAT
GGTCAAGC CCTGGGCC ATCTTGGCTGTGAAGC AGATCCCACAAC AC CAGTGACTTGTG
TGGAAATGC AC TGTGAGTTTGGC GC C TC C TGC GTAGAGGAGGC TGGTTTTGC C C AGTG
TGTCTGCCCAACTCTCACATGTCCAGAGGCTAACTCTACCAAGGTCTGTGGATCAGATG
GTGTCACATACGGCAATGAATGCCAGCTGAAGACCATTGCCTGCCGCCAGCGTCTGGA
C ATC TC CATTCAGAGTCTTGGTCC ATGC CGGGAGAGTGTTGCTCCTGGGGTTTC CCC TA
C ATCTGC ATCTATGACC A CCC C A AGGC ATATCCTGAGC AGGAC ACTGGC GTCTCC CC A
C AGCAGCC TTCC TCTGTC TC CCAGC ACTACTGC CC ATGATTGGCCC ACC CCATTACC CA
CATCACCTCAGACCGTAGTCGGCACCCCCAGGAGCAC TGCAGCCACACCCTCTGATGT
GGC CAGTCTTGCTAC AGC GATCTTCAGGGAATC TGGCAGCAC CAACGGC AGTGGC GAT
GAGGAGCTCAGTGGCGATGAGGAGGCCAGTGGGGGCGGGTCTGGGGGACTTGAGCCC
C C GGT GGGC AGCGTTGTGGTGACC CAC GGGCCAC C CATC GAGAGGGCTTCCTGTTACA
AC TC ACCTTTGGGCTGCTGCTCAGATGGCAAGACACC CTCACTGGACTCAGAAGGC TC
CAACTGTCCAGCTACCAAGGCATTCCAGGGCGTGCTGGAGCTTGAGGGGGTCGAGGGA
CAGGAACTGTTCTACACACCAGAGATGGCTGACCCCAAGTCAGAGTTGTTTGGGGAGA
C TGC AAGGAGCATTGAGAGC AC GCTGGACGAC CTGTTC CGGAATTCGGATGTTAAGAA
GGACTTCTGGAGCATCCGCCTACGGGAACTGGGGCCTGGCAAATTAGTCCGTGCCATT
GTGGATGTTCACTTTGACCCCACCACAGCCTTCCAGGCACC AGATGTGGGTCAGGCCTT
GC TC CAACAGATCCAGGTATCC AGGC CGTGGGCCCTGGC AGTGAGGAGGCC TCTGC GG
GAGC ATGTGC GATTC TT GGAC TTTGAC TGGTTTC C C AC TTTTTTTAC GGGAGC TGC AAC
AGGA ACC AC A GCTGCTGTGGCC AC AGCC A GAGCC ACC ACTGTGAGCCGACTGTCTGCC
TCTTCTGTCACCCCACGAGTCTACCCCAGTTACACCAGCCGGCCTGTTGGCAGAACTAC
GGCACCGCTAACCACTCGCCGGCCACCAACCACTACCGCCAGTATTGACCGACCTCGG
ACTCCAGGCCCGCAACGGCCCCCAAAGTCCTGTGATTCCCAGCCTTGCCTCCACGGAG
GTACCTGCCAGGACCTGGATTCTGGCAAGGGTTTCAGCTGCAGCTGTACTGCAGGCAG
GGC TGGCACTGTCTGTGAGAAAGTGCAGCTCC CC TCTGTGC CAGCTTTTAAGGGCC ACT
CCTTCTTGGCCTTCCCCACCCTCCGAGCCTACCACACGCTGCGTCTGGCACTAGAATTC
C GGGCGC TGGAGAC AGAGGGAC TGC TGC TC TAC AATGGC AATGC AC GTGGC AAAGAT
TTCCTGGCTCTGGCTCTGTTGGATGGTCATGTACAGTTCAGGTTCGACACGGGCTCAGG
GC C GGC GGTGC TAAC AAGC TTAGTGC C AGTGGAAC C GGGAC GGTGGC AC C GC C TC GA
GTTGTCAC GGCATTGGC GGCAGGGC AC ACTTTCTGTGGATGGC GAGGC T CC TGTTGTA
GGTGAAAGTCCGAGTGGCACTGATGGCCTCAACTTGGACACGAAGCTCTATGTGGGTG
GTCTCCC AGAAGAACAAGTTGC CAC GGTGCTTGAT CGGAC CTCTGTG G GCATCGGCCT
GAAAGGATGCATTCGTATGTTGGACATCAACAACCAGCAGCTGGAGCTGAGCGATTGG
C AGAGGGCTGTGGTTC A A AGCTC TGGTGTGGGGGA ATGC GGAGAC C ATCCC TGC TC AC
CTAACCCCTGCCATGGCGGGGCCCTCTGCCAGGCCCTGGAGGCTGGCGTGTTCCTCTGT
C AGTGC C C AC C TGGC C GCTTTGGC C C AAC TTGTGC AGATGAAAAGAAC CC C TGC C AAC
C GAACCC CTGCCACGGGTC AGC CC CCTGC CATGTGCTTTCCAGGGGTGGGGCCAAGTG
TGCGTGCCCCCTGGGACGCAGTGGTTCCTTCTGTGAGACAGTCCTGGAGAATGCTGGC
TCCCGGCCCTTCCTGGCTGACTTTAATGGCTTCTCCTACCTGGAACTGAAAGGCTTGCA
C A CCTTCGA GA GA GA CCT A GGGGA GA A GATGGCGCTGGA GATGGTGTTCTTGGCTCGT
GGGCCCAGTGGCTTACTCCTCTACAATGGGCAGAAGACGGATGGCAAGGGGGACTTTG
TATCCCTGGCCCTGC ATAAC C GGC AC C TAGAGTTC C GC TATGAC C TTGGC AAGGGGGC
TGCAATCATCAGGAGCAAAGAGCCCATAGCCCTGGGCACCTGGGTTAGGGTATTCCTG
GAACGAAATGGCCGCAAGGGTGCCCTTCAAGTGGGTGATGGGCCCCGTGTGCTAGGG
GAATCTCCGAAATCCCGCAAGGTCCCGCACACCATGCTCAACCTCAAGGAGCCCCTCT
ATGTGGGGGGAGCTCCTGACTTCAGCAAGCTGGCTCGGGGCGCTGCAGTGGCCTCCGG
CTTTGATGGTGCCATCCAGCTGGTGTCTCTAAGAGGCCATCAGCTGCTGACTCAGGAG
C ATGTGTTGCGGGCAGTAGATGTAGCGCCTTTTGC AGGC CAC CC TTGTACC CAGGCCGT
GGACAACCCCTGCCTTAATGGGGGCTCCTGTATCCCGAGGGAAGCCACTTATGAGTGC
C TGTGTCC TGGGGGC TTCTCTGGGCTGC ACTGCGAGAAGGGGATAGTT GAGAAGTC AG
TGGGGGACCTAGA A AC ACTGGCCTTTGATGGGCGGACCTAC ATCGAGTACCTC A ATGC
TGTGACTGAGAGC GAGCTGACCAATGAGATC CCAGC C CC CGAAACTCTGGATTC CCGG

GCCCTTTTCAGTGAGAAAGCGCTGCAGAGCAACCACTTTGAGCTGAGCTTACGCACTG
AGGCCACGCAGGGGCTGGTGCTGTGGATTGGAAAGGTTGGAGAACGTGCAGACTACA
TGGCTCTGGCCATTGTGGATGGGCACCTACAACTGAGCTATGACCTAGGCTCCCAGCC
AGTTGTGCTGCGCTCCACTGTGAAGGTCAACACCAACCGCTGGCTTCGAGTCAGGGCT
CACAGGGAGCACAGGGAAGGTTCCCTTCAGGTGGGCAATGAAGCCCCTGTGACTGGCT
CTTCCCCGCTGGGTGCCACACAATTGGACACAGATGGAGCCCTGTGGCTTGGAGGCCT
ACAGAAGCTTCCTGTGGGGCAGGCTCTCCCCAAGGCCTATGGCACGGGTTTTGTGGGC
TGTCTGCGGGACGTGGTAGTGGGCCATCGCCAGCTGCATCTGCTGGAGGACGCTGTCA
CCAAACCAGAGCTAAGACCCTGCCCCACTCTCTGA
Mu Agrin (SEQ ID NO: 113) MPPLPLEHRPRQQPGASVLVRYFMIPCNICLILLATSTLGFAVLLELSNYKPGIHETAAPSMP
PDVCRGMLCGFGAVCEPSVEDPGRASCVCKKNVCPAMVAPVCGSDASTYSNECELQRAQ
CNQQRRIRLLRQGPCGSRDPCANVTCSEGSTCVPSADGQTASCLCPTTCFGAPDGTVCGSD
GVDYPSECQLLRHACANQEHIEKKEDGPCDPCQGSMSDLNHICRVNPRTRHPEMLLRPEN
CPAQHTPICGDDGVTYENDCVMSRIGAARGLLLQKVRSGQCQTRDQCPETCQFN SVCLSR
RGRPHCSCDRVTCDGAYRPVCAQDGHTYDNDCWRQQAECRQQQTIPPKHQGPCDQTPSP
CRGAQCAFGATCTVKNGKAVCECQRVCSGGYDPVCGSDGVTYGSVCELESMACTLGREI
RVARRGPCDRCGQCREGSLCEVETGRCVCPSECVESAQPVCGSDGHTYASECELHVHACT

AACQQQVQIEEARAGPCEPAECGSGGSGSGEDNACEQELCRQHGGVWDEDSEDGPCVCD
FSCQSVLKSPVCGSDGVTYSTECHLKKARCEARQELYVAAQGACRGPTLAPLLPMASPHC
AQTPYGCCQDNVTAAQGVGLAGCPSTCHCNPHGSYSGTCDPVTGQCSCRPGVGGLRCDR
CEPGEWNERGIVTDGHSGCTPCSCDPRGAVRDDCEQMTGLCSCRPGVAGPKCGQCPDGQ
ALGHLGCEADPTTPVTCVEMHCEFGASCVEEAGFAQCVCPTLTCPEANSTKVCGSDGVTY
GNECQLKTIACRQRLDISIQSLGPCRESVAPGVSPTSASMTTPRHILSRTLASPHSSLPLSPST
TAHDWPTPLPTSPQTVVGTPRSTAATPSDVASLATAIFRESGSTNGSGDEELSGDEEASGGG
SGGLEPPVGSVVVTHGPPIERASCYNSPLGCCSDGKTPSLDSEGSNCPATKAFQGVLELEGV
EGQELFYTPEMADPKSELFGETARSIESTLDDLERNSDVKKDEWSIRLRELGPGKLVRAIVD
VHFDPTTAFQAPDVGQALLQQIQVSRPWALAVRRPLREHVRELDFDWEPTETTGAATGTT
AAVATARATTVSRL SAS SVTPRVYP SYTSRPVGRTTAPLTTRRPPTTTASIDRPRTPGPQRPP
KSCDSQPCLHGGTCQDLDSGKGESCSCTAGRAGTVCEKVQLPSVPAFKGHSFLAFPTLRAY
HTLRLALEFRALETEGLLLYNGNARGKDELALALLDGHVQEREDTGSGPAVLTSLVPVEPG
RWHRLELSRHWRQGTLSVDGEAPVVGESPSGTDGLNLDTKLYVGGLPEEQVATVLDRTS
V GI GLKGC IRMLDINNQ QLEL SDWQRAVVQS S GV GE C GDHP C S PNP CHGGAL C QAL EA GV

FL C QCPPGRFGPTCADEKNPCQPNPCHGS AP CHVL SRGGAKC ACPL GRSGSFCETVL ENAG
S RPFADFNGF S YLELKGLHTFERDL GEKMALEMV FLARGP S GLLLYNGQKTD GKGDF V S L
ALHNRHLEF RYDL GKGAAII RS KEPIALGTWVRVFLERNGRKGAL QV GD GPRVL GE S PKS R
KVPHTMLNLKEPLYVGGAPDFSKLARGAAVASGFDGAIQLV SLRGHQLLTQEHVLRAVD
V APF AGHPCTQ AVDNPCT AIGGS CIPRE A TY-EC T ,CP GGFS GI ,HCEK GIVEK SVGDI ,FTI
,AFD
GRTYIEYLNAVTE S ELTNEIP APETLD S RALF S EKAL Q SNHFELSLRTEATQGLVLWIGKVG
ERADYMALAIVDGHLQL SYDLGSQPVVLRSTVKVNTNRWLRVRAHREHREGSLQVGNEA
PVTGS SPLGATQLDTDGALWLGGLQKLPVGQALPKAYGTGFVGCLRDVVVGHRQLHLLE
DAV TKPELRP CPTL
Mu IL10 (SEQ ID NO: 114) ATGCCTGGCTCAGCACTGCTATGCTGCCTGCTCTTACTGACTGGCATGAGGATCAGCAG
GGGCCAGTACAGCCGGGAAGACAATAACTGCACCCACTTCCCAGTCGGCCAGAGCCA
CATGCTCCTAGAGCTGCGGACTGCCTTCAGCCAGGTGAAGACTTTCTTTCAAACAAAG
GACCAGCTGGACAACATACTGCTAACCGACTCCTTAATGCAGGACTTTAAGGGTTACT
TGGGTTGCCAAGCCTTATCGGAAATGATCCAGTTTTACCTGGTAGAAGTGATGCCCCA

