WO2023020475A1

WO2023020475A1 - Cd40-targetting antibodies and uses thereof

Info

Publication number: WO2023020475A1
Application number: PCT/CN2022/112730
Authority: WO
Inventors: Yangbing Zhao; Jie Wang; Xiaojun Liu; Gengzhen ZHU
Original assignee: Utc Therapeutics (Shanghai) Co., Ltd.
Priority date: 2021-08-16
Filing date: 2022-08-16
Publication date: 2023-02-23

Abstract

Disclosed herein are novel anti-CD40 antibodies and antigen-binding fragments. Disclosed herein are also fusion proteins comprising a first domain that activates an antigen-presenting cell (APC) (e.g., a dendritic cell) and a second domain that activates an immune effector cell (e.g., a T cell), wherein the first domain comprises an anti-CD40 antibody and antigen-binding fragment disclosed herein. Polynucleotides encoding the antibodies and antigen-binding fragments, and polynucleotides encoding the fusion proteins the fusion proteins are also disclosed. Pharmaceutical compositions comprising the antibodies or antigen-binding fragments or fusion proteins, genetically engineered immune effector cells expressing such fusion protein, methods of their production, and their uses in treatment of diseases such as cancers are also provided herein.

Description

CD40-TARGETTING ANTIBODIES AND USES THEREOF

1. Field

The present invention relates to molecular biology and immuno-oncology. Provided herein include anti-CD40-antibodies and uses thereof in treating tumors or cancers.

2. Background

T cells can be engineered to express T cell receptors (TCRs) (Morgan RA et al., Science (2006) 314 (5796) : 126-129; Robbins PF et al., J Clin Oncol (2011) 29 (7) : 917-924; Rapoport AP et al., Nature Medicine (2015) 21 (8) : 914-921) or chimeric antigen receptor (CAR) (Kochenderfer JN et al., Blood (2010) 116 (20) : 4099-4102; Kalos M et al., Science Translational Medicine (2011) 3 (95) : 95ra73) that recognize disease-specific antigens for the treatment of cancers and other diseases. Although T cells engineered with CARs specific to the B cell markers, such as CD19, showed dramatic clinical responses in hematological malignancies, effective immunotherapy in solid cancers has proven to be challenging, mainly due to the immune escape caused by complex, dynamic tumor microenvironment (TME) that induces T cell hypofunction and exhaustion and limits the antitumor immune response (Anderson KG et al, Cancer Cell (2017) 31 (3) : 311-325) . Thus, strategies to circumvent suppressive pathways without causing systemic toxicities represent unmet need.

Human cancers and chronic infections can be treated with agents that modulate the patient’s immune response to malignant or infected cells. Anti-CD40 antibodies have been tried for treating cancer because they can enhance immune responses. See, e.g., Kirkwood et al. (2012) CA Cancer J. Clin. 62: 309; Vanderheide &Glennie (2013) Clin. Cancer Res. 19: 1035. The need exists for improved agonistic anti-human CD40 antibodies for treatment of cancer and chronic infections in human subjects.

The anti-CD40 antibodies, fusion proteins, and related compositions and methods provided herein meet these needs and provide other relative advantages.

3. Summary

Provided herein are antibodies and antigen-binding fragments thereof that specifically bind human CD40, comprising: (a) a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6; (2) a light chain CDR2 (VL CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12; and (3) a light chain CDR3 (VL CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VH CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-30; and (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments of the antibodies and antigen-binding fragments provided herein, (a) the VL CDR1, CDR2 and CDR3 have (1) the amino acid sequences of SEQ ID NOs: 1, 7, and 13, respectively; (2) the amino acid sequences of SEQ ID NOs: 2, 8, and 14, respectively; (3) the amino acid sequences of SEQ ID NOs: 3, 9, and 15, respectively; (4) the amino acid sequences of SEQ ID NOs: 4, 10, and 16, respectively; (5) the amino acid sequences of SEQ ID NOs: 5, 11, and 17, respectively; or (6) the amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) the VH CDR1, CDR2 and CDR3 have (1) the amino acid sequences of SEQ ID NOs: 19, 25, and 31, respectively; (2) the amino acid sequences of SEQ ID NOs: 20, 26, and 32, respectively; (3) the amino acid sequences of SEQ ID NOs: 21, 27, and 33, respectively; (4) the amino acid sequences of SEQ ID NOs: 22, 28, and 34, respectively; (5) the amino acid sequences of SEQ ID NOs: 23, 29, and 35, respectively; or (6) the amino acid sequences of SEQ ID NOs: 24, 30, and 36, respectively; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments of the antibodies and antigen-binding fragments provided herein, (1) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 7, and 13, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 19, 25, and 31, respectively; (2) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 8, and 14, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 20, 26, and 32, respectively; (3) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 3, 9, and 15, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 21, 27, and 33, respectively; (4) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 10, and 16, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 22, 28, and 34, respectively; (5) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 5, 11, and 17, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 23, 29, and 35, respectively; or (6) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 24, 30, and 36, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 10, 16, 22, 28, and 34, respectively. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3 having the amino acid sequences of SEQ ID NOs: 6, 12, 18, 24, 30, and 36, respectively

Provided herein are antibodies and antigen-binding fragments thereof that specifically bind human CD40, comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and/or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48.