GGCAGAGAAGCATGGCCCAGAAATCAAGGAGCATTTGAATTCCCTGGGTGAGAAGCT
GAAGACCCTCAGGATGCGGCTGAGGCGCTGTCATCGATTTCTCCCCTGTGAAAATAAG
AGCAAGGCAGTGGAGCAGGTGAAGAGTGATTTTAATAAGCTCCAAGACCAAGGTGTC
TACAAGGCCATGAATGAATTTGACATCTTCATCAACTGCATAGAAGCATACATGATGA
TCAAAATGAAAAGCTAA
Mu IL10 (SEQ ID NO: HS) MPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQ
LDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLR
MRLRRCHRFLP CENKSKAVEQVKSDFNKLQDQ GVYKAMNEFDIFINCIEAYMMIKMKS
Mu MYDGF (C19orf10) (SEQ ID NO: 116) ATGGCAGCCCCCAGCGGAGGCTTCTGGACTGCGGTGGTCCTGGCGGCCGCAGCGCTGA
AATTGGCCGCCGCTGTGTCCGAGCCCACCACCGTGCCATTTGACGTGAGGCCCGGAGG
GGTCGTGCATTCGTTCTCCCAGGACGTAGGACCCGGGAACAAGTTTACATGTACATTC
ACCTACGCTTCCCAAGGAGGGACCAACGAGCAATGGCAGATGAGCCTGGGGACAAGT
GAAGACAGCCAGCACTTTACCTGTACCATCTGGAGCTCCCCAGGGGAAATCCTACCTCT
AC TTC AC AC AGTTCAAGGC TGAGTTGC GAGGTGC TGAGATC GAGTATGC C ATGGC C TA
CTCC A A AGCCGCATTTGAGAGAGAGAGTGATGTCCCCCTGA A A AGTGAGGAGTTTGA A
GTGACCAAGACAGCAGTGTCTCACAGGCCTGGGGCCTTCAAAGCTGAGCTCTCCAAGC
TGGTGATCGTAGCCAAGGCGGCACGCTCGGAGCTGTGA
Mu MYDGF (C19orf10) (SEQ ID NO: 117) MAAPSGGFWTAVVLAAAALKLAAAVSEPTTVPFDVRPGrGVVHSF S QDV GP GNKF TC TFT
YASQGGTNEQWQMSLGTSEDSQHFTCTIWRPQGKSYLYFTQFKAELRGAEIEYAMAYSK
AAFERESDVP LKSEEFEVTKTAV SHRP GAF KAEL SKLVIVAKAARS EL
pWF-521 (SEQ ID NO: 118) C AGTC TGTGTTGACGC AGCC GCCC TCAGTGTC TGC GGCCC C AGGACAGAGGGTCACC A
TCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAACAC
CTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGGGA
TTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGA
C TC C AGAC TGGGGAC GAGGC C GATTATTAC TGCGGC AC ATGGGATTAC ACC C TGAATG
GTGTGGTGTTC GGCGGAGGGACCAAGCTGACC GTC CTAGGTCAGCCC AAGGC CAAC CC
CACTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACTA
GTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCAGATG
GCAGCCCCGTCAAGGCGGGAGTGGAGACC ACCA A ACCCTCCAA ACAGAGCA ACAACA
AGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAA
GC TAC AGC TGC C AGGTC AC GC ATGAAGGGAGC AC C GTGGAGAAGAC AGTGGC C C C TA
CAGAATGTTCAGGCGCCGGATCTGGTGGAAACTGGAGTCATCCCCAATTCGAGAAGGG
CGGAAGCGGTGGGAGTGGCGGGTCCGGTGGAAGCAACTGGTCACACCCACAATTCGA
GAAAGGCGGTTCTGGCGGATCTGGTGGATCTGGC GGAAGTAACTGGTCTCATCCTCAA
TTC GAAAAGGGCGGAAGCGGTGGCGGC AGGC TAGGTGGAGGCTC AGTGCAGGTGC AG
CTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGG
GAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCACATACTATGCA
GACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAACACGCTGTATC
TGC A A ATGA AC AGCCTGAGAGC CGAGGAC ACGGCCGTATATTACTGTGCGCGC ACTTC
TTACCTGAACCATGGTGATTACTGGGGTCAAGGTACTCTGGTGACCGTGTCTAGCGCCT

CCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG
C AC AGCGGC CCTGGGCTGC CTGGTCAAGGAC TAC TTCC CC GAACCGGTGAC GGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA
GAC CTACATCTGCAACGTGAACC ACAAGCC CAGCAAC ACC AAGGTGGAC AAGAGAGT
GGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTG
CTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCC A AGCCC A AGGACACCCTGATGATC A
GC AGGACCC CCGAGGTGACCTGC GTGGTGGTGGAC GTGAGC CAC GAGGACCCAGAGG
TGAAGTTC AAC TGGTAC GTGGAC GGC GTGGAGGTGC AC AAC GC C AAGAC C AAGC C CA
GAGAGGAGCAGTACAAC AGCACCTACAGGGTGGTGTCC GTGC TGACCGTGC TGC ACC A
GGACTGGC TGAAC GGCAAGGAATAC AAGTGC AAGGTCTC C AACAAGGC CCTGCC AGC
C C CCATC GAAAAGACC ATC AGCAAGGC CAAGGGC CAGCC ACGGGAGCC CC AGGTGTA
CACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTG
GTGAAGGGCTTC TAC CCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGC CAGC CC
GAGAACAAC TAC AAGACCAC CCC CC CAGTGCTGGACAGC GACGGC AGC TTCTTCCTGT
AC AGCAAGCTGAC CGTGGACAAGTCCAGGTGGCAGC AGGGCAACGTGTTC AGCTGCA
GC GTGATGCAC GAGGC CC TGC ACAACC AC TAC ACC CAGAAGAGCCTGAGCC TGTC CCC
CGAGCTGCAACTGGAGGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGACGGGCT
GTGGACGACC ATC ACC ATC TTCATC ACACTCTTCC TGTTAAGCGTGTGC TAC AGTGC CA
C CGTC AC C TTC TTC AAGGTGAAGTGGATC TTC TC C TC GGTGGTGGAC C TGAAGC AGAC C
ATCATCCCCGACTACAGGAACATGATCGGACAGGGGGCCTGA
pWF-521 (SEQ ID NO: 119) Q S VLTQP P S V SAAP GQRVTI S C S GTRS N IGSDY V S WY QHLP GTAPKLL V Y GDN LRP
SGIPDR
FSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGQPKANPTVTLF
PPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYL
SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSGAGSGGNWSHPQFEKGGSGGSGGSGG
SNWSHPQFEKGGSGGSGGSGGSNWSHPQFEKGGSGGGRLGGGSVQVQLVESGGGLVQPG
GSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSK_ NTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
V TC V V V DV SHEDPEVKFN WY VDGVEVHN AKTKPREEQ Y N STYRV VS V LTVLHQDWLN G
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSATVTFFKVKWIFSSVVDL
KQTIIPDYRNMIGQGA
pWF-533 (SF() ID NO: 120) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCACCA
TC TC C TGC TC TGGAAC C AGGTC C AAC ATT GGGAGTGATTATGTTTC C TGGTAC C AAC AC
CTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGGGA
TTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGA
CTCCAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAATG
GTGTGGTGTTCGGCGGAGGGACCAAGCTGACC GTC CTAtcttcaGCCTC CACC AAGGGC CC
ATCGGTCTTCCC CC TGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC CCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCG
CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC
CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCA
ACGTGA ACCAC A AGCCC AGC A AC ACC A AGGTGGACA AGAGAGTGGAGCCCAAGAGCT
GC

pWF-533 (SEQ ID NO: 121) QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWWQHLPGTAPKLLVYGDNLRPSGIPDR
FSASKSGTSATLGITGLQTGDEADYYCGTVVDYTLNGVVFGGGTKLTVLSSASTKGPSVFPL
APS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVP
SS SLGTQTYICNVNHKPSNTKVDKRVEPKSC
pWF-534 (SEQ ID NO: 122) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAC
TCTCCTGTGCAGCCTCTGGATTC ACC TTTAGCAGCTATGCCATGAGCTGGGTCCGCCAG
GC TCCAGGGAAGGGGCTGGAGTGGGTC TCAGTTATTTATAGCGGTGGTAGTAGCACAT
ACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAACAC
GCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCG
CGCACTTCTTACCTGAACCATGGTGATTACTGGGGTCAAGGTACTCTGGTGACCGTGTC
TAGCGCCTCCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA
AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA
GTACAGTGGAAGGTGGATAACGC CCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC AG
AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC
GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGCCCCCCC
TGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAA
GGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGC
CACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAAC
GCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTG
CTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCA
ACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGC AAGGCCAAGGGCCAGCC AC
GGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGG
TGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGA
GAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGA
CGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGC
AACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGA
GCCTGAGCCTGTCCCCCGAGCTGCAACTGGAGGAGAGCTGTGCGGAGGCGCAGGACG
GGGA GCTGGA CGGGC TGTGGA CGA C C ATC A C C ATCTTC ATC A C A C TC TTCC TGTT A
AG
CGTGTGCTACAGTGCCACCGTCACCTTCTTCAAGGTGAAGTGGATCTTCTCCTCGGTGG
TGGACCTGAAGCAGACCATCATCCCCGACTACAGGAACATGATCGGACAGGGGGCCT
GA
pWF-534 (SEQ ID NO- 123) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYY
ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARTSYLNHGDYWGQGTLVTVSSA
SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECDKTHTCPPCPAPELLG
GP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPP SREE
MTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSC SVMHEALHNHYTQKSLSLS PELQLEES CAEAQDGELDGLWTTITIFITLFLLSV
CYSATVTFFKVKWIFSSVVDLKQTIIPDYRNMIGQGA
mu IL15 (SEQ ID NO: 124) ATGAAAATTTTGAAACCATATATGAGGAATACATCCATCTCGTGCTACTTGTGTTTCCT
TCTAAACAGTCACTTTTTAACTGAGGCTGGCATTCATGTCTTCATTTTGGGCTGTGTCA
GTGTAGGTCTC CCTAAAACAGAGGC CAACTGGATAGATGTAAGATATGACCTGGAGAA
AATTGAAAGCCTTATTCAATCTATTCATATTGACACCACTTTATACACTGACAGTGACT
TTCATCCCAGTTGCAAAGTTACTGCAATGAACTGCTTTCTCCTGGAATTGCAGGTTATT
TTACATGAGTACAGTAACATGACTCTTAATGAAACAGTAAGAAACGTGCTCTACCTTG
C A A AC AGC A CTCTGTCTTCT A AC A AGA ATGT AGC A GA ATCTGGCTGCAAGGA ATGTGA
GGAGC TGGAGGAGAAAAC C TTC AC AGAGTTTTTGC AAAGC TTTATAC GCATTGTC CAA
ATGTTC ATC AAC AC GTC C
mu IL15 (SEQ ID NO: 125) MK ILKPYMRNTS I S CYLCFLLN SHF LTEAGIHVF IL GCV S V GLP KTEANWIDVRYDLEKIE SL
I Q S IHIDTTLYTD S DFHP S CKVTAMN CFL L EL QVILHEY SNMTLNETV RNVLYLAN S TL S
SN
KNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS
anti-human GPC3 CAR (79a) (SEQ ID NO: 126) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCACCA
TCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAACAC
CTC CCAGGAACAGCCC CCAAACTC CTCGTTTATGGCGATAATCTGCGAC CCTCAGGGA
TTCCTGACC GATTCTCTGCCTCC A AGTCTGGC AC GTC AGC C A CCCTGGGC ATC ACC GGA
CTCCAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAATG
GTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTC CTAGGTTCTAGAGGTGGTGGTGG
TAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGC,TG
GTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG
CCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGC TGGAGTGGGTC TC AGTTATTTATAGC GGTGGTAGTAGC AC ATAC TATGCAGAC
TCC GTGAAGGGC C GGTTC AC C ATC TC C AGAGATAATTC C AAGAAC AC GC TGTATC TGC
AAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCACTTCTTA
C C TGAAC C ATGGTGATTAC TGGGGTC AAGGTACT C TGGTGAC C GTGTC TAGC GC C OCT
GCAGTGGTCCCCGTGCTGCAGAAAGTTAATAGCACCACCACTAAACCTGTCCTGAGGACTCC
TAGTCCAGTGCACCCAACAGGGACCAGTCAGCCACAGAGACCGGAAGACTGCAGACC,4AGA
GGTTCAGTGAAGGGAACCGGCCTGGATTTCGCCTGCGATTTTTGGGCCCTGGTCGTCGTC
GC AGGAGTTTTGTTTTGC TATGGACTGC TC GTCAC AGTT GCTTTGTGTGTTATCTGGAC
AAGGAAACGGTGGCAAAATGAGAAGTTTGGGGTGGACATGCCAGATGACTATGAAGATGAA
AATCTCTATGAGGGCCTGAACCTTGATGACTGTTCTATGTATGAGGACATCTCCAGGGGACT
CCAGGGC,4CCT,4CC4GGATGTGGGCAACCTCC4C,4TTGGAGATGCCCAGCTGGAA,4AGCC,4 TGA
anti-human GPC3 CAR (79a) (SEQ ID NO: 127) QSVLTQPPSVSAAPGQRVTTSCSGTRSNTGSDYVSWYQHLPGTAPKLLVYGDNT ,RP SGIPDR
FSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGSRGGGGSGGG
GS GGGGS LEMAQV QLVE S GGGLV QP GGS LRL S C AAS GF TF S SYAMSWVRQAPGKGLEWV
SVIYSGGS STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYW
GQGTL V TV S S AAA VVPVLOKVNS177KPVLR1P5PVHP1G1SOPORPLDC'RPRGSVKG1'GLDF
ACDFWALVVVAGVLFCYGLLVTVALCVIWTRKR WQNEKFGVDMPDDYEDENLYEGLNLD
DCSMYEDISRGLQGTYQDVGNLHIGDAQLEKP
anti-human PSMA(XENP14484) CAR 79a (SEQ ID NO: 128) GAGGTTCAACTTGTTCAATCTGGGGCAGAAGTGAAGAAGCCCGGGGCATCTGTGAAAG
TATCATGCAAAACATCCGGCTATACGTTTACCGAATACACCATTCACTGGGTCAGACA
GGCTC CC GGTC A A AGCCTC GA A TGGATGGGA A ATATTA ACCCTA AC A ATGGCGGA AC C
ACATATAATCAGAAATTCCAAGGCCGAGTGACGATAACTGTCGATAAGAGTACGTCCA

CAGCTTACATGGAACTCAGCTCTTTGAGATCCGAAGACACTGCAGTTTATTATTGTGCA
GCTGGATGGAACTTCGACTATTGGGGACAAGGGACTCTTGTTACGGTGTCCAGTGGCA
AAC CAGGTAGTGGTAAACC CGGAAGCGGCAAGCC CGGGAGCGGTAAACCTGGTAGCG
ACATC GTCATGACTCAAAGCCCTGACTCAC TC GC C GTGAGC C TGGGAGAGC GTGCAAC
GCTATCTTGTCGGGCCTCTCAGGATGTCGGAACTGCTGTAGACTGGTATCAACAGAAA
C C TGAC C AATC AC CAAAAC TC CTGATTTATTGGGC CTCAAC ACGTCACACAGGAGTGC
C A GATA GGTTC AC AGGTAGTGGC A GTGGA AC TGA TTTT ACTTTGAC A A TTA GC AGC CT
GC AAGC C GAAGATGTAGC CGTTTACTTCTGTC AAC AATATAACTCATACCC AC TAAC G
TTCGGTGCCGGGACGAAGGT AGAGATT AAAGTGGTCCCCGTGCTGCAGAAAGTTAATAG
CACCACCACTAAACCTGTCC TGAGGACTCCTAGTCCAGTGCACCCAACAGGGACCAGTCAG
CCACAGAGACCGGAAGACTGCAGACCAAGAGG I
_________________________________________________ I
CAGTGAAGGG,4ACCGGCCTGGATTTCG
CCTGCGA TITTTGGGCCCTGGTCGTCGTCGCAGGAGTTTTGTTTTGCTATGGACTGCTCG
TCACAGTTGCTTTGTGTGTTATCTGGACAAGGAAACGGTGGCAAAA TGAGAAGTTTGGGG
TGGACATGCCAGATGACTATG,4AGATGAAAATCTCTATGAGGGCCTG,4ACCTTG,4TGACTGTT
CTATGTATGAGGACATCTCCAGGGGACTCCAGGGCACCTACCAGGATGTGGGC,4ACCTCCA
CATTGGAGATGCCCAGCTGGA,4AAGCCATGA
anti-human PSMA(XENP14484) CAR 79a (SEQ ID NO: 129) EV QLV Q S GAEVKKP GAS VKV SCKTSGYTFTEYTIHWVRQAPGQSLEWMGNINPNNGGTT
YNQKF Q GRVTITVD KS T S TAYMEL S SLRSEDTAVYYCAAGWNFDYWGQGTLVTVS SGKP
GS GKP GS GKP GS GK P GSDIVMTQ SP D SL AV S LGER ATL SCR AS QDVGTAVDWYQQKPDQS
PKLLIYWAS TRHTGVPDRF TGS GS GTDFTLTI S SLQAEDVAVYFCQQYNSYPLTFGAGTKV
EIKVVP VLOKVNSTTTKPVL1?TPSPVHPTGTSOPORPEDCRPRGSVKGTGLDFACDFW ALVVV
AGVLFCYGLLVTVALCVIWTRKR WQNEKFG VDMPDDYEDENL YEGLNLDDCSMYEDLSRGL
QGIT QD VGNLHIGDAQLEKP
mouse IL 12a-mouse IgG2a Fc (SEQ ID NO: 130) ATGTGTCAGTCACGCTATCTTCTCTTCCTTGCTACTCTGGCCTTGCTCAATCACTTGTCC
CTTGCTCGTGTGATTCCTGTGTCCGGCCCAGCTAGGTGTCTCTCCCAGTCACGGAATCT
C C TGAAAAC C AC GGATGAC ATGGTAAAGAC AGC TAGGGAGAAACTC AAGC AC TAC TC
CTGCACAGCTGAGGATATCGATCATGAGGACATCACCAGGGACCAGACATCCACTCTG
AAAAC TTGC C TGC CTTTGGAAC TC CACAAGAACGAATC TTGTC TGGCAAC GC GTGAAA
CGAGTTCTACTACAAGAGGGTCCTGTCTTCCCCCTCAAAAGACAAGCCTTATGATGAC
CTTGTGTCTCGGTAGCATTTATGAGGACCTAAAGATGTATCAAACCGAGTTTCAGGCTA
TC A A TGC A GCGCTCC AGA ATC AT A ACC ATC A GC A GATC A TTCTTGAC A A A GGA
ATGCT
CGTGGCCATTGATGAAC TAATGCAGAGC C TAAAC CAC AATGGCGAGACTCTTCGACAG
AAAC C GC CTGTGGGC GAGGCCGATCC ATATAGAGTC AAAATGAAAC TGTGTATTCTCC
TGCATGCATTTAGTACTCGTGTAGTGACTATTAACAGAGTGATGGGTTACCTTTCCTCA
GCTCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACC
TCTTGGGTGGACCATCC GTCTTCATC TTCCCTCCAAAGATCAAGGATGTACTCATGATC
TCC CTGA GCCC C A T A GT C AC A TGTGTGGTGGTGGATGTGA GCGA GGA TGA C CC A GA TG
TCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCA
TAGAGAGGATTACAACAGTAC TC TC CGGGTGGTC AGTGC CCTCCC CATC C AGC AC C AG
GACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCG
CCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATG
TCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGT
CACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGA
GC TAAAC TAC AAGAAC AC TGAAC C AGTCCTGGAC TCTGATGGTTCTTACTTCATGTAC A
GCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAG
TGGTC CAC GAGGGTCTGCAC AATCACCACACGACTAAGAGCTTCTC CC GGACTC C GGG
TAAATAG
mouse IL 12a-mouse IgG2a Fc (SEQ ID NO: 131) MCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSC
TAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETS STTRGSCLPPQKTSLMMTLCLGSI
YEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQ SLNHNGETLRQKPPVGEAD
PYRVKMKLCILLHAFSTRVVTINRVMGYLS SAPRGPTIKPCPP CKCPAPNLLGGP SVFIFPPK
IKDVLMISL SPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS ALP
IQHQDWMS GKEFKCKVNNKDLP APIERTISKPKGSVR APQVYVLPPPEEEMTKKQVTLTC
MVTDEMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSVFMYSKLRVEKKNWVERNSYSCS
VVHEGLHNHHTTKSFSRTP GK*
mouse IL 12b-mouse IgG2a Fc (SEQ ID NO: 132) ATGTGCCCACAGAAACTCACAATTTCTTGGTTCGCAATCGTCCTGCTGGTGTCACCCCT
GATGGCAATGTGGGAGTTGGAAAAGGATGTATACGTCGTC GAGGTC GACTGGAC ACC T
GACGCTCCGGGTGAAACTGTCAACCTCACTTGC GATACTCCTGAAGAGGAC GA CATCA
CGTGGACGAGCGACCAGCGACATGGAGTGATAGGGTCTGGCAAGACGCTTACTATCAC
GGTTAAGGAATTTCTCGACGCAGGGCAGTACACATGTCACAAGGGCGGCGAGACTCTG
AGCCACTCCCATTTGCTGCTGCACAAGAAGGAGAATGGTATCTGGTCTACCGAAATCC
TGAAGAATTTTAAGAACAAGACTTTTCTGAAATGCGAGGCCCCAAATTATTCCGGACG
TTTCACTTGCAGTTGGCTCGTTCAAAGAAATATGGACTTGAAATTTAACATTAAATCCA
GC TC TTCATCTCC TGACAGCAGGGC CGTAAC TTGTGGAATGGCTTC ATTGTCAGC TGAG
AAAGTTACGCTTGACCAAAGGGATTATGAGAAATACAGCGTGAGTTGCCAGGAAGAT
GTGACATGTCCAACGGCAGAGGAAACGTTGCCAATTGAGCTCGCTTTGGAAGCTCGTC
AACAAAACAAGTATGAAAACTATAGTACTAGCTTCTTCATACGGGACATCATCAAACC
AGATCCACCTAAGAATTTGCAGATGAAGCCTCTGAAGAATTCACAAGTCGAGGTATCC
TGGGAATACCCAGATTCATGGTCCACTCC TCATAGTTACTTTAGCCTGAAATTCTTTGT
ACGCATACAGCGGAAGAAGGAGAAAATGAAGGAGACGGAAGAAGGCTGCAATCAGA
AAGGC GCTTTTCTTGTTGAAAAGACGAGCACTGAGGTTCAATGCAAAGGCGGGAATGT
ATGTGTTCAAGCCC AAGATAGGTATTATAATAGCTCC TGC TC TAAGTGGGCTTGCGTAC
CATGCAGAGTTAGAAGTCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATG
CCCAGC AC C TAAC CTCTTGGGTGGAC CATC CGTCTTCATCTTCC CTCCAAAGATCAAGG
ATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGA
GGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCT
CAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCC
CCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACA
AAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAG
CTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCAC
TCTGACC TGC ATGGTC AC AGACTTC ATGC CTGAAGACATTTAC GTGGAGTGGACC AAC
AAC GGGAAAAC AGAGCTAAAC TACAAGAAC AC TGAAC CAGTC C TGGAC TCTGATGGT
TCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAAT
AGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCT
TCTCCCGGACTCCGGGTA A ATAG
mouse IL 12b-mouse IgG2a Fc (SEQ ID NO: 133) MCP QKLTI S WFAIVLLV S P LMAMWELEKDVYVV EVDWTP DAP GETVNLTC DTPEEDDI T
W TS DQ RHGVIGS GKTLTITV KEFLD AGQY TCHKGGETL SHSHLLLHKKEN GIW S TEILKN F
KNKTFLKCEAPNYS GRFTC SWLVQRNMDLKFNIKS S S S SP D SRAVTCGMASL SAEKVTLD
QRDYEKYS V S CQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMK
PLKNSQVEVSWEYPDSWSTPHSYF SLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTE
V Q CKGGNVCV QAQDRYYNS SCSKWACVPCRVRSPRGPTIKP CPP CKCPAPNLLGGP SVFIF
PPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS
AL PI QHQDWMS GKEFKCKVNNKDLP AP IERTI S KPKGSV RAP QVYVLPPP EEEMTKKQV TL
TCMVTDEMPEDIYVEW'TNNGKTELNYKNTEPVLDSDGSVFMYSKLRVEKKNWVERNSYS
C SVVHEGLHNHHTTKSFSRTPGK

mouse IL 12a-mouse IgG2a Fc (Silent) (SEQ ID NO: 134) ATGTGTCAGTCACGCTATCTTCTCTTCCTTGCTACTCTGGCCTTGCTCAATCACTTGTCC
CTTGCTCGTGTGATTCCTGTGTCCGGCCCAGCTAGGTGTCTCTCCCAGTCACGGAATCT
CCTGAAAACCACGGATGACATGGTAAAGACAGCTAGGGAGAAACTCAAGCACTACTC
CTGCACAGCTGAGGATATCGATCATGAGGACATCACCAGGGACCAGACATCCACTCTG
AAAACTTGCCTGCCTTTGGAACTCCACAAGAACGAATCTTGTCTGGCAACGCGTGAAA
C GAGTTC TAC TAC AAGAGGGT C C TGTC TTC CC CC TC AAAAGACAAGCCTTATGATGAC
CTTGTGTCTCGGTAGCATTTATGAGGACCTAAAGATGTATCAAAC CGAGTTTC AGGC TA
TCAATGCAGCGCTCCAGAATCATAACCATCAGCAGATCATTCTTGACAAAGGAATGCT
C GTGGC CATTGATGAAC TAATGCAGAGC C TAAAC CAC AATGGCGAGACTCTTCGACAG
AAAC C GC CTGTGGGC GAGGC C GATC C ATATAGAGTC AAAATGAAACTGTGTATTCTCC
TGCATGCATTTAGTACTCGTGTAGTGACTATTAACAGAGTGATGGGTTACCTTTCCTCA
GC TC C CAGAGGGC C CAC AATCAAGC C CTGTC CTC CATGCAAATGC C CAGC AC CTAAC G
CTGCCGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATC
TCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGAC CCAGATG
TCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCA
TAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCC CATC CAGCACC AG
GACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCGGAGCG
C CCATC GAGAGAACCATCTC AAAACC CAAAGGGTC AGTAAGAGCTC C AC AGGTATAT G
TCTTGCCTCC ACC AGA AGA A GA GA TGACT A AGA A AC A GGTC A CTCTGACC TGC ATGGT
CACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGA
GC TAAACTACAAGAACACTGAACC AGTC CTGGAC TCTGATGGTTCTTACTTCATGTAC A
GC AAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTC C TGTTCAG
TGGTC CAC GAGGGTCTGCAC AATCAC C ACAC GACTAAGAGCTTC TC CC GGACTC C GGG
TAAATGA
mouse IL 12a-mouse IgG2a Fc (Silent) (SEQ ID NO: 135) MCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSC
TAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETS STTRGSCLPPQKTSLMMTLCLGSI
YEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQ SLNHNGETLRQKPPVGEAD
PYRVKMKLCILLHAFSTRVVTINRVMGYLS S APRGPTIKP CPP C KCPAPNAAGGP S VFIF PP
KIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSAL
PIQHQDWMSGKEFKCKVN NKDL GAPIERTISKPKGS VRAPQVY LPPP EEEMTKKQ TLT
CMVTDFMPEDIYVEW'TNNGKTELNYKNTEPVLD S D GSYFMYS K LRVEKKNWVERN SY S C
SVVHEGLHNHHTTKSFSRTP GK*
mouse IL 12b-mouse IgG2a Fc (Silent) (SEQ ID NO: 136) ATGTGCCCACAGAAACTCACAATTTCTTGGTTCGCAATCGTCCTGCTGGTGTCACCCCT
GATGGCAATGTGGGAGTTGGAAAAGGATGTATACGTCGTCGAGGTCGACTGGACACCT
GA C GCTCCGGGTGA A A CTGTC A A C CTC A CTTGC GA T A CTC CTGA AGAGGAC GA C A TC
A
CGTGGACGAGCGACCAGCGACATGGAGTGATAGGGTCTGGCAAGACGCTTACTATCAC
GGTTAAGGAATTTC TC GAC GC AGGGC AGTAC AC ATGTC AC AAGGGC GGC GAGAC TC TG
AGCCACTCCCATTTGCTGCTGCACAAGAAGGAGAATGGTATCTGGTCTACCGAAATCC
TGAAGAATTTTAAGAACAAGACTTTTCTGAAATGCGAGGCCCCAAATTATTCCGGACG
TTTCACTTGCAGTTGGCTCGTTCAAAGAAATATGGACTTGAAATTTAACATTAAATCCA
GCTCTTCATCTCCTGACAGCAGGGCCGTAACTTGTGGAATGGCTTCATTGTCAGCTGAG
AAAGTTAC GC TTGAC CAAAGGGATTATGAGAAATACAGCGTGAGTTGCCAGGAAGAT
GTGACATGTCCAACGGCAGAGGAAACGTTGCCAATTGAGCTCGCTTTGGAAGCTCGTC
AACAAAACAAGTATGAAAACTATAGTACTAGCTTCTTCATACGGGACATCATCAAACC
AGATCCACCTAAGAATTTGCAGATGAAGCCTCTGAAGAATTCACAAGTCGAGGTATCC
TGGGA ATAC CC A GATTC ATGGTCC ACTCCTC AT A GTT ACTTT A GCC TGA A ATTCTTTGT
ACGCATACAGCGGAAGAAGGAGAAAATGAAGGAGACGGAAGAAGGCTGCAATCAGA