In some embodiments of the antibodies and antigen-binding fragments provided herein, the VL and VH each have at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequences of (1) SEQ ID NOs: 37 and 43, respectively; (2) SEQ ID NOs: 38 and 44, respectively; (3) SEQ ID NOs: 39 and 45, respectively; (4) SEQ ID NOs: 40 and 46, respectively; (5) SEQ ID NOs: 41 and 47, respectively; or (6) SEQ ID NOs: 42 and 48, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH, wherein the VL and VH each have at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequences of SEQ ID NOs: 40 and 46, respectively. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise g a VL and a VH, wherein the VL and VH each have at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequences of SEQ ID NOs: 42 and 48, respectively.

Provided herein are antibodies and antigen-binding fragments thereof that specifically bind human CD40, comprising (a) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and/or (b) a VH comprising VH CDR1, CDR2, and CDR3 from a VH having an amino acid sequence selected from group consisting of SEQ ID NOs: 43-48.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise (1) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 37, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 43; (2) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 38, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 44; (3) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 39, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 45; (4) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 40, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 46; (5) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 41, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 47; or (6) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 42, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 48.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 40, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 42, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 48.

Provided herein are also antibodies and antigen-binding fragments thereof that compete with an antibody or antigen-binding fragment described herein for binding to human CD40.

In some embodiments, the antibody or antigen-binding fragment provided herein is a monoclonal antibody or antigen-binding fragment. In some embodiments, the antibody provided herein selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody. In some embodiments, the antibody or antigen-binding fragment provided herein is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) . In some embodiments, the antibody or antigen-binding fragment provided herein is a scFv.

In some embodiments, the antibody or antigen-binding fragment provided herein is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment provided herein is a human antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment provided herein is a bispecific antibody or a multispecific antibody.

Provided herein are also polynucleotides encoding an antibody or antigen-binding fragment described herein. Provided herein are also vectors comprising a polynucleotide described herein.

Provided herein are fusion proteins comprising a first domain and a second domain, wherein (i) the first domain comprises the anti-CD40 antibody or antigen-binding fragment described herein; and (ii) the second domain activates an immune effector cell and comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments of fusion proteins provided herein, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, the N-terminus of the second domain is linked to the C-terminus of the first domain. In some embodiments, the first domain and the second domain are linked via a linker.

In some embodiments of fusion proteins provided herein, the second domain comprises a cytoplasmic domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain comprises a cytoplasmic domain of CD28. In some embodiments, the second domain comprises a cytoplasmic domain of 4-1BB. In some embodiments, the second domain further comprises the transmembrane domain of the co-stimulatory receptor.

In some embodiments of fusion proteins provided herein, the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.

In some embodiments of fusion proteins provided herein, the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain is an antibody that binds CD28. In some embodiments, the antibody that binds CD28 is a scFv having the amino acid sequence of SEQ ID NO: 161.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 67. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 68. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 69. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 70. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 71. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 72.

Provided herein are polynucleotides that encode a fusion protein described herein. Provided herein are also vectors that comprise the polynucleotide described herein. The vector can be a viral vector.

Provided herein are genetically engineered immune effector cells that recombinantly express the fusion protein described herein, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.

In some embodiments, the cells further recombinantly express a chimeric antigen receptor (CAR) , a T cell receptor (TCR) or a Bi-specific T-cell engager (BiTE) , wherein the CAR, TCR or BiTE binds a tumor antigen or a viral antigen.

Provided herein are also genetically engineered immune effector cells comprising the polynucleotide that encode a fusion protein described herein or the vector that comprise the polynucleotide described herein, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.

In some embodiments, the cells further comprise a polynucleotide that encodes a CAR, a TCR, or BiTE, wherein the CAR, TCR or BiTE binds a tumor antigen or a viral antigen.

In some embodiments, the CAR, TCR or BiTE binds a viral antigen selected from the group consisting of HPV, EBV, and HIV. In some embodiments, the CAR, TCR or BiTE binds a tumor antigen selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin.

In some embodiments, the CAR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-93 and 169. In some embodiments, the TCR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-101. In some embodiments, the BiTE has an amino acid sequence selected from the group consisting of SEQ ID NO: 102, 103, and 167.

In some embodiments, the cells provided herein are derived from cells isolated from peripheral blood or bone marrow. In some embodiments, the cells provided herein are derived from cells differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.

In some embodiments, the cell provided herein is a T cell. In some embodiments, the T cell is a cytotoxic T cell, a helper T cell, or a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, an effector memory TEMRA cell, or a gamma delta T cell.

Provided herein are populations of the genetically engineered immune effector cell described herein, which are derived from cells isolated from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .

Provided herein are pharmaceutical compositions comprising the antibody or antigen-binding fragment described herein, and a pharmaceutically acceptable excipient. Provided herein are also pharmaceutical compositions comprising the fusion protein described herein, and a pharmaceutically acceptable excipient. Provided herein are also pharmaceutical compositions comprising the cell or population of cells described herein, and a pharmaceutically acceptable excipient.