AAGGCGCTTTTCTTGTTGAAAAGACGAGCACTGAGGTTCAATGCAAAGGCGGGAATGT
ATGTGTTCAAGCCCAAGATAGGTATTATAATAGCTCCTGCTCTAAGTGGGCTTGCGTAC
CATGCAGAGTTAGAAGTCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATG
CCCAGCACCTAACGCTGCCGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGG
ATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGA
GGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCT
CAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCC
CCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACA
AAGACCTCGGAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAG
CTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCAC
TCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAAC
AACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGT
TCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAAT
AGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCT
TCTCCCGGACTCCGGGTAAATGA
mouse IL 12b-mouse IgG2a Fc (Silent) (SEQ ID NO: 137) MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDIT
WTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNF
KNKTFLKCEAPNYSGRFTC SWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLD
QRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMK
PLKNSQVEVSWEYPDSWSTPHSYF SLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTE
VQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSPRGPTIKPCPPCKCPAPNAAGGPSVFI
FPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVV
SALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGS VRAPQVYVLPPPEEEMTKKQV
TLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS
YSCSVVHEGLHNHHTTKSFSRTPGK
CD3 zeta cytoplasmic domain-human (SEQ ID NO. 13g) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGL
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CD3 zeta cytoplasmic domain-human (SEQ ID NO: 139) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG
CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC
GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAA
GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGG
ATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT
ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
pWF-506 (SEQ ID NO: 140) METDTLLLWVLLLWVPGSTGQSVLTQPP SVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLP
GTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVF
GGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESGGGLVQPGGSLRLSCAASGFT
FS SYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA
EDTAVYYCARTSYLNHGDYWGQGTLVTVSSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSATVTFF
KVKWIFSSVVDLKQTIIPDYRNMIGQGA
pWF-506 (SEQ ID NO: 141) ATGGAAACCGATACACTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGATCTACCG
GTCAGTCTGTGTTGACGC AGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTC AC
CATCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAAC
ACCTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGG
GATTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCG
GACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGCrGATTACACCCTGAA
TGGTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTTCTAGAGGTGGTGGT
GGTAGCGGCGGCGGC,GGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGC
TGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGC
AGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGG
AAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCACATACTATGCAG
ACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAACACGCTGTATCT
GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCACTTCT
TACCTGAACCATGGTGATTACTGGGGTCAAGGTACTCTGGTGACCGTGTCTAGCCCCA
AGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGG
ACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACC
CCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTC
AACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAG
CAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGC
TGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCG
AAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGC
CCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGG
CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAA
CTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAG
CTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGC
ACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGAGCTGCA
ACTGGAGGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGACGGGCTGTGGACGAC
CATCACCATCTTCATCACACTCTTCCTGTTAAGCGTGTGCTACAGTGCCACCGTCACCT
TCTTCAAGGTGAAGTGGATCTTCTCCTCGGTGGTGGACCTGAAGCAGACCATCATCCCC
GACTACAGGAACATGATCGGACAGGGGGCCTGA
pWF-507:
(SEQ ID NO: 142) METDTLLLWVLLLWVPGSTGQSVLTQPP SVSAAPGQRVTIS CS GTRSNIGSDYVSWYQHLP
GTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVF
GGGTKI.TVI.GQPK ANPTVTI ,FPP S SFR ,Q ANK ATI.VCIISDFYPGAVTVAWK ADGSPVK A
GVETTKPSKQSNNKYAAS SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
pWF-507:
(SEQ ID NO: 143) ATGGAAACCGATACACTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGATCTACCG
GTCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCAC
CATCTCCTGCTCTGGAACCAGGTCCAACATTGGGAGTGATTATGTTTCCTGGTACCAAC
ACCTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGG
GATTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCG
GACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAA
TGGTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCCAAC
CCCACTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGCCACACT
AGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCAGAT

GGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCAAACCCTCCAAACAGAG CAACAAC
AAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGA
AGC TAC AGC TGC C AGGTC AC GC ATGAAGGGAGC AC C GTGGAGAAGAC AGTGGC C C CT
ACAGAATGTTCATAG
pWF-508:
(SEQ ID NO: 144) MVFTPQILGLMLFWISASRGQVQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQA
PGKGLEWVSVIYSGGS S TYYAD SVKGRFTI S RDN S KNTLYLQMN S LRAEDTAVYYC ARTS
YLNHGDYWGQ GTLV TV S S AS TKGP S VFPLAP S S KS T S GGTAAL GCLVKDYFPEPVTV SWN
SGALTSGVHTFPAVLQS SGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNS TYRVV S VLTVLHQDWLN GKEYKC KV SNKALPAPIEKTI S KAKG
QP REP QVYTLPP S REEMTKNQV S LTC LVKGFYP S DIAVEWE SN CTQP ENNYKTTP PVLD S D G

SFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELD

pWF-508:
(SEQ ID NO: 145) ATGGTGTTTACACCGCAAATATTGGGGCTCATGCTTTTCTGGATCAGTGCAAGCAGGG
GA C A GGTGC A GCTGGTGGA GTCTGGGGGA GGC TTGGT A C AGC C TGGGGGGTC C CTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGC
CAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCA
CATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT
GCGCGCACTTCTTACCTGAACCATGGTGATTACTGGGGTCAAGGTACTCTGGTGACCGT
GTCTAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCA
C C TC TGGGGGC AC AGC GGCCCTGGGCTGC CTGGTCAAGGACTACTTCCC CGAACC GGT
GACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC
CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT
GGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGA
CAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGC
CCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACC
CTGATGATCAGCAGGACCC CC GAGGTGAC CTGC GTGGTGGTGGAC GTGAGC C ACGAG
GAC CC A GAGGTGA AGTTC A ACTGGTACGTGGACGGCGTGGAGGTGC AC A ACGC C A AG
ACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACC
GTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAG
GC CCTGCC AGCC CCC ATC GAAAAGACC ATC AGC AAGGC CAAGCTGC CAGCC AC GGGAG
CCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCC
TGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
CGGCCACTC,CCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAG
CTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTG
TTC AGC TGC AGC GTGATGC AC GAGGC C C TGC ACAAC C AC TAC AC C C AGAAGAGC C TGA
GCCTGTCCCCCGAGCTGCAACTGGAGGAGAGCTGTGC GGAGGCGCAGGACGGGGAGC
TGGACGGGCTGTGGACGACCATCACCATCTTCATCACACTCTTCCTGTTAAGCGTCITGC
TACAGTGCCACCGTCACCTTCTTCAAGGTGAAGTGGATCTTCTCCTCGGTGGTGGACCT
GAAGCAGACCATCATCCCCGACTACAGGAACATGATCGGACAGGGGGCCTGA
pWF-509:
(SEQ ID NO: 146) METDTLLLWVLLLWVPGSTGQSVLTQPPSVSAAPGQRVTIS CS GTRSNIGSDYVSWYQHLP
GT APKLLVYGDNLRP SGIPDRFS A SKS GTS ATLGITGLQTGDEADYYCGTWDYTLNGVVF
GGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESGGGLVQPGGSLRLSCAASGFT

FS SYAMSWVRQAPGKGLEWV SVIYSGGS STYYADSVKGRFTISRDNSK_NTLYLQMNSLRA
EDTAVYYC ART SYLNHGDYVVGQ GTLVTV S S AAAFV PVF LPAKP TTTPAPRP PTP AP TIAS Q
PLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVLDKDDS
KAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE
pWF-509:
(SEQ ID NO: 147) ATGGAAACCGATACACTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGATCTACCG
GTC AGTC TGTGTTGAC GC AGC C GC CCTCAGTGTCTGC GGCCC C AGGAC AGAGGGTC AC
CATCTC CTGCTCTGGAACCAGGTC CAACATTGGGAGTGATTATGTTTC CTGGTACCAAC
ACCTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGG
GATTCCTGACCGATTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCG
GACTC CAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAA
TGGTGTGGTGTTC GGCGGAGGGACCAAGCTGACCGTCCTAGGTTCTAGAGGTGGTGGT
GGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCC CTCGAGATGGCCCAGGTGCAGC
TGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTC CTGTGC
AGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGG
AAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCACATACTATGCAG
ACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAACACGCTGTATCT
GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCACTTCT
TACCTGAAC CATGGTGATTACTGGGGTCAAGGTACTCTGGTGAC C GT GTC TAGC GC C G
CTGCATTCGTGCCTGTGTTCCTCCCAGCTAAGCCCACTACCACCCCCGCTCCAAGGCCG
CCCACGCCCGCTCCTACTATTGCTAGTCAGCCTTTAAGTTTACGACCCGAAGCTTGCAG
GCCCGCCGCCGGCGGCGCTGTGCACACCAGGGGGCTTGATTTTGCCTGCGACTTTTGG
GTATTGGTAGTGGTGGGCGGAGTTTTAGCCTGCTACAGCCTCCTGGTAACAGTGGCTTT
TATCATCTTTTGGGTGCTGGACAAGGATGACAGCAAGGCTGGCATGGAGGAAGATCAC
ACCTACGAGGGCCTGGACATTGACCAGACAGCCACCTATGAGGACATAGTGACGCTGC
GGACAGGGGAAGTGAAGTGGTCTGTAGGTGAGCACCCAGGCCAGGAGTGA
pWF-510:
(SEQ ID NO: 148) METDTLLLWVLLLWVPGSTGQSVLTQPPSVSAAPGQRVTIS CS GTRSNIGSDYVSWYQHLP
GTAPKLLVYGDNLRP S GIPDRF S AS KS GT S ATLGITGL QTGDEADYYC GTWDYTLNGVVF
GGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESGGGLVQPGGSLRLSCAASGFT
FS SYAMSWVRQAPGKGLEWV SVIYSGGS STYYADSVKGRFTISRDNSKNTLYLQMNSLRA
EDTAVYYCARTSYLNHGDYWGQGTL V TV S SAAAF V P VFLPAKP TTTPAPRPPTP AP TIAS Q
PLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRKRWQ
NEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP
pWF -510 :
(SEQ ID NO: 149) ATGGAAACCGATACACTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGATCTACCG
GTCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAGGGTCAC
C A TCTC CTGCTCTGGA ACC A GGTC C A ACA TTGGGA GTGA TT A TGTTTC CTGGT A CC A AC

ACCTCCCAGGAACAGCCCCCAAACTCCTCGTTTATGGCGATAATCTGCGACCCTCAGG
GATTCCTGAC CGATTCTCTGCCTCC AAGTCTGGC AC GTC AGC CAC CCTGGGCATC AC C G
GACTC CAGACTGGGGACGAGGCCGATTATTACTGCGGCACATGGGATTACACCCTGAA
TGGTGTGGTGTTC GGCGGAGGGACCAAGCTGACCGTCCTAGGTTCTAGAGGTGGTGGT
GGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCC CTCGAGATGGCCC AGGTGC AG C
TGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC CCTGAGACTCTC CTGTGC
AGC C T C TGGATTC AC C TTTAGC AGC TATGC C ATGAGC TGGGTC C GC C AGGC TC C AGGG
AAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGTAGCACATACTATGCAG
ACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGATAATTCCAAGAACACGCTGTATCT
GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCACTTCT
T ACCTGA AC C ATGGTGATT ACTGGGGTC A AGGTACTCTGGTGACCGTGTCTAGCGCC G
CTGCATTCGTGCCTGTGTTCCTCCCAGCTAAGCCCACTACCACCCCCGCTCCAAGGCCG

CCCACGCCCGCTCCTACTATTGCTAGTCAGCCTTTAAGTTTACGACCCGAAGCTTGCAG
GC C C GC C GC C GGC GGC GCTGTGCACAC CAGGGGGCTTGATTTTGC C TGC GACTTTTGG
GTATTGGTAGTGGTGGGCGGAGTTTTAGCCTGCTACAGCCTCCTGGTAACAGTGGCTTT
TATCATCTTTTGGGTGAGGAAACGATGGCAGAACGAGAAGCTCGGGTTGGATGCCGGG
GATGAATATGAAGATGAAAACCTTTATGAAGGCCTGAACCTGGACGACTGCTCCATGT
ATGAGGACATCTCCCGGGGCCTCCAGGGCACCTACCAGGATGTGGGCAGCCTCAACAT
AGGAGATGTCCAGCTGGAGAAGCCGTGA
INCORPORATION BY REFERENCE
[0168] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and definitions control.
EQUIVALENTS
[0169] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof [0170] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.
EXAMPLES
Example 1 ¨ Chimeric Antigen Receptor for B Cells (CAR-B) Constructs to Bind PSMA.
[0171] DNA Constructs. Exemplary CAR-B constructs were designed to recognize Prostate Specific Membrane Antigen (-PSMA"). PSMA is an antigen that is expressed more highly on prostate cancer cells than on other non-cancerous cells. Various construct were made comprising an extracellular domain that comprised an scFv specific for PSMA, an extracellular hinge region from CD8, a CD28 transmembrane domain, and various intracellular signaling domains. A list of the constructs is provided in Table 6:

ConstEuct pTLPW-SFFV-XENP14484 scFv-hCD8H-hCD28M-hCD19E (SEQ ID NOS. 39 pWF-82 and 40) pTLPW-SFFV-XENP14484 scFv-heD8H-heD28M-heD4OE (SEQ ID NOS. 41 pWF-83 and 42) pTLPW-SFFV-XENP14484 scFv-hCD8H-hCD28M-h(CD4O+CD79b)E (SEQ ID
pWF-84 NOS. 43 and 44) pTLPW-SFFV-XENP14484 scFv-hCD8H-hCD28M-h(CD4O+CD137)E (SEQ ID
pWF-85 NOS. 45 and 46) pTLPW-SFFV-XENP14484 scFv-hCD8H-hCD28M-h(CD4O+Fcyr2a)E (SEQ ID
pWF-86 NOS. 47) JLPW-SFFV-XENP14484 scFv-hCD8H-hCD28M-h(hMyd88+CD40)E (SEQ ID
pWF-87 NOS. 48 and 49) pTLPW-SFFV-XENP14484 scFv-hCD8H-hCD28M-hCD79aE (SEQ ID NOS. 50 pWF-88 and 51) pTLPW-SFFV-XENP14484 scFv-hCD8H-hCD28M-hCD79bE (SEQ ID NOS. 52 pWF-89 and 53) [0172] Expression of anti-PSMA CAR-B on HEK-293 Cells. The constructs encoding pWF82 to pWF89 were used to prepare lentivirus in Lentix cells using the Takara lentivirus preparation kit.
Expression of the various CAR-B constructs was measured using flow cytometry using antibodies specific for PSMA (biotin-PSMA, Sinobiological and is depicted in FIG. 5.
[0173] Expression of anti-PSMA CAR-B in Human B Cells. To measure expression and binding of anti-PSMA CAR-B's in B cells, two additional constructs were made:

1MA-0We MITTIMMITETTIMMETEMEtiiiiiiiiiitiAfEMITMETTITIMMITIME
pMMLV(LTR)-hEF1 a promoter-anti hPSMA(XENP14484)-CBCR (SEQ ID NOS.
pWF-391 54 and 55) pWF-394 )MMLV(LTR)-hEFla promoter-anti sarcoglycan CBCR1 (SEQ ID NOS. 56 and 57;
[0174] A MMLV based vector was used for the preparation of the retrovirus. The retrovirus was used to infect mouse B cells isolated from the spleen. After transduction, B
cells were further expanded on feeder cells expressing CD4OL and soluble IL-4. The expression of anti-PSMA CAR-B was detected by using recombinant biotin-PSMA. PE-labeled streptavidin was used to detect PSMA binding in HEK-293 cells.
[0175] Results. The results of this experiment are depicted in FIG. 6 and demonstrate that it is possible to create mouse B cell that expresses a CAR-B that can bind with specificity to an antigen.
For example, B cells expressing pWF396 or pWF397 bound to PSMA whereas the B
cells expressing pWF394 did not bind PSMA pWF398 was designed to bind sarcoglycan not PSMA).
Example 2¨ Chimeric Antigen Receptor on B cells (CAR-B) Constructs to Bind GPC3.
[0176] DNA Construct. Exemplary CAR constructs were designed to recognize glypican-3 (GPC-3). Glypican-3 is expressed on hepatocellular carcinoma cells among other tumor types, but not on most non-cancer cells. GPC3 can be used to target an anti-GPC3 CAR to hepatocellular carcinoma, as well as other cancers in which GPC3 is expressed (e.g. ovarian clear cell carcinoma, pediatric cancers, lung cancers (i.e. lung adenocarcinoma and lung squamous cell carcinoma), urothelial carcinoma, thyroid cancer, gastric cancer, and others). Various construct were made comprising an extracellular domain that comprised an scFy specific for GPC-3, an extracellular hinge region from CD8, a CD28 transmembrane domain, and various intracellular signaling domains. An additional anti-PSMA CAR-B was constructed as a control for these experiments. A
list of the constructs is provided in Table 8.