Provided herein are uses of the antibodies or antigen-binding fragments described herein or the fusion protein described herein in cancer treatment. Provided herein are also uses of the antibodies or antigen-binding fragments described herein or the fusion proteins described herein for the preparation of a medicament for the treatment of cancer. In some embodiments, the fusion protein is used in combination with an immune effector cell. In some embodiments, the immune effector cell is selected from the group consisting of a CAR T cell, a TCRT cell, a TIL, a CIK, a LAK, and a MIL.

Provided herein are uses of the cells or populations of cells described herein in cancer treatment. Provided herein are also uses of the cell or population of cells described herein for the preparation of a medicament for the treatment of cancer.

In some embodiments of the uses described herein, the antibody or antigen-binding fragment, the fusion protein, the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.

In some embodiments, provided herein are methods of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment described herein or the fusion protein described herein to the subject. In some embodiments, the methods further comprise administering a cell therapy to the subject. In some embodiments, the cell therapy is selected from the group consisting of a CAR T therapy, a TCRT therapy, a TIL therapy, a CIK therapy, a LAK therapy, and a MIL therapy.

Provided herein are methods of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the cell or population of cells described herein to the subject. In some embodiments, the methods further comprise administering an additional therapy to the subject.

In some embodiments of the methods provided herein, the subject is a human.

In some embodiments of the methods or uses described herein, the fusion protein, cell, population of cells, or pharmaceutical composition reduces cancer-induced immunosuppression.

In some embodiments of the methods or uses described herein, the cancer is a hematological cancer. In some embodiments, the cancer is a solid tumor.

In some embodiments, provided herein are methods of genetically engineering an immune effector cell comprising transferring the polynucleotide described herein into the cell. The immune effector cell can be selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell. In some embodiments, the polynucleotide is transferred via electroporation. In some embodiments, the polynucleotide is transferred via viral transduction. In some embodiments, the polynucleotide is transferred using a transposon system. In some embodiments, the polynucleotide is transferred using gene-editing. In some embodiments, the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.

4. Brief Description of Drawings

FIG. 1 provides results of five representative 96-well plate of anti-human CD40-Fc monoclonal phage ELISA. Colony 18#, 37#, 38#, 45#, 47#and 52#produced the scFv (s) designated as 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52, which were selected for further studies.

FIG. 2 provides FACS staining results showing the binding of the anti-CD40 scFv (s) expressed in CAR-T cells to CD40-Fc protein.

FIG. 3 provides the killing curves of different mRNA-based CD40 scFv + anti-Her2 CART cells against A549-GFP tumor cells at different E/T ratio.

FIGs. 4A-4C provide CD107a staining of CART cells in the coculture and killing assay with A549 cells (FIG. 4A) , PC-3 (FIG. 4B) and SK-OV3 (FIG. 4C) .

5. Detailed Description

Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting.

The present disclosure provides novel antibodies, including antigen-binding fragments that specifically bind CD40 (e.g., human CD40) . Pharmaceutical compositions comprising a therapeutically effective amount of such antibodies or antigen-binding fragments are also disclosed herein. Also disclosed herein are uses of such pharmaceutical compositions for treating cancer (e.g., CD40-expressing cancer) and methods of cancer treatment.

The term “CD40” includes any variants or isoforms of CD40 which are naturally expressed by cells. Accordingly, antibodies described herein can cross-react with CD40 from species other than human (e.g., cynomolgus CD40) . Alternatively, the antibodies can be specific for human CD40 and do not exhibit any cross-reactivity with other species. CD40 or any variants and isoforms thereof, can either be isolated from cells or tissues which naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.

CD40 is a 48 kD transmembrane glycoprotein surface receptor that is a member of the Tumor Necrosis Factor Receptor superfamily (TNFRSF) . Exemplary amino acid sequences of human CD40 are described (see, e.g., Accession: ALQ33424.1 GI: 957949089; SEQ ID NO: 108) , CD40 was initially characterized as a co-stimulatory receptor expressed on APCs that played a central role in B and T cell activation. The ligand for CD40, CD154 (also known as TRAP, T-BAM, CD40 Ligand or CD40L) is a type II integral membrane protein. CD40L has been reported to promote induction of dendritic cells and facilitate development of immunogenic responses. See, e.g., Elgueta R et al., Immunol Rev. (2009) 229 (1) : 10.1111; Ma D &Clark EA, Semin Immunol. 2009 21 (5) : 265–272; Borges L et al., J Immunol. (1999) 163: 1289-1297; Grewal I, Immunol Res. (1997) 16: 59-70. Exemplary polynucleotides that encode CD40 ligand and equivalents are described (see, e.g., Genbank Accession Nos. X65453 and L07414) , as are preparations, compositions, and methods of use (U.S. Pat. No. 6,290,972) .

5.1 Definitions

Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

The term “a” or “an” entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies.

The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B, ” “A or B, ” “A” (alone) , and B” (alone) . Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .

The term “antibody, ” and its grammatical equivalents as used herein refer to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule comprising an antigen-binding site (e.g., dual variable domain immunoglobulin molecules) as long as the antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. Unless expressly indicated otherwise, the term “antibody” as used herein include “antigen-binding fragment” of intact antibodies. The term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenic determining variable region of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F (ab’) 2, Fv, linear antibodies, single chain antibody molecules (e.g., scFv) , heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , disulfide-linked scFv (dsscFv) , diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD) , single variable domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , and single variable domain of heavy chain antibodies (VHH) , and bispecific or multispecific antibodies formed from antibody fragments. A “bispecific” antibody is an artificial hybrid antibody having two different antigen binding sites, which recognize and specifically bind two different targets. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai &Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990) ; Kostelny et al., J. Immunol. 148, 1547-1553 (1992) .

The term “humanized antibody” as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulin. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. In some instances, residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and/or binding capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (a) , delta (δ) , epsilon (ε) , gamma (γ) and mu (μ) , based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “variable domain” or “variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR) . The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. Numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C. ) 5thed. A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by a variety of methods/systems. These systems and/or definitions have been developed and refined over years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the regions of most hypervariability within the antibody variable (V) domains (Kabat et al, J. Biol. Chem. 252: 6609-6616 (1977) ; Kabat, Adv. Prot. Chem. 32: 1-75 (1978) ) . The Chothia definition is based on the location of the structural loop regions, which defines CDR region sequences as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) . Both terminologies are well recognized in the art. Additionally, the IMGT system is based on sequence variability and location within the structure of the variable regions. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Software programs (e.g., abYsis) are available and known to those of skill in the art for analysis of antibody sequence and determination of CDRs. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al, J. Mol. Biol. 273: 927-948 (1997) ; Morea et al, Methods 20: 267-279 (2000) ) . Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997) ) . Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable) or Chothia

(structural) designations, are set forth in the table below.

	Kabat ¹	Chothia ²	Loop Location
VHCDRl	31-35	26-32	linking B and C strands
VHCDR2	50-65	53-55	linking C’ and C” strands
VHCDR3	95-102	96-101	linking F and G strands
VLCDRl	24-34	26-32	linking B and C strands
VLCDR2	50-56	50-52	linking C’ and C” strands
VLCDR3	89-97	91-96	linking F and G strands

¹Residue numbering follows the nomenclature of Kabat et al., supra

²Residue numbering follows the nomenclature of Chothia et al., supra

One or more CDRs also can be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin can incorporate the CDR (s) as part of a larger polypeptide chain, can covalently link the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest. The CDR regions can be analyzed by, for example, abysis website (http: //abysis. org/) .

The terms “epitope” and “antigenic determinant” are used interchangeably herein an refer to the site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, such as a localized region on the surface of an antigen. The target molecule can comprise, a protein, a peptide, a nucleic acid, a carbohydrate, or a lipid. An epitope having immunogenic activity is a portion of a target molecule that elicits an immune response in an animal. An epitope of a target molecule having antigenic activity is a portion of the target molecule to which an antibody binds, as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. The term, “epitope” includes linear epitopes and conformational epitopes. A region of a target molecule (e.g., a polypeptide) contributing to an epitope can be contiguous amino acids of the polypeptide or the epitope can come together from two or more non-contiguous regions of the target molecule. The epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The term “specifically binds, ” as used herein, means that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. A binding moiety (e.g., antibody) that specifically binds a target molecule (e.g., antigen) can be identified, for example, by immunoassays, ELISAs, Bio-Layer Interferometry ( “BLI” ) , SPR (e.g., Biacore) , or other techniques known to those of skill in the art. Typically, a specific reaction will be at least twice background signal or noise and can be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity. A binding moiety that specifically binds a target molecule can bind the target molecule at a higher affinity than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a target molecule can bind the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a particular target molecule binds a different molecule at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art. In some embodiments, “specifically binds” means, for instance, that a binding moiety binds a molecule target with a K _D of about 0.1 mM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a K _D of at about 10 μM or less or about 1 μM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a K _D of at about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a binding moiety (e.g., antibody) that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target. Thus, a binding moiety (e.g., antibody) can, in some embodiments, specifically bind more than one target. For example, an antibody can, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities.

The term “binding affinity” as used herein generally refers to the strength of the sum total of noncovalent interactions between a binding moiety and a target molecule (e.g., antigen) . The binding of a binding moiety and a target molecule is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (K _D) . K _D is the ratio of a dissociation rate (k _off or k _d) to the association rate (k _on or k _a) . The lower the K _D of a binding pair, the higher the affinity. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In some embodiments, the “K _D” or “K _D value” can be measured by assays known in the art, for example by a binding assay. The K _D may be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293: 865-881) . The K _D or K _D value can also be measured by using biolayer interferometry (BLI) using, for example, the Gator system (Probe Life) , or the Octet-96 system (Sartorius AG) . The K _D or K _D value can also be measured by using surface plasmon resonance assays by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ) .

The term “variant” as used herein in relation to a protein or a polypeptide with particular sequence features (the “reference protein” or “reference polypeptide” ) refers to a different protein or polypeptide having one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to the reference protein or reference polypeptide. The changes to an amino acid sequence can be amino acid substitutions. The changes to an amino acid sequence can be conservative amino acid substitutions. A functional fragment or a functional variant of a protein or polypeptide maintains the basic structural and functional properties of the reference protein or polypeptide.