wei pMMLV(LTR)-hEF1 a promoter-anti-GPC3 scFv-hCD8H-hCD28M-hCD79aE (SEQ ID
pWF-396 NOS. 58 and 59) pMMLV(LTR)-hEFla promoter-anti-GPC3 scFv-hCD8H-hCD28M-hCD79bE (SEQ ID
pWF-397 NOS. 60 and 61) pMMLV(LTR)-hEFla promoter-anti-hPSMA(XENP14484) scFv-hCD8H-hCD28M-pWF-398 hCD79aE (SEQ ID NOS. 62 and 63) [0177] Expression of anti-GPC-3 on HEK-293 Cell. Lentiviral transductions were used to express GPC3 CAR-B proteins on the surface of HEK293 cells. Expression was determined by flow cytometry with an anti-idiotype antibody specific for GPC-3 (Eureka Therapeutics).
[0178] Expression of anti-GPC-3 CAR-B in Human B Cells. pWF 396, 397 and 398 encoding CAR constructs were used to prepare MMLV retrovirus. This retrovirus was used to transduce mouse B cells isolated by negative selection (Stem Cell Technologies) and activated for 24 hours by co-culture with HeLa cells expressing CD4OL and the addition of soluble IL-4. 48 hours post-transduction, expression was confirmed using flow cytometry. The expression of the CAR-B was detected using an anti-idiotype antibody against human GPC3. The anti-idiotype antibody was obtained from Eureka Therapeutics.
101791 Results. Mouse B cells expressing anti-GPC-3 CAR-Bs, pWF-396 and 397, were expressed and specifically bound by anti-GPC3 idiotype antibody.
Example 3¨ Adenovirus Variant F35 Expressing GFP
[0180] Adenovirus variant F35 expressing GFP was demonstrated to efficiently infect human B
cells. Human B cells were isolated from the peripheral blood. The B cells were infected with adenovirus encoding GFP at volumes of 0, 1, 3, 10 L. The titer of the adenovirus preparations were approximately 1 x ell particles/ml.

Example 4- Delivering Payloads to Tumor Cells 101811 A large screening study was conducted to examine the effect of payloads on NIH3T3 fibroblasts in a CT26 Model. Payloads included various immunomodulators, including cytokines and chemokines. First, BALB/c mice were injected with CT26 tumors into their left and right flank.
See FIG. 8. Twelve and sixteen days later, mice were injected into the right flank tumor, with various combinations of 4-5 payloads. Tumor volume was measured for up to 35 days.
[0182] Generation of the BALB/C CT26 tumor model. A total of 139 mice were injected with CT26 tumors into their left and right flanks.
[0183] Selection of Payload. Twelve peptides were identified for their potential to (i) recruit and activate dendritic cells; (ii) initiate homing and guidance of dendritic cells and T cells into the tumor site; and (iii) activate effector T cells. The payloads screened are listed in Table 9.

pRBSBPMrailVadAMBBNMWMEMKSEQTDNOAMSM
FLT3L 70, 71 XCL1 72,73 TIM4-Fc 74, 75 CXCL13 68,69 mCCL21 92, 93 mCD80 - membrane bound 86, 87 mCD4OL - membrane bound 88, 89 m1FNa A2 84,85 m1L-12 80, 81 m1L-21 90, 91 mLIGHT mutant 78,79 M4-1BBL-membrane bound 76, 77 mIL-15 124, 125 [0184] Each was either given a combination of 4-5 payloads, all 12 payloads, or 3T3 cells (without payload) or saline as a control. In total, there were twenty-seven groups (n = 5 mice/group). The experimental groups are identified in Table 10.

groupTreatment 1 FLT3L, XCL1, CXCL13, TIM4-Fc, TLR
2 FLT3L, XCL1, CXCL13, CD80-MB
3 FLT3L, XCL1, CXCL13, CD4OL-MB, TLR
4 FLT3L, XCL1, CXCL13, IL-12 and TM
FLT3L, XCL1, CXCL13, 4-1BBL-MB
6 FLT3L, XCL1, CXCL13, IFNa A2 7 FLT3L, XCL1, LIGHT, TIM4-Fc 8 FLT3L, XCL1, LIGHT, CD80-MB
9 FLT3L, XCL1, LIGHT, CD4OL-MB, TLR

FLT3L, XCL1, LIGHT, IL-12 and TM
11 FLT3L, XCL1, LIGHT, 4-1BBL-MB
12 FLT3L, XCL1, LIGHT, IFNa A2 13 FLT3L, XCL1, IL-21, TIM4-Fc 14 FLT3L, XCL1, IL-21, CD80-MB
FLT3L, XCL1, IL-21, CD4OL-MB
16 FLT3L, XCL1, IL-21, IL-12 and TM
17 FLT3L, XCL1, IL-21, 4-1BBL-MB
18 FLT3L, XCL1, IL-21, IFNa A2 19 FLT3L, XCL1, CCL21, TIM4-Fc FLT3L, XCL1, CCL21, CD80-MB
21 FLT3L, XCL1, CCL21, CD4OL-MB
22 FLT3L, XCL1, CCL21, IL-12 and TM
23 FLT3L, XCL1, CCL21, 4-1BBL-MB
24 FLT3L, XCL1, CCL21, IFNa A2 All Payloads 26 Saline 27 3t3 cells (no payload) [0185] Dosing. Tumor volume was between 100 mm3 and 150 mm3 at the time of the first injection. For the groups receiving 4 payloads, each pay load was delivered at 2.5 x 105 cells per injection for a total of 106 cells. For the groups receiving 5 payloads, each pay load was delivered at [x] cells per injection for a total of 3 x 106 cells. The fifth payload was co-administered with Poly(I:C), which is a ds-RNA analog. Payloads were administered by intra-tumor injection. The volume of administration was 50 1,11_, for all groups except the poly (I:C) group and the large 12-way group, where the volume was 150 L.
[0186] Payload Administration Procedures. Cells were harvested with versene (not in the presences of trypsin). Once collected, the cells were counted, spun and resuspended in a volume that could be adjusted to 20 x 106/m1 after the cells are recounted. TLR
agonist (Invivogen Cat#
ODN:1826) by resuspending lyophilized powder in water provided. TLR agonist was resuspended at 10 mg/m1 and heated to 70degC and then let to sit at RT for 1 hour prior to using. The dose of TLR agonist is 50 [tg in 50 1.
[0187] Results. The results are depicted in FIGS 9-11. Several combinations of payloads injected ipsilaterally demonstrated antitumor activity in the contralateral tumors manifested as delayed tumor growth in this model. Groups 3, 8 and 21 showed the most significant impairment of tumor growth over 30 days.
Example 5- Modified B Cells that Express and Secrete Payloads [0188] Experimental Design. A BALB/c mouse CT26 tumor model was used to evaluate the efficacy of modified B cells expressing various payload on tumor volume and survival. Mice were injected with tumor cells at a volume of 100 L. On day 6 once tumors had reached a volume of 175mm3, mice were injected with modified B cells expressing various payloads as described below_ Tumor volume and survival were measured for 17 days.
101891 Isolation of Mouse PBMCs. Mouse PBMCs or splenocytes are isolated from blood or spleen, respectively. PBMCs are isolated using Lympholyte-M (CedarLane, Cat#CL5030).
Splenocytes are isolated by manual cell separation through a 70 micron nylon cell strainer. B cells are then isolated from PBMCs or splenocytes via immunomagnetic negative selection using EasySep Mouse B cell Isolation Kit (Stem Cell Technologies, Cat #19854).
[0190] Selection of Payloads. Nucleic acid sequences expressing payload peptides or proteins are transfected or transduced into isolated B cells. The following twelve peptides were identified for their potential to (i) recruit and activate dendritic cells; (ii) initiate homing and guidance of dendritic cells and T cells into the tumor site; and (iii) activate effector T
cells. The payloads screened are listed in Table 9.
[0191] Each mouse was either given a combination of 4-5 payloads, or isolated B cells (without payload) or saline as a control. In total, there were twenty-seven groups (n =
5 mice/group). The experimental groups are identified in Table 11.

3 FLT3L, XCL1, CXCL13, CD4OL-MB, TLR
8 FLT3L, XCL1, mLIGHT, CD80-MB
21 FLT3L, XCL1, CCL21, CD4OL-MB
26 Saline 27 B cells (no payload) [0192] Generation of Payload Expressing B Cells. For transfection, purified or cultured B cells are washed and suspended in Cytoporation Medium T (BTX, Cat # 47-0002) at 5 x 106 to 25 x 106 cells per ml and mixed with 7.5 lag to 50 lag RNA (RNA constructs are designed and prepped in house or purchased from TriLink using CleanCapk and fully substituted with Pseudo-U). 200 p.1_, cell/RNA suspension electroporated using BTX Agilpulsek Electroporation System.
[0193] Dosing. Tumor volume was between 100 mm3 and 150 mm3 at the time of the first injection. For the groups receiving 4 payloads, each payload was delivered at 2.5 x 105 cells per injection for a total of 106 cells delivered. For the groups receiving 5 payloads, each pay load was delivered at 2.5 x 105 cells per injection for a total of 1.25 x 106 cells delivered. Payloads were injected intra-tumor. The volume of administration was 501.11_, for groups receiving 4 payloads, the volume of administration was 1001,IL for groups receiving 5 payloads.
[0194] Payload Administration Procedures. Cells were harvested with versene (not in the presence of trypsin). Once collected, the cells were counted, spun and resuspended in a volume that could be adjusted to 20 x 106/ml. TLR agonist (InvivoGen Cat# ODN:1826) by resuspending lyophilized powder in water provided. TLR agonist was resuspended at 10 mg/ml and heated to 70 C and then let to sit at RT for 1 hour prior to using. The dose of TLR
agonist is 50 mg in 50 ill.
Example 6 ¨ Anti-tumor activity of intratumorally injected B cells [0195] Mouse splenocytes were obtained and isolated via manual cell separation utilizing a 70 micron nylon cell strainer. Autologous (BALB/c) or allogeneic (C57B1/6) donor mice were used (data shown utilized allogeneic B cells). B cells were isolated from the splenocytes above using immunomagnetic negative selection via the Easy Sep Mouse B Cell Isolation Kit (Stem Cell Technologies , Cat #19854).
[0196] B cells were then injected either (i) fresh or (ii) first stimulated for 16-24 hours in growth media (RPMI, 10% FBS, 1% Pen/Strep, 5ng/m1 recombinant mouse IL-4, 100uM beta-mercaptoethanol) with 5 jig/m1 Lipopolysaccharide. 5X106 B cells were then intratumorally injected into the CT26 mouse model, and anti-tumor responses in the distal (abscopal) tumor where measured. Tumors were implanted at day 0, and at day 6 palpable tumor mass was observed.
Treatment was initiated on day 6 intratumorally. The results are set forth in Figure 12.
Example 7 ¨ Expression of chimeric antigen receptor (CAR) in B cells using RNA
electroporation to make CAR B cells [0197] Mouse PBMCs or splenocytes were isolated from blood or spleen as follows. Mouse PBMCs were isolated using Lympholyte-M (CedarLane, Cat #CL5030), and splenocytes were isolated by manual cell separation via passage through a 70 micron nylon cell strainer. B cells were then isolated from PBMCs or splenocytes, respectively, via immunomagnetic negative selection using the EasySep Mouse B Cell Isolation Kit (Stem Cell Technologies, Cat #19854).
[0198] B cells were then stimulated for 16-24 hours in growth media (RPMI, 10%
FBS, 1%
Pen/Strep, 5ng/m1 recombinant mouse IL-4, and 100uM beta-mercaptoethanol) with 5-15 ug/ml lipopolysaccharide. B cells were then transduced or transfected using known techniques (viral transfection or electroporation) to achieve either stable or transient expression of CAR-B. A strep II tag was incorporated for post-translational detection. Representative CAR-Bs depicted are as follows:
1. XENP PSMA CBCR (3X strep II tag) 2. HyHEL10 CBCR (3X strep II tag) 3. D1.3-M3 HEL CBCR (3X strep II tag) [0199] For transfection, purified or cultured B cells were washed and suspended in Cytoporation Medium T (BTX, Cat #47-0002) at 5x106 to 25x106 cells per ml and mixed with 7.5ug to 50ug RNA (RNA constructs were designed and prepped either in-house or purchased from TriLink using CleanCap and fully substituted with Pseudo-U). A 200u1 cell/RNA suspension was obtained and el ectroporated using the BTX AgilePul se El ectroporati on System. Cells were then washed in PBS and prepped for IV injection into immune-incompetent mice with established HepG2 tumor cells that express respective antigen (e.g. GPC3, HEL, PSMA). Translation and expression of protein of interest was then measured using an anti-Strep II tag antibody. The results are set forth in Figure 13. In Figure 13, the X axis shows strength of expression signal as measured by flow cytometry, and the Y axis sets forth percent of cells expressing the desired protein of interest (PSMA, HEL).
[0200] This experiment demonstrates that the desired RNA sequence/s are successfully transfected or transduced (accordingly), the RNA is successfully translated, and the desired protein of interest is expressed on the cell surface.
Example 8¨ Modified B cells expressing integrins and homing receptors [0201] Nucleic acid constructs expressing an integrin, a homing receptor, or both are constructed using known techniques. Mouse and Human B cells are transfected or transduced (accordingly) with the nucleic acid constructs to express the integrin, the homing receptor, or both. These modified cells are administered intravenously into mice or a human host. Time-lapse imaging will measure accumulation of the modified B cells at the site/target of interest, such as a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment, to establish that expression of an integrin and/or a homing receptor of defined homing specificity endows the B cells with the ability to home to and accumulate at the site/target of interest where delivery of therapeutic payloads is desirable. A screening study is conducted according to the techniques of Example 5 to examine delivery and effect of payloads at the site/target of interest.
Example 9¨ Altering B cell trafficking [0202] Isolated B cells are cultured with a specific concentration of all-trans-retinoic acid (ATRA) or derivatives thereof that induce expression of a4137 integrin and the homing receptor CCR9. Thereafter, the B cells are harvested and administered intravenously into mice. There are two experimental groups of the recipient mice. The first group of mice are pre-treated with DSS or TNBS to induce gut inflammation. The second group of mice are not treated with DSS or TNBS.
Inflammation similar to that observed in human intestinal bowl diseases is induced by pretreatment with DSS or TNBS. Administered B cells treated with ATRA or derivative thereof will home to areas of inflammation consistent with their homing potential due to increased expression of a4fl7 integrin and the homing receptor CCR9.
Example 10 ¨ Modified B cells expressing immune inhibitory molecules [0203] Nucleic acid constructs expressing an immune inhibitory molecule selected from IL-10, TGF-fl, PD-L1, PD-L2, LAG-3, and TIM-3, or any combinations thereof, are constructed using known techniques. Mouse and Human B cells are transfected or transduced (accordingly) with the nucleic acid constructs to express one or more of the immune inhibitory molecules listed above.