The terms “polypeptide, ” “peptide, ” “protein, ” and their grammatical equivalents as used interchangeably herein refer to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids. A polypeptide, peptide, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

The terms “polynucleotide, ” “nucleic acid, ” and their grammatical equivalents as used interchangeably herein mean polymers of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

The terms “identical, ” percent “identity, ” and their grammatical equivalents as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

The term “vector, ” and its grammatical equivalents as used herein refer to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product (e.g., an anti-CD40 antibody or antigen-binding fragment as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

As used herein, the term “encode” and its grammatical equivalents refer to the inherent property of specific sequences of nucleotides in a polynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

A polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

The term “treat” and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated. For example, when used in reference to a cancer or tumor, the term “treat” and its grammatical equivalents refer to an action that reduces the severity of the cancer or tumor, or retards or slows the progression of the cancer or tumor, including (a) inhibiting the growth, or arresting development of the cancer or tumor, (b) causing regression of the cancer or tumor, or (c) delaying, ameliorating or minimizing one or more symptoms associated with the presence of the cancer or tumor.

The term “administer” and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art. The therapeutic can be a compound, a polypeptide, an antibody, a cell, or a population of cells. Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV) , intramuscular (IM) , or intraperitoneal (IP) ; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

The terms “effective amount, ” “therapeutically effective amount, ” and their grammatical equivalents as used herein refer to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required vary from subject to subject, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a material that is suitable for drug administration to an individual along with an active agent without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.

The term “subject” as used herein refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can have a particular disease or condition.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention.

Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOS. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to

GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .

5.2 Anti-CD40 antibodies and antigen-binding fragments

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 (e.g., human CD40) . In some embodiments, provided herein are anti-CD40 antibodies. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibodies provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

In some embodiments, provided herein are antigen-binding fragments of an anti-CD40 antibody. In some embodiments, antigen-binding fragments provided herein can be a single domain antibody (sdAb) , a heavy chain antibody (HCAb) , a Fab, a Fab’, a F (ab’) ₂, a Fv, a single-chain variable fragment (scFv) , or a (scFv) ₂. In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a single domain antibody (sdAb) . In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a heavy chain antibody (HCAb) . In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a Fab. In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a Fab’. In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a F (ab’) ₂. In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a Fv. In some embodiments, the antigen-binding fragment of an anti- CD40 antibody is a scFv. In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a disulfide-linked scFv [ (scFv) ₂] . In some embodiments, the antigen-binding fragment of an anti-CD40 antibody is a diabody (dAb) .

In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein comprise recombinant antibodies or antigen-binding fragments. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein comprise monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein comprise polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein comprise camelid (e.g., camels, dromedary and llamas) antibodies or antigen-binding fragments. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein comprise chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein comprise humanized antibodies or antigen-binding fragments. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein comprise human antibodies or antigen-binding fragments. In some embodiments, provided herein are anti-CD40 human scFvs.

In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein are isolated. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments provided herein are substantially pure.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment provided herein comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-CD40 antibody or antigen-binding fragment provided herein comprises a bispecific antibody or antigen-binding fragment. In some embodiments, provided herein is a Bi-specific T-cell engager (BiTE) . BiTEs are bispecific antibodies that bind to a T cell antigen (e.g., CD3) and a tumor antigen. BiTEs have been shown to induce directed lysis of target tumor cells and thus provide great potential therapies for cancers and other disorders. In some embodiments, provided herein are BiTEs that specifically bind CD3 and CD40. In some embodiments, the BiTEs comprises an anti-CD40 antibody or antigen-binding fragment provided herein. In some embodiments, the BiTEs comprises an anti-CD40 scFv provided herein.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment provided herein comprises a monovalent antigen-binding site. In some embodiments, an anti-CD40 antibody or antigen-binding fragment comprises a monospecific binding site. In some embodiments, an anti-CD40 antibody or antigen-binding fragment comprises a bivalent binding site.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those of skill in the art. One exemplary approach is screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228: 1315-1317; and WO 92/18619. In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable regions or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a human protein or a fragment thereof.

Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies directed to a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, BLI, SPR (e.g., Biacore) , and radioimmunoassay) . Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution or other techniques. The hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal. The monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

In some embodiments, monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art. For example, the polynucleotides encoding an antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

In some embodiments, a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region (s) is used to optimize specificity and/or affinity of a monoclonal antibody.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment. Various methods for generating humanized antibodies are known in the art. Methods are known in the art for achieving high affinity binding with humanized antibodies. A non-limiting example of such a method is hypermutation of the variable region and selection of the cells expressing such high affinity antibodies (affinity maturation) . In addition to the use of display libraries, the specified antigen (e.g., recombinant CD40 or an epitope thereof) can be used to immunize a non-human animal, e.g., a rodent. In certain embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be generated and isolated using methods known in the art and/or disclosed herein. In some embodiments, a mouse can be immunized with an antigen (e.g., recombinant CD40 or an epitope thereof) .

In some embodiments, an anti-CD40 antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In any case, cells that produce an antibody directed against a target antigen can be generated and isolated. In some embodiments, a human antibody is selected from a phage library, where that phage library expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well-known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, can be employed to generate higher affinity human antibodies. In some embodiments, human antibodies are produced in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

The specific CDR sequences defined herein are generally based on a combination of Kabat and Chothia definitions. However, it is understood that reference to a heavy chain CDR or CDRs and/or a light chain CDR or CDRs of a specific antibody encompass all CDR definitions as known to those of skill in the art.