These modified cells are administered intravenously into mice or a human host or elsewhere near or at sites of inflammation. Time-lapse imaging will measure accumulation of the modified B cells at a site/target of interest, such as a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment, to establish that inflammation at the site and autoimmune activity of the B cells localized to the site are decreased, thereby leading to a positive therapeutic response.
Example 11 ¨ Activation of B cells with TLRs [0204] B cells are treated with TLR agonists and/or modified to express a constitutively active TLR for use in potentiating B cells for immune responses and producing potent effector B cells to increase antigen-specific immune responses in a subject. Isolated mouse or human B cells are treated in vitro with a TLR agonist at the same time or in advance of the administration of the B
cells. In some instances, the mouse or human B cells are treated with more than one TLR agonists.
[0205] A modified B cell, transfected or transduced with or without a CAR-B
construct of the foregoing examples, is engineered to express one or more constitutively active TLRs. Each TLR
is introduced into the modified B cell (transduced or transfected using known techniques) as a DNA
construct under the control of a constitutively activated transcriptional pathway. A modified B cell, expressing one or more constitutively active TLRs (with or without a CAR-B
construct), is also treated with one or more TLR agonists at the same time or in advance of the administration of the modified B cells to a subject or patient in need thereof Time-lapse imaging and other known techniques will measure accumulation of the modified B cells in the desired location and confirm expression of the TLR(s) and any expressed CAR-B of a defined specificity.
[0206] This experiment will demonstrate that the desired DNA sequence/s encoding specific TLR(s) of interest are successfully transfected or transduced (accordingly) into B cells with or without a CAR-B construct and treated with or without TLR agonist(s), the RNA
is successfully translated, the desired TLR(s) are expressed in the B cells for producing potent effector B cells potentiating B cells for immune responses.
Example 12 ¨ Antigen presentation both in HLA Class I and Class II molecules using RNA
electroporated B cells [0207] mRNA Constructs. Exemplary mRNA constructs are designed by fusing a specific antigen, e.g., a tumor antigen or an infectious disease antigen, to the targeting signal of a the lysosomal protein LAMP1, to target the specific antigen to the lysosomes and present the antigen simultaneously and efficiently in both HLA class I and class II molecules.
Tumor antigens and infectious disease antigens are well known in the art and can include any antigen of interest against which an immune response is desired. Various mRNA constructs are made encoding at least one specific antigen of interest fused to the targeting signal of LAMP1 that is capable of presenting the specific antigen simultaneously and efficiently by both HLA class I and class II molecules when transfected into a suitable immune cell.
102081 Experimental Design. Isolated mouse or human B cells are electroporated in vitro with an mRNA construct described above (i.e., encoding a specific antigen of interest fused to the targeting signal of LAMP1) using known mRNA electroporati on techniques. In some instances, the mouse or human B cells are also transduced or transfected using known techniques with a CAR-B construct according to any of the foregoing examples. The mRNA
electroporated B cells, transduced with or without a CAR-B construct of interest, are introduced intravenously into mice or a human host. Time-lapse imaging will measure accumulation of the modified B cells in the desired location and also confirm expression of CAR-B of a defined specificity. Translation and expression of the specific tumor antigens or infectious disease antigens of interest are measured using known techniques to establish that the antigens of interest are targeted to the lysosomes and presented simultaneously and efficiently by both HLA class I and class II
molecules.
[0209] This experiment will demonstrate that the desired mRNA sequence/s encoding specific antigens of interest fused to a targeting signal are successfully transfected into B cells (which, if desired, are also transduced with a CAR-B construct), the mRNA is successfully translated, and the electroporated and modified B cells simultaneously and efficiently present the specific antigen of interest by both HLA class I and class II molecules for increasing antigen-specific immune responses in a subject.
Example 13 ¨ B Cells Expressing a PSMA-specific CAR Reduce Tumor Growth in PSMA Tumors [0210] Mouse Tumor Model. A BALB/c CT26-PSMA tumor model engineered to express human PSMA was used to evaluate the efficacy of PSMA-specific CAR engineered murine B cells on tumor volume and survival. Eight-week-old BALB/c mice were injected on one hind flank with 1.0x106CT26-PSMA tumor cells in a volume of 50 On day 5 when the tumor volume reached approximately 60 mm-3 the mice were distributed equally into 3 groups of 10 mice. Treatment of mice was started on day 6 using murine B cells engineered with mRNA encoding two different PSMA-specific CAR formats or un-engineered B cells administered intravenously at a dose of 1.5x106 cells in 100n1, or saline on day 6. Tumor volume was measured using calipers on day 5, 9, 11, and 13. There was a statistically significant tumor reduction of 57% in the PSMA-CAR group (format 79a) relative to saline on day 13. There was not a significant reduction of tumor volume on day 13 in the PSMA-CAR treatment group (format 79b) relative to saline (FIG. 14).
[0211] Engineering of Murine B Cells. Mouse splenocytes were isolated from BALB/c donor spleens by manual cell separation through a 70 micron nylon cell strainer. B
cells were then isolated from splenocytes via immunomagnetic negative selection using EasySep Mouse B
cell Isolation Kit (Stem Cell Technologies, Cat #19854). B cells were stimulated for 24 hours in growth media (RPMI, 10% FBS, 25 mM HEPES, 1% Pen/Strep, 5ng/m1 recombinant mouse IL-4, 100jtM beta-mercaptoethanol) with anti-CD40 (250 ng/ml). Cells were then electroporated with 20 g CAR
mRNA construct per 3.6x106 B cells using BTX AgilePulse electroporation system set at 280V for lms. Cells were washed and resuspended in PBS at a concentration of 15x106 B
cells /ml. 100 11 of cell suspension were used per dose.
PSMA construct CD79a: pmRNA d7 13 anti hPSMA(XENP14484) scFv-mCD8H-mCD28M-mCD79aE #ab-1 PSMA construct CD79b: pmRNA d7 13 anti hPSMA(XENP14484) scFv-mCD8H-mCD28M-mCD79bE #ac-1 Example 14 ¨ Allogenic B Cells Expressing a PSMA-specific CAR Reduce Tumor Growth in CT26-PSMA Tumors [0212] Mouse Tumor Model. A BALB/c CT26-PSMA tumor model engineered to express human PSMA was used to evaluate the efficacy of PSMA-specific CAR engineered allogeneic murine B cells on tumor volume and survival. Eight-week-old BALB/c mice were injected on one hind flank with 1.0x106 CT26-PSMA tumor cells in a volume of 50 1. On day 5 when the tumor volume reached approximately 70 mm3 the mice were distributed equally into 3 groups of 10 mice.
Treatment of mice was started on day 6 using autologous murine B cells engineered with mRNA
encoding a PSMA-specific CAR and an mRNA encoding CCR7 or allogeneic murine B
cells engineered with mRNA encoding a PSMA-specific CAR administered intravenously at a dose of 1.5x106 cells in 100 1, or saline. Tumor volume was measured using calipers on day 5, 8, and 10.
There was a statistically significant tumor reduction of 51% in the allogeneic and autologous engineered B cell groups relative to saline on day 10 (FIG. 15). (p<0.005).
[0213] Engineering of Murine B Cells. Mouse splenocytes were isolated from autologous BALB/c and allogeneic C57B1/6 donor spleens by manual cell separation through a 70 micron nylon cell strainer. B cells were then isolated from splenocytes via immunomagnetic negative selection using EasySep Mouse B cell Isolation Kit (Stem Cell Technologies, Cat #19854). B cells were stimulated for 24 hours in growth media (RPMI, 10% FBS, 25 mM HEPES, 1%
Pen/Strep, 5ng/m1 recombinant mouse IL-4, 100 M beta-mercaptoethanol) with anti-CD40 (250 ng/ml). Cells were then electroporated with 20 ug CAR mRNA construct per 3.6x106 B cells using BTX
AgilePulse el ectroporation system set at 280V for lms. Cells were washed and resuspended in PBS
at a concentration of 15x106 B cells /ml. 100 I of cell suspension were used per dose.
Example 15 ¨ The Antitumor Activity of PSMA-CAR-Engineered B Cells Depends on an Intact Host Immune System [0214] Mouse Tumor Models. The effect of antitumor PSMA-CAR B cells was studied in WT
and immunocompromised NSG mice.
[0215] WT Mice. A BALB/c CT26-PSMA tumor model engineered to express human PSMA
was used to evaluate the efficacy of PSMA-specific CAR engineered murine B
cells on tumor volume and survival in WT mice. Eight-week-old BALB/c mice were injected on one hind flank with 1.0x106 CT26-PSMA tumor cells in a volume of 50 IA On day 5 when the tumor volume reached approximately 60 mm3 the mice were distributed equally into 4 groups of 10 mice.
Treatment of mice was started on day 6 using murine B cells engineered with mRNA encoding two different PSMA-specific CAR formats or un-engineered B cells administered intravenously at a dose of 1.5x106 cells in 10014 or saline on day 6. Tumor volume was measured using calipers on day 5, 9, 11, and 13. There was a statistically significant tumor reduction of 57% in the PSMA-CAR group (format 79a) relative to saline on day 13. There was not a significant reduction of tumor volume on day 13 in the PSMA-CAR treatment group (format 79b) or un-engineered B cells, relative to saline (FIG. 14).
102161 NSG Mice. A BALB/c CT26-PSMA tumor model engineered to express human PSMA
was used to evaluate the efficacy of PSMA-specific CAR engineered murine B
cells on tumor volume and survival in immunocompromised mice. Eight-week-old NSG mice were injected on one hind flank with 1.0x106 CT26-PSMA tumor cells in a volume of 50 IA On day 5 when the tumor volume reached approximately 60 min3 the mice were distributed equally into 2 groups of mice. Treatment of mice was started on day 6 using murine B cells engineered with mRNA
encoding a PSMA-specific CAR format administered intravenously at a dose of 1.5x106 cells in 1041, or saline on day 6. Tumor volume was measured using calipers on day 5, 8, 10, and 13.
There was no significant reduction in tumor volume in the PSMA-CAR group (format 79a) relative to saline on day 13 (FIG. 16B).
[0217] Engineering of Murine B Cells. Mouse splenocytes were isolated from autologous BALB/c and allogeneic C57B1/6 donor spleens by manual cell separation through a 70 micron nylon cell strainer. B cells were then isolated from splenocytes via immunomagnetic negative selection using EasySep Mouse B cell Isolation Kit (Stem Cell Technologies, Cat #19854). B cells were stimulated for 24 hours in growth media (RPMI, 10% FBS, 25 mM HEPES, 1%
Pen/Strep, 5ng/m1 recombinant mouse IL-4, 1001.tM beta-mercaptoethanol) with anti-CD40 (250 ng/ml). Cells were then electroporated with 20 ug CAR mRNA construct per 3.6x106 B cells using BTX
AgilePulse electroporation system set at 280V for lms. Cells were washed and resuspended in PBS
at a concentration of 15x106 B cells / ml. 1001,11 of cell suspension were used per dose.
PSMA construct CD79a: pmRNA d7 13 anti hPSMA(XENP14484) scFv-mCD8H-mCD28M-mCD79aE #ab-1 PSMA construct CD79b: pmRNA d7 13 anti hPSMA(XENP14484) scFv-mCD8H-mCD28M-mCD79bE #ac-1 Example 16¨ B Cells Expressing a GPC3-specific CAR Reduce Tumor Growth in HEPA

GPC3 Tumors [0218] Mouse Tumor Model. A C57B1/6 HEPA 1-6 tumor model engineered to express human GPC3 (HEPA 1-6-GPC3) was used to evaluate the efficacy of murine B cells on tumor volume and survival. Eight-week-old C57B1/6 mice were injected on one hind flank with 5.0x106 HEPA 1-6-GPC3 tumor cells at a volume of 200 IA. On day 19 when the tumors volume reached approximately 250 mm3 the mice were distributed equally into 3 groups of 10 mice. Treatment of mice was started on day 20 using murine B cells engineered with mRNA encoding a GPC3-specific CAR or a PSMA-specific CAR administered intravenously at a dose of 1.5x106 cells in 100 1, or saline on day 20 and day 27. Tumor volume was measured using calipers on day 19, 23, 26, and 30. There was a statistically significant tumor reduction of 68% in the GPC3-CAR group relative to saline on day 30. There was not a significant reduction of tumor volume on day 30 in the PSMA-CAR
treatment group relative to saline. (note: this study is still in progress on 1-19-2021) (FIG. 17).
[0219] Engineering of Murine B Cells. Mouse splenocytes were isolated from C57B1/6 donor spleens by manual cell separation through a 70 micron nylon cell strainer. B
cells were then isolated from splenocytes via immunomagnetic negative selection using EasySep Mouse B
cell Isolation Kit (Stem Cell Technologies, Cat #19854). B cells were stimulated for 24 hours in growth media (RPMI, 10% FBS, 25 mA/1 HEPES, 1% Pen/Strep, 5ng/m1 recombinant mouse IL-4, 100jiM beta-mercaptoethanol) with anti-CD40 (250 ng/ml). Cells were then electroporated with 20 jig CAR
mRNA construct per 3.6x106 B cells using BTX AgilePulse electroporation system set at 280V for lms. Cells were washed and resuspended in PBS at a concentration of 15x106 B
cells /ml. 100 il of cell suspension were used per dose.
GPC3 mRNA construct: pmRNA d7 13 anti-hGPC3 scFv-mCD8H-mCD28M-mCD79aE #15-1 PSMA construct: pmRNA d7 13 anti hPSMA(XENP14484) scFv-mCD8H-mCD28M-mCD79aE #ab-1 Example 17 ¨ Multimerized GPC3 can activate NFKB expression of luciferase in cells expressing a GPC3 CAR in a dose-responsive manner [0220] CAR-B Construct Design. Five CAR-B constructs were designed using three basic formats (i) CAR 2 (an scFv, a hinge domain, a transmembrane domain and a signaling domain (see FIG XA)); (ii) CAR 3 (a multimerized receptor complex with 2 of each of the following: an scFv, a hinge domain, an FC domain, a transmembrane domain and a cytoplasmic tail (see FIG XB));
(iii) CAR 4 (a multimerized receptor complex with 2 of each of the following:
(a FAB domain, a hinge domain, an FC domain, a transmembrane domain and a cytoplasmic tail (see FIG XC). The five CAR-B constructs are as follows:

pWF-506 pmRNA d7 13 anti-hGPC3 scFv-hIgG1 Fc [TM + cyto] A-1 (SEQ ID NO. 140/141) (CAR 3) pWF-507 pmRNA d7 13 anti-hGPC3 vl-hcLamda /
pmRNA d7 13 anti-hGPC3 vH-hlgHgl [TM+ cyto]

(SEQ ID NO. 142/143) / (CAR 4) pWF-508 (SEQ ID NO. 144/145) pWF-509 pmRNA d7 13 anti-hGPC3 scFv-hCD8H-hCD28M-hCD79bE
(SEQ ID NO. 146/147) (CAR 2) pWF-510 pmRNA d7 13 anti-hGPC3 scFv -hCD8H-hCD28M-hCD79aE
(SEQ ID NO. 148/149) (CAR 2) [0221] NFKB Reporter Assay: Antigen induced signaling. Ramos NFx13-luciferase reporter cells were transduced with mRNA coding for one of the CAR-B constructs listed above.
Ramos NFI<B-luciferase reporter cells were transfected at 280 V and 1 msec with 10 ug of RNA in 200 uL of electroporation buffer followed by culturing overnight in growth medium. The cells were left at room temperature for 4 hours to quiesce the cells to reduce background. 30,000 of the transfected cells were transferred to each well in a multi-well plate in a volume of 30 !IL per well. The transfected Ramos cells were then incubated with GPC3 protein multimerized with streptavidin, streptavidin control or GPC3-Fc protein for 3 hours in growth medium. 30 tiL
of Bioglo substrate (Promega) was added to each well and the plate was read within 5 minutes using a luminometer.
As demonstrated in FIG. 18, multimerized GPC3 was capable of activating NFKB
expression of luciferase in cells expressing three of the four GPC3 CAR-Bs except pWF-509 (GPC3-CD79b).
All four constructs displayed good binding to GPC3 in FACS assays. Therefore, CD79b was an example where a CAR, which had good binding affinity, did not signal.
[0222] NEKB Reporter Assay: Tonic Signaling. Tonic signaling was also assessed, using the NFid3 Reporter Assay. CAR constructs, which induced elevated tonic signaling in the absence of cognate antigen binding, were generated. FIG. 19 shows that the four CAR-B
constructs were expressed in a human B cell reporter line and NFKB luciferase activity was measured in the absence of cognate target antigen. Each construct displayed significant tonic signaling activity. Engineered B cells with tonic signaling CAR Bs remained at a high number in vivo and led to high and durable expression of replacement factors or other payloads.
Example 18¨ A CD80 payload enhances the antitumor activity of Anti-GPC3CAR-CD79a B
cells [0223] Experimental Design. A syngeneic C57B1/6 mouse HEPA1-6GPC3 tumor is a model of human HCC engineered to express human GPC3. This model was used to evaluate the efficacy of murine B cells electroporated with anti-GPC3CAR-CD79a and a CD80 payload mRNAs. Mice were injected on one hind flank with 5.0 X 106 HEPA1-6GPC3 tumor cells at a volume of 200 ul in matrigel. On day 11,14, and 17 the mice were administered a 200 ul IV dose of 1.5x106 B cells, B cells engineered with anti-GPC3CAR-CD79a. B cells engineered with anti-GPC3CAR-CD79a and CD80, or saline as indicated in FIG. 20. The B cells were engineered with mRNA as described below. Tumor volume was monitored on multiple days as indicated in FIG. 20.
[0224] In FIG. 20, both the anti-GPC3CAR-CD79a, and anti-GPC3CAR-CD79a plus combo displayed a statistically significant effect relative to saline or un-engineered B cells on day 44 and at multiple earlier time points. Additionally, by day 44 there were no complete responses in the saline control or B cell control groups but the anti-GPC3CAR-CD79a, and anti-GPC3CAR-CD79a plus CD80 combo resulted in 4 and 7 complete responses, respectively, as indicated in FIGS. 21A-21C. These data demonstrate that inclusion of the CD80 payload as mRNA potentiated the antitumor activity of B cells co-electroporated with an antigen-specific GPC3 CAR.
102251 B Cell preparation. Mouse splenocytes were isolated from C57B1/6 donor spleens by mechanical cell separation through a 70 micron nylon cell strainer. B cells were then isolated from splenocytes via immunomagnetic negative selection using EasySep Mouse B cell Isolation Kit (Stem Cell Technologies, Cat #19854). B cells were stimulated for 24 hours in growth media (RPMI, 10% FBS, 1% Pen/Strep, 5ng/m1 recombinant mouse IL-4, 100uM beta-mercaptoethanol) with 250 ng/ml CD40 antibody (anti-murine CD40 Ab). Cells were then electroporated with 2Oug mRNA per 1.0x107 B cells using BTX AgilePulse electroporation system set at 400V for lms, 2ms interval for 5 pulses_ When two mRNA's were cotransfected, 20ug of each mRNA
was used.
Immediately after electroporation, the cells were washed in PBS and prepared for IV administration at a dose of 1.0x107 per 200u1. The electroporated cells were administered to mice within 90 minutes after electroporation.
[0226] Twelve hours after electroporation, a small aliquot of the cells were stained for expression of anti-GPC3CAR-CD79a and CD80 expression. For detection of anti-GPC3CAR-CD79a expression, GPC3-Avitag and Streptavidin-BV421 were used. CD80 expression was measured with an anti-CD8O-PE FACS antibody. The FACS plots in FIGS. 22A-22C show expression of the GPC3 CAR post-electroporation. CD80 was expressed at a basal level in un-engineered B cells, thus accounting for the ¨10% positivity. This level remained in the CAR
sample, but was increased dramatically in the CAR + CD80 sample. The latter suggested efficient expression of CD80.

Claims

What is Claimed

1. An isolated modified B cell, capable of expressing a chimeric receptor (CAR-B), wherein said chimeric receptor comprises a) an extracellular domain, wherein the extracellular domain comprises an extracellular binding domain and a hinge domain;
b) a transmembrane domain; and c) a cytoplasmic domain that comprises at least one signaling domain.

2. The isolated modified B cell of claim 1, wherein said extracellular binding domain(s), recognizes at least one antigen or protein expressed on the surface of a target cell.

3. The isolated modified B cell of claim 1, wherein the extracellular binding domain(s) recognizes at least one antigen that is a secreted protein.

4. The isolated modified B cell of claim 3, wherein the secreted protein accumulates in the extracellular matrix.

5. The isolated modified B cell of claim 2, wherein said target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell, an endothelial cell, a hepatocyte, a pulmonary epithelial cell, and a fibroblast cell.

6. The isolated modified B cell of claim 1, wherein said B cell expresses more than one CAR-B receptor construct.

7. The isolated modified B cell of claim 1, wherein said CAR-B receptor comprises more than one extracellular binding domain.

8. The isolated modified B cell of claim 1, wherein said extracellular binding domain is a single chain variable fragment (scFv), or a full-length antibody or an antibody fragment, or the extracellular domain of a receptor or ligand.

9. The isolated modified B cell of claim 1, wherein said extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GPC3, ASGR1, ASGR2, Sarcoglycan, Corin, FAP, MUC1, CEA153, JAM-1, LAF-1 and Her2.

10. The isolated modified B cell of claim 1, wherein said hinge domain is derived from the group consisting of an immunoglobulin (e.g., IgG, IgM), CD28 and CD8.

11. The isolated modified B cell of claim 1, wherein said cytoplasmic domain comprises at least one signaling domain native to B cells receptors.

12. The isolated modified B cell of claim 1, wherein said cytoplasmic domain comprises at least one signaling domain that is an immunoglobulin.

13. The isolated modified B cell of claim 1, wherein said cytoplasmic domain comprises a domain that is selected from the group consisting of: CD79a (Immunoglobulin a), CD4O, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, CDX and BLNK.

14. The isolated modified B cell of claim 13, wherein said cytoplasmic domain comprises domain that is CD79a.

15. The isolated modified B cell of claim 13, wherein said cytoplasmic domain further comprises a costimulatory domain.

16. An isolated modified B cell, wherein said B cell is capable of expressing and secreting a payload, wherein the payload is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell.

17. The isolated modified B cell of claim 16, wherein the payload is an antibody or fragment thereof

18. The isolated modified B cell of claim 17, wherein the antibody is a secreted antibody or fragment thereof

19. The isolated modified B cell of claim 17, wherein the antibody is membrane bound.

20. The modified B cell of claim 16, wherein said payload is selected from a group of cytokines, chemokines, T cell costimulatory molecules, and checkpoint molecules, the group consisting of: IL-1, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, IL-21, interferon a, interferon [3, interferon y, TSLP, CCL21, FLT3L, XCL1, LIGHT(TNFSF14), OX4OL, CD137L, CD4OL, ICOSL, anti-CD3 antibody, CD47, TIM4-FC, CXCL13, CCL21, CD80, CD4OL, IFNa A2, LIGHT
, 4-1BBL, MDGF (C19orf10), FGF10, PDGF, agrin. TNF-a, GM-CSF, an anti-FAP
antibody, an anti-TGF-fl antibody; a TGF-0 trap, decoy or other inhibitory molecule; an anti-BMP antibody; a BMP trap, decoy or other inhibitory molecule.

21. The modified B cell of claim 16, wherein said B cell is capable of expressing more than one payload.

22. The modified B cell of claim 16, wherein said B cell is capable of expressing more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 payloads.

23. A method of treating a patient comprising administering the modified B
cell of claim 14.

24. The method of claim 23, wherein the modified B cell is administered intra-tumorally, intravenously, subcutaneously, intradermally, or within an inflammatory lesion

25. The method of claim 23, further comprising administering one or more checkpoint inhibitors, with or without an additional chemotherapeutic agent.

26. The method of claim 25, wherein the checkpoint inhibitor is capable of inhibiting a checkpoint molecule selected from the group consisting of: PD-1, PD-L1, CTLA-4, LAG3, TIM-3 and NKG2A.

27. The method of claim 23, further comprising administering a T cell costimulatory molecule.

28. The method of claim 27, wherein the T cell costimulatory molecule is selected from the group consisting of- CDRO, CD86, ICOSL, 4-1BBL, OX401., CD27, and LIGHT

29. An isolated modified B cell, capable of expressing a chimeric receptor, wherein said chimeric receptor comprises a) an extracellular domain, wherein the extracellular domain comprises an extracellular binding domain and a hinge domain;
b) a transmembrane domain; and c) a cytoplasmic domain that comprises at least one signaling domain wherein said modified B cell is further capable of expressing a payload, wherein the payload is not naturally expressed in a B cell.

30. The isolated modified B cell of claim 29, wherein said extracellular binding domain, recognizes an antigen or protein expressed on the surface of a target cell.

31. The isolated modified B cell of claim 29, wherein said extracellular binding domain, recognizes an antigen or protein that is secreted.

32. The isolated modified B cell of claim 29, wherein said target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell, an endothelial cell, a hepatocyte, a pulmonary epithelial cell and a fibroblast.

33. The isolated modified B cell of claim 29, wherein said B cell expresses more than one CAR-B receptor construct.

34. The isolated modified B cell of claim 29, wherein said CAR-B receptor comprises more than one extracellular binding domain.

35. The isolated modified B cell of claim 29, wherein said extracellular binding domain is a single chain variable fragment (scFv), a full-length antibody, an antibody fragment or an extracellular domain of a receptor or ligand.

36. The isolated modified B cell of claim 29, wherein said extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GP3, ASGR1, ASGR2, Sarcoglycan, Corin, and Her2, FAP, MUC1, CEA153, JAM-1, and LFA-1.

37. The isolated modified B cell of claim 29, wherein said hinge domain is derived from the group consisting of an immunoglobulin (for example, IgG or IgM), CD28 and CD8.

38. The isolated modified B cell of claim 29, wherein said cytoplasmic domain comprises at least one signaling domain native to B cells.

39. The isolated modified B cell of claim 29, wherein said cytoplasmic domain comprises at least one signaling domain that is an immunoglobulin.

40. The isolated modified B cell of claim 29, wherein said cytoplasmic domain comprises a domain selected from the group consisting of: CD79a (Immunoglobulin a)õ CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLC72, CDR, and BLNK.

41. The isolated modified B cell of claim 40, wherein the cytoplasmic domain comprises a domain that is CD79a (Immunoglobulin a).

42. The isolated modified B cell of claim 41, wherein said cytoplasmic domain further comprises a costimulatory domain.

43. The isolated modified B cell of claim 29, wherein the payload is a secreted or membrane bound antibody or fragment thereof

44. The isolated modified B cell of claim 29, wherein said payload is selected from a group of cytokines, chemokines, T cell costimulatory molecules, and checkpoint molecules, the group consisting of: IL-1, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, IL-21, interferon a, interferon [3, interferon y, TSLP, CCL21, FLT3L, XCL1, LIGHT(TNFSF14), OX4OL, CD137L, CD4OL, ICOSL, anti-CD3 antibody, CD47, TIM4-FC, CXCL13, CCL21, CD80, CD4OL, IFNa A2, LIGHT
, 4-1BBL, MDGF (C19orf10), FGF10, PDGF, agrin, GM-CSF, an anti-FAP
antibody, an anti-TGF-I3 antibody; a TGF-I3 trap, decoy or other inhibitory molecule; an anti-BMP antibody; a BMP trap, decoy or other inhibitory molecule.

45. The isolated modified B cell of claim 29, wherein said B cell is capable of expressing more than one payload.

46. The isolated modified B cell of claim 29, wherein said B cell is capable of expressing more than 1, 2, 3, 4, 5, 6, 7, g, 9, 10, 11, or 12 payloads

47. The isolated modified B cell of claim 29, wherein the modified B cell further encodes at least one protein selected from the group consisting of: the cytoplasmic domains of CD79a (Immunoglobulin a), CD79b (Immunog1obulin13), CD40, CD19, CD137, Fc7r2a, CD3c;
and MyD88.

48. A method of treating a patient comprising administering the modified B
cell of claim 29.

49. The method of claim 48, further comprising administering a checkpoint inhibitor.

50. The method of claim 48, wherein the checkpoint inhibitor is capable of inhibiting a checkpoint molecule selected from the group consisting of: PD-1, PD-L1, CTLA-4, LAG3, TIM-3 and NKG2A.

51. The method of claim 50, wherein the checkpoint inhibitor is a monoclonal antibody.

52. An isolated modified B cell, capable of expressing a chimeric receptor, wherein said chimeric receptor comprises an extracellular domain, wherein said extracellular domain comprises a hinge domain and an extracellular binding domain, wherein said extracellular binding domain is not naturally expressed on a B cell; and wherein said extracellular binding domain is capable of recognizing a target of interest.

53. The isolated modified B cell of claim 52, wherein said binding domain is a single chain variable fragment (scFv), a full length antibody, an antibody fragment, an extracellular domain of a ligand or receptor.

54. The isolated modified B cell of claim 53, wherein said binding domain is an scFv.

55. The isolated modified B cell of claim 52, wherein herein said B cell is capable of expressing more than one chimeric receptor.