Anti-CD40 antibodies or antigen-binding fragments provided herein include the followings scFv clones: 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52. The sequence features are described below.

In some embodiments, anti-CD40 antibodies or antigen-binding fragments provided herein comprise one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein. In some embodiments, anti-CD40 antibodies or antigen-binding fragments provided herein comprise one, two, three, four, five, and/or six CDRs of 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52. In some embodiments, anti-CD40 antibodies or antigen-binding fragments provided herein comprise a VL comprising one, two, and/or three, VL CDRs from Table 1. In some embodiments, anti-CD40 antibodies or antigen-binding fragments provided herein comprise a VH comprising one, two, and/or three VH CDRs from Table 2. In some embodiments, anti-CD40 antibodies or antigen-binding fragments provided herein comprise one, two, and/or three VL CDRs from Table 1 and one, two, and/or three VH CDRs from Table 2.

Table 1 Amino acid sequences of light chain variable region CDRs (VL CDRs) of anti-CD40 Abs

Table 2 Amino acid sequences of heavy chain variable region CDRs (VH CDRs) of anti-CD40 Abs

In some embodiments, an anti-CD40 antibody or antigen-binding fragment thereof comprises a humanized antibody or antigen-binding fragment. In some embodiments, an anti-CD40 antibody or antigen-binding fragment thereof comprises a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 from an antibody or antigen-binding fragment described herein. In some embodiments, an anti-CD40 antibody or antigen-binding fragment thereof comprises a variant of an anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises one to 30 amino acid substitutions, additions, and/or deletions in the anti-CD40 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises one to 25 amino acid substitutions, additions, and/or deletions in the anti-CD40 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises one to 20 substitutions, additions, and/or deletions in the anti-CD40 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises one to 15 substitutions, additions, and/or deletions in the anti-CD40 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises one to 10 substitutions, additions, and/or deletions in the anti-CD40 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises one to five conservative amino acid substitutions, additions, and/or deletions in the anti-CD40 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises one to three amino acid substitutions, additions, and/or deletions in the anti-CD40 antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and/or deletions are conservative amino acid substitutions. In some embodiments, the conservative amino acid substitution (s) is in a CDR of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution (s) is not in a CDR of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution (s) is in a framework region of the antibody or antigen-binding fragment.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40, comprising a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6; (2) a light chain CDR2 (VL CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40, comprising a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VH CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-30; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-30; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-30; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL, wherein the VL comprises VL CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 1, 7, and 13, respectively; (2) SEQ ID NOs: 2, 8, and 14, respectively; (3) SEQ ID NOs: 3, 9, and 15, respectively; (4) SEQ ID NOs: 4, 10, and 16, respectively; (5) SEQ ID NOs: 5, 11, and 17, respectively; or (6) SEQ ID NOs: 6, 12, and 18, respectively; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VH, wherein the VH comprises VH CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 19, 25, and 31, respectively; (2) SEQ ID NOs: 20, 26, and 32, respectively; (3) SEQ ID NOs: 21, 27, and 33, respectively; (4) SEQ ID NOs: 22, 28, and 34, respectively; or (5) SEQ ID NOs: 23, 29, and 35, respectively; or (6) SEQ ID NOs: 24, 30, and 36, respectively; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL and a VH. In some embodiments, the VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=3, 4, or 5 (SEQ ID NO: 104) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=3, 4, or 5 (SEQ ID NO: 105) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 106) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 107) .

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL and a VH, wherein (a) the VL comprises VL CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 1, 7, and 13, respectively; (2) SEQ ID NOs: 2, 8, and 14, respectively; (3) SEQ ID NOs: 3, 9, and 15, respectively; (4) SEQ ID NOs: 4, 10, and 16, respectively; (5) SEQ ID NOs: 5, 11, and 17, respectively; or (6) SEQ ID NOs: 6, 12, and 18, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and (b) the VH comprises VH CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 19, 25, and 31, respectively; (2) SEQ ID NOs: 20, 26, and 32, respectively; (3) SEQ ID NOs: 21, 27, and 33, respectively; (4) SEQ ID NOs: 22, 28, and 34, respectively; or (5) SEQ ID NOs: 23, 29, and 35, respectively; or (6) SEQ ID NOs: 24, 30, and 36, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 1, 7, 13, 19, 25, and 31, respectively; (2) SEQ ID NOs: 2, 8, 14, 20, 26, and 32, respectively; (3) SEQ ID NOs: 3, 9, 15, 21, 27, and 33, respectively; (4) SEQ ID NOs: 4, 10, 16, 22, 28, and 34, respectively; (5) SEQ ID NOs: 5, 11, 17, 23, 29, and 35, respectively; or (6) SEQ ID NOs: 6, 12, 18, 24, 30, and 36, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 7, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 13. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 7, and 13, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 25, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 31. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: , respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of NOs: 1, 7, and 13; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 19, 25, and 31.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 2, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 14. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 2, 8, and 14, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 20, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 26, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 32. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 20, 26, and 32, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of NOs: 2, 8, and 14; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 20, 26, and 32.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 3, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 9, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 15. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 3, 9, and 15, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 27, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 33. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 21, 27, and 33, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of NOs: 3, 9, and 15; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 21, 27, and 33.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 10, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 16. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 4, 10, and 16, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 22, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 34. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 22, 28, and 34, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of NOs: 4, 10, and 16; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 22, 28, and 34.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 5, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 17. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 5, 11, and 17, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 23, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 29, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 35. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 23, 29, and 35, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of NOs: 5, 11, and 17; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 23, 29, and 35.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 6, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 12, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 18. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 24, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 30, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 36. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 24, 30, and 36, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of NOs: 6, 12, and 18; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 24, 30, and 36.