56. The isolated modified B cell of claim 52, wherein said B cell is further capable of expressing a payload.

57. A method of treating a patient comprising administering the modified B
cell of claim 52.

58. A nucleic acid capable of expressing a chimeric B cell receptor, wherein said chimeric receptor comprises:
a) an extracellular domain, wherein said extracellular domain comprises an extracellular binding domain and a hinge domain;
b) a transmembrane domain; and c) a cytoplasmic domain that comprises at least one signaling domain.

59. The nucleic acid of claim 58, wherein said extracellular binding domain, recognizes an antigen or protein expressed on the surface of a target cell.

60. The nucleic acid of claim 58, wherein said extracellular binding domain, recognizes an antigen or protein that is a secreted protein.

61. The nucleic acid of claim 58, wherein the secreted protein accumulates in the extracellular matrix.

62. The nucleic acid of claim 58, wherein said extracellular binding domain is a single chain variable fragment (scFv), a full length antibody, an antibody fragment or an extracellular domain of a receptor or ligand.

63. The nucleic acid of claim 58, wherein said target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell, an endothelial cell, a hepatocyte, a pulmonary epithelial cell or a fibroblast.

64. The nucleic acid of claim 58, wherein said vector expresses more than one CAR-B
receptor.

65. The nucleic acid of claim 58, wherein said CAR-B receptor expresses more than one extracellular binding domain.

66. The nucleic acid of claim 58, wherein said extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GP3, ASGR1, AGSR2, Sarcoglycan, Corin, Her2, PAFT, MUCT, CEA153, JAM-1, and LFA-1.

67. The nucleic acid of claim 58, wherein said hinge domain is derived from the group consisting of an immunoglobulin domain (for example, TgG or IgM), CD28 and CDR.

68. The nucleic acid of claim 58, wherein said cytoplasmic domain comprises at least one signaling domain native to B cell receptors.

69. The nucleic acid of claim 58, wherein said cytoplasmic domain comprises a domain selected from the group consisting of: CD79a (Immunoglobulin a), CD79b (Immunoglobulin 13), CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, C.D3,: and BLNK.

70. The nucleic acid of claim 69, wherein said cytoplasmic domain comprises a domain that is CD79a (Immunoglobulin a).

71. The nucleic acid of claim 69, wherein said cytoplasmic domain further comprises a costimulatory domain.

72. A vector comprising a nucleic acid capable of expressing a chimeric B
cell receptor, wherein said chimeric receptor comprises:
a) an extracellular domain, wherein said extracellular domain comprises an extracellular binding domain and a hinge domain;
b) a transmembrane domain; and c) a cytoplasmic domain that comprises at least one signaling domain.

73. The vector of claim 72, wherein said extracellular binding domain recognizes an antigen or protein expressed on the surface of a target cell.

74. The vector of claim 72, wherein said extracellular binding domain, recognizes an antigen or protein that is a secreted protein.

75. The vector of claim 72, wherein the secreted protein accumulates in the extracellular matrix.

76. The vector of claim 72,wherein said target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell , an endothelial cell, a hepatocyte, a pulmonary epithelial cell or a fibroblast.

77. The vector of claim 72, wherein said vector expresses more than one CAR-B receptor.

78. The vector of claim 72, wherein said CAR-B expresses more than one extracellular binding domain.

79. The vector of claim 72, wherein said extracellular binding domain is a single chain variable fragment (scFv), a full-length antibody, an antibody fragment, an extracellular domain of a receptor or ligand.

80. The vector of claim 72, wherein said extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GPC3, ASGR1, AGSR2, Sarcoglycan, Corin, Her2, PAF1, MUC1, CEA153, JAM-1, and LFA-1.

81. The vector of claim 72, wherein said hinge domain is derived from the group consisting an immunoglobulin domain (for example, IgG or IgM), CD28 and CD8.

82. The vector of claim 72, wherein said cytoplasmic domain comprises at least one signaling domain native to B cells.

83. The vector of claim 72, wherein said cytoplasmic domain comprises a signaling domain selected from the group consisting of: CD79a (Immunoglobulin a), CD79b (Immunog1obu1inf3), CD40, CD19, CD137, Fcyr2a, MyD88, CD21, Syk, FYN, LYN, PI3K, BTK, PLCy2, CD.V:
and BLNK.

84. The vector of claim 72, wherein said cytoplasmic domain further comprises a costimulatory domain.

85. An isolated modified B cell, capable of expressing an integrin, a homing antibody, protein, a receptor, or combinations thereof, wherein said integrin, homing antibody, protein, or receptor is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell; and wherein said integrin, homing antibody, protein, receptor, or combinations thereof is attracted to a site or target of interest.

86. The isolated modified B cell of claim 85, wherein the integrin, homing antibody, protein, and receptor is selected from CLA (PSGL-1 glycoform), CLA (PSGL-1 glycoform), CCRIO, CCR3, CCR4, CCR5, CCR6, CCR9, CD43E, CD44, c-Met, CXCR3, CXCR4, LFA-1, (aL132), selectin ligands, VLA-4, VLA-4 (a4f31), and a4137, or combinations thereof

87. The isolated modified B cell of claim 86, wherein the site of interest is a homing or target tissue, an inflammatory site in a specific location or tissue, or a tumor or tumor microenvironment, where delivery of payloads is desirable.

88. The isolated modified B cell of claim 87, wherein the homing or target tissue is selected from skin, gut (intestine, colon, mesenteric lymph nodes (rnLN), Peyer's Patch (PP), small intestine), liver, lung, bone marrow, heart, peripheral lymph node (LN), CNS, thymus, and bone marrow.

89. The isolated modified B cell of claim 88, wherein the target of interest is selected from CXCL16, CCL17, CCL17(22), CCL2O (MIP-3a), CCL21, CCL25, CCL27, CCL28, CCL4, CCL5, CD62E, CD62P, CXCL10, CXCL12, CXCL13, CXCL16, CXCL9/CXCL10, CXCR3, E/P-selectin, E-selectin, GPR15L, HGF, Hyaluronate, ICAM-1, ligands for CCR1, 2, 5, MAdCAM, MAdCAM-1, PNAd, VAP-1, VCAM, and VCAM-1, or combinations thereof

90. A method of treating a patient comprising administering the isolated modified B cell of claim 82.

91. The method of claim 90, further comprising administering a compound or a derivative thereof, wherein the compound or derivative thereof is capable of modulating the expression of the integrin, homing antibody, protein, and receptor, or combinations thereof

92. The method of claim 91, wherein the compound or a derivative thereof is capable of altering trafficking of B cells to a site or target of interest in the patient.

93. The method of claim 92, wherein the compound is all-trans-retinoic acid (ATRA) or a derivative thereof

94. An isolated modified B cell, capable of expressing an immune inhibitory molecule, wherein said immune inhibitory molecule is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell.

95. The isolated modified B cell of claim 94, wherein said immune inhibitory molecule is selected from IL-10, TGF-f3, PD-L1, PD-L2, LAG-3, and TIM-3, or combinations thereof.

96. The isolated modified B cell of claim 95, wherein said immune inhibitory molecule is capable of decreasing inflammation and autoimmune activity of B cells at a site or target of interest in a patient.

97. A method of treating a patient comprising administering the isolated modified B cell of claim 94.

98. The method of claim 97, wherein said immune inhibitory molecule is selected from IL-10, TGF-f3, PD-L1, PD-L2, LAG-3, and TIM-3, or combinations thereof

99. The method of claim 98, wherein said immune inhibitory molecule is capable of decreasing inflammation and autoimmune activity of B cells at a site or target of interest in the patient.

100. The method of claim 99, further comprising administering a compound or a derivative thereof capable of increasing the expression of an integrin, a homing antibody, a protein, a receptor, or combinations thereof in the B cell.

101. The method of claim 100, wherein said compound or derivative thereof is capable of altering trafficking of B cells to a site or target of interest in the patient.

102. The method of claim 101, wherein said compound is all-trans-retinoic acid (ATRA) or a derivative thereof

103. An isolated modified B cell, wherein the isolated modified B cell is treated with a compound or a derivative thereof, wherein said compound or derivative thereof is capable of increasing the expression of an integrin, a homing antibody, a protein, a receptor, or combinations thereof in B cells.

104. The isolated modified B cell of claim 103, wherein said compound or derivative thereof is capable of altering trafficking of B cells to a site or target of interest in the patient.

105. The isolated modified B cell of claim 104, wherein said compound is all-trans-retinoic acid (ATRA) or a derivative thereof

106 A method of treating a patient comprising administering the isolated modified B cell of claim 100, wherein said compound or derivative thereof is capable of (i) modulating the expression of an integrin, a homing antibody, a protein, a receptor, or combinations thereof in B cells, and (11) altering trafficking of B cells to a site or target of interest in the patient.

107. The method of claim 106, wherein said compound is all-trans-retinoic acid (ATRA) or a derivative thereof

108. An isolated modified B cell, capable of expressing at least one or more of a constitutively active Toll-like receptor (TLR), wherein said TLR is not naturally expressed in a B cell or is expressed at higher levels than is naturally expressed in a B cell.

109. The isolated modified B cell of claim 108, wherein said TLR is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, or combinations thereof.

110. The isolated modified B cell of claim 109, wherein said TLR is capable of potentiating B
cells for increasing immune responses in a patient.

111. The isolated modified B cell of claim 110, wherein said TLR is capable of producing potent effector B cells for increasing immune responses in a patient.

112. A method of treating a patient comprising administering the isolated modified B cell of claim 108, wherein said immune inhibitory molecule is capable of decreasing inflammation and autoimmune activity of B cells at a site or target of interest in a patient.

113. The method of claim 112, wherein said TLR is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, or combinations thereof

114. The method of claim 113, wherein said TLR is capable of (i) potentiating B cells, and (ii) producing potent effector B cells, for increasing immune responses in a patient.

115. The method of claim 114, further comprising administering at least one or more of a TLR
agonist to the patient.

116. An isolated modified B cell, wherein the isolated modified B cell is treated with at least one or more of a TLR agonist

117. The isolated modified B cell of claim 116, wherein said TLR agonist is capable of (i) potentiating B cells, and (n) producing potent effector B cells, for increasing immune responses in a patient.

118. The isolated modified B cell of claim 117, wherein said TLR agonist hinds -to one or more TLRs selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, or combinations thereof

119. The isolated modified B cell of claim 118, wherein said TLR agonist is selected from CpG-rich oligonucleotides, double-stranded RNA mimic, polyinosinic acid:polycytidylic acid (poly-I:C).

120. The isolated modified B cell of claim 119, wherein said TLR agonist comprises CpG
oligonucleotides.

121. A method of treating a patient comprising administering the isolated modified B cell of claim 116.

122. The method of claim 121, wherein said TLR agonist is capable of is capable of (i) potentiating B cells, and (ii) producing potent effector B cells, for increasing immune responses in the patient.

123. The method of claim 122, wherein said TLR agonist binds to one or more TI_Rs selected from TLRI, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, or combinations thereof

124. The method of claim 123, wherein said TLR agonist is selected from CpG-rich oligonucleotides, double-stranded RNA mimic, polyinosinic acid:polycytidylic acid (poly-I:C).

125. The method of claim 124, wherein said TLR agonist comprises CpG
oligonucleotides.

126. An isolated modified B cell, wherein said B cell is electroporated with an mRNA
encoding at least one or more of an antigen fused to a targeting signal.

127. The isolated modified B cell of claim 126, wherein said antigen is (i) not naturally presented by a B cell, (ii) not presented by a B cell simultaneously in both HLA class I and class II
molecules naturally, or (iii) not presented by a B cell with high efficiencies in both HLA class I and class II molecules naturally.

128. The isolated modified B cell of claim 127, wherein said targeting signal is targeting signal of a lysosomal protein.

129. The isolated modified B cell of claim 128, wherein said targeting signal is a targeting signal of lysosome-associated membrane protein-1 (LAMP1).

130. The isolated modified B cell of claim 129, wherein said antigen is capable of being targeted to the lysosomes and presented simultaneously and efficiently in both HLA class I and class II molecules.

131. The isolated modified B cell of claim 130, wherein said B cells is capable of increasing antigen-specific immune responses in a patient.

132. A method of treating a patient comprising administering the isolated modified B cell of claim 126.

133. The method of claim 132, wherein said antigen is (i) not naturally presented by a B cell, (ii) not presented by a B cell simultaneously by both HLA class 1 and class 11 molecules naturally, or (iii) not presented by a B cell with high efficiencies by both HLA class I
and class II molecules naturally.

134. The method of claim 133, wherein said targeting signal is targeting signal of a lysosomal protein.

135. The method of claim 134, wherein said targeting signal is a targeting signal of lysosome-associated membrane protein-1 (LAMP1).

136. The method of claim 135, wherein said antigen is capable of being targeted to the lysosomes and presented simultaneously and efficiently in both HLA class I and class II molecules.

137. The method of claim 136, wherein said B cells is capable of increasing antigen-specific immune responses in the patient.

138. The method of any of the claims 132-137, wherein the patient is treated with more than one differently modified B cells

139. The method of any one of the claims 132-137, wherein the patient is treated with more than two differently modified B cells.

140. An isolated modified B cell, capable of expressing a chimeric receptor (CAR-B), wherein said chimeric receptor comprises a) an extracellular domain, wherein the extracellular domain comprises an extracellular binding domain and a hinge domain;

b) a transmembrane domain; and c) a cytoplasmic domain.

141. The isolated modified B cell of claim 140, wherein the extracellular domain comprises an immunoglobulin domain.

142. The isolated modified B cell of claim 140, wherein the chimeric receptor is capable of forming a homologous dimer.

143. The isolated modified B cell of claim 140, wherein the extracellular binding domain, recognizes an antigen or protein expressed on the surface of a target cell.

144. The isolated modified B cell of claim 143, wherein said extracellular binding domain, recognizes an antigen or protein that is secreted.

145. The isolated modified B cell of claim 143, wherein said target cell is selected from the group consisting of a tumor cell, a cardiac muscle cell, a skeletal muscle cell, a bone cell, a blood cell, a nerve cell, a fat cell, a skin cell, an endothelial cell, a hepatocyte, a pulmonary epithelial cell and a fibroblast.

146. The isolated modified B cell of claim 143, wherein said extracellular binding domain is a single chain variable fragment (scFv), a full-length antibody, an antibody fragment or an extracellular domain of a receptor or ligand.

147. The isolated modified B cell of claim 143, wherein said extracellular binding domain is capable of binding to an antigen or protein selected from the group consisting of: PSMA, GP3, ASGR1, ASGR2, Sarcoglycan, Corin, and Her2, FAP, MUC1, CEA153, JAM-1, and LFA-1.

148. The isolated modified B cell of claim 14, wherein said hinge domain is derived from the group consisting of an immunoglobulin (for example, IgG or IgM), CD28 and CD8.

149. An isolated modified B cell, capable of expressing a chimeric receptor (CAR-B), wherein said chimeric receptor comprises the amino acid sequence SEQ ID NO: 146.

150. An isolated modified B cell, capable of expressing a chimeric receptor (CAR-B), wherein said chimeric receptor comprises the amino acid sequence SEQ ID NO: 142, SEQ ID NO:
144 or both.