In some embodiments, anti-CD40 antibodies or antigen-binding fragments provided herein comprise the VL and/or the VH of any one of the antibodies described herein. In some embodiments, anti-CD40 antibodies or antigen-binding fragments provided herein comprise the VL and/or the VH of the scFv designated as 40-18, 40-37, 40-38, 40-45, 40-47, or 40-52.

Table 3 Amino acid sequences of light chain variable regions (VLs) and heavy chain variable region (VHs) of anti-CD40 antibodies

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has the amino acid sequence of SEQ ID NO: 37, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has the amino acid sequence of SEQ ID NO: 38, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has the amino acid sequence of SEQ ID NO: 39, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has the amino acid sequence of SEQ ID NO: 40, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has the amino acid sequence of SEQ ID NO: 41, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has the amino acid sequence of SEQ ID NO: 42, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42, and the VH has the amino acid sequence of SEQ ID NO: 43. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42, and the VH has the amino acid sequence of SEQ ID NO: 44. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42, and the VH has the amino acid sequence of SEQ ID NO: 45. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42, and the VH has the amino acid sequence of SEQ ID NO: 46. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42, and the VH has the amino acid sequence of SEQ ID NO: 47. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42, and the VH has the amino acid sequence of SEQ ID NO: 48.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 37. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 37. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 37. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 37. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 37. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL having the amino acid sequence of SEQ ID NO: 37.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 38. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 38. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 38. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 38. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 38. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL having the amino acid sequence of SEQ ID NO: 38.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 39. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 39. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 39. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 39. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 39. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL having the amino acid sequence of SEQ ID NO: 39.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 40. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 40. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 40. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 40. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 40. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL having the amino acid sequence of SEQ ID NO: 40.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 41. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 41. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 41. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 41. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 41. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL having the amino acid sequence of SEQ ID NO: 41.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 42. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 42. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 42. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 42. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 42. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL having the amino acid sequence of SEQ ID NO: 42.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 43. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 43. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 43. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 43. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 43. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH having the amino acid sequence of SEQ ID NO: 43.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 44. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 44. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 44. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 44. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 44. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH having the amino acid sequence of SEQ ID NO: 44.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 45. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 45. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 45. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 45. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 45. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH having the amino acid sequence of SEQ ID NO: 45.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 46. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 46. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 46. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 46. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 46. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH having the amino acid sequence of SEQ ID NO: 46.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 47. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 47. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 47. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 47. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 47. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH having the amino acid sequence of SEQ ID NO: 47.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 48. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 48. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 48. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 48. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 48. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH having the amino acid sequence of SEQ ID NO: 48.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 37 and 43, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 38 and 44, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 39 and 45, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 40 and 46, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 41 and 47, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 42 and 48, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising (a) a VL comprising

VL CDRs

1, 2, and 3 from a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and/or (b) a VH comprising

VH CDRs

1, 2, and 3 from a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL, wherein the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 40. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 42.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VH, wherein the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 44. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 45. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 46. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 48.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD40 comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 37, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 38, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 44. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 39, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 45. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 40, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 46. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 41, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 42, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 48.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is the scFv designated as 40-18 (SEQ ID NO: 61) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL from 40-18 (SEQ ID NO: 37) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH from 40-18 (SEQ ID NO: 43) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from 40-18. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from 40-18 (SEQ ID NO: 37) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from 40-18 (SEQ ID NO: 43) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of 40-18, respectively. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is a variant of 40-18 (SEQ ID NO: 61) . The 40-18 variant can have a VL that is a variant of the VL of 40-18 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 37. The 40-18 variant can have a VH that is a variant of the VH of 40-18 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 43. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of 40-18 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of 40-18 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is the scFv designated as 40-37 (SEQ ID NO: 62) . In some embodiments, the anti- CD40 antibody or antigen-binding fragment thereof provided herein has a VL from 40-37 (SEQ ID NO: 38) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH from 40-37 (SEQ ID NO: 44) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from 40-37. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from 40-37 (SEQ ID NO: 38) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from 40-37 (SEQ ID NO: 44) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of 40-37, respectively. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is a variant of 40-37 (SEQ ID NO: 62) . The 40-37 variant can have a VL that is a variant of the VL of 40-37 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 38. The 40-37 variant can have a VH that is a variant of the VH of 40-37 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 44. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of 40-37 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of 40-37 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is the scFv designated as 40-38 (SEQ ID NO: 63) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL from 40-38 (SEQ ID NO: 39) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH from 40-38 (SEQ ID NO: 45) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from 40-38. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from 40-38 (SEQ ID NO: 39) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from 40-38 (SEQ ID NO: 45) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of 40-38, respectively. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is a variant of 40-38 (SEQ ID NO: 63) . The 40-38 variant can have a VL that is a variant of the VL of 40-38 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 39. The 40-38 variant can have a VH that is a variant of the VH of 40-38 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 45. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of 40-38 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of 40-38 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is the scFv designated as 40-45 (SEQ ID NO: 64) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL from 40-45 (SEQ ID NO: 40) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH from 40-45 (SEQ ID NO: 46) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from 40-45. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from 40-45 (SEQ ID NO: 40) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from 40-45 (SEQ ID NO: 46) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of 40-45, respectively. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is a variant of 40-45 (SEQ ID NO: 46) . The 40-45 variant can have a VL that is a variant of the VL of 40-45 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 40. The 40-45 variant can have a VH that is a variant of the VH of 40-45 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 46. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of 40-45 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of 40-45 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is the scFv designated as 40-47 (SEQ ID NO: 65) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL from 40-47 (SEQ ID NO: 41) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH from 40-47 (SEQ ID NO: 47) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from 40-47. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from 40-47 (SEQ ID NO: 41) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from 40-47 (SEQ ID NO: 47) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of 40-47, respectively. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is a variant of 40-47 (SEQ ID NO: 65) . The 40-47 variant can have a VL that is a variant of the VL of 40-47 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 41. The 40-47 variant can have a VH that is a variant of the VH of 40-47 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 47. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of 40-47 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of 40-47 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is the scFv designated as 40-52 (SEQ ID NO: 66) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL from 40-52 (SEQ ID NO: 42) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH from 40-52 (SEQ ID NO: 48) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from 40-52. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from 40-52 (SEQ ID NO: 42) . In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from 40-52 (SEQ ID NO: 48) . The anti-CD40 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of 40-52, respectively. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof provided herein is a variant of 40-52 (SEQ ID NO: 66) . The 40-52 variant can have a VL that is a variant of the VL of 40-52 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 42. The 40-52 variant can have a VH that is a variant of the VH of 40-52 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 48. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of 40-52 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of 40-52 has up to 3 conservative amino acid substitutions.

In some embodiments, provided herein are also antibodies or antigen-binding fragments that compete with an antibody or antigen-binding fragment provided above for binding to CD40 (e.g., human CD40) . Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition experiments, e.g., BLI analysis, or

surface plasmon resonance (SPR) analysis. In some embodiments, an anti-CD40 antibody or antigen-binding fragment competes with, and inhibits binding of another antibody or antigen-binding fragment to CD40 (e.g., human CD40) by at least 50%, 60%, 70%, 80%, 90%or 100%. The level of inhibition or competition can be different depending on which antibody is the “blocking antibody” (i.e., the cold antibody that is incubated first with the target) . Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Haib Protoc; 2006; doi: 10.1101/pdb. prot 4277 or in Chapter 11 of USING ANTIBODIES by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies “cross-compete” if antibodies block each other both ways by at least 50%, i.e., regardless of whether one or the other antibody is contacted first with the antigen in the competition experiment.

Competitive binding assays for determining whether two antibodies compete or cross-compete for binding include: competition for binding to T cells expressing CD40, e.g., by flow cytometry, such as described in the Examples. Other methods include: biolayer interferometry (BLI) , SPR (e.g.,

) , solid phase direct or indirect radioimmunoassay (RIA) , solid phase direct or indirect enzyme immunoassay (EIA) , sandwich competition assay (see Stahli et al., Methods in Enzymology 9: 242 (1983) ) ; solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137: 3614 (1986) ) ; solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press (1988) ) ; solid phase direct label RIA using 1-125 label (see Morel et al., MoI. Immunol. 25 (1) : 7 (1988) ) ; solid phase direct biotin-avidin EIA (Cheung et al., Virology 176: 546 (1990) ) ; and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32: 77 (1990) ) .

In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with 40-18 for binding to CD40 (e.g., human CD40) . In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with 40-37 for binding to CD40 (e.g., human CD40) . In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with 40-38 for binding to CD40 (e.g., human CD40) . In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with 40-45 for binding to CD40 (e.g., human CD40) . In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with a 40-47 for binding to CD40 (e.g., human CD40) . In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with a 40-52 for binding to CD40 (e.g., human CD40) .

In some embodiments, provided herein are also antibodies or antigen-binding fragments that bind to the same epitope on CD40 (e.g., human CD40) as does an antibody or antigen-binding fragment provided above. In some embodiments, provided herein are antibodies or antigen-binding fragments that bind to the same epitope on CD40 (e.g., human CD40) as does 40-18. In some embodiments, provided herein are antibodies or antigen-binding fragments that bind to the same epitope on CD40 (e.g., human CD40) as does 40-37. In some embodiments, provided herein are antibodies or antigen-binding fragments that bind to the same epitope on CD40 (e.g., human CD40) as does 40-38. In some embodiments, provided herein are antibodies or antigen-binding fragments that bind to the same epitope on CD40 (e.g., human CD40) as does 40-45. In some embodiments, provided herein are antibodies or antigen-binding fragments that bind to the same epitope on CD40 (e.g., human CD40) as does 40-47. In some embodiments, provided herein are antibodies or antigen-binding fragments that bind to the same epitope on CD40 (e.g., human CD40) as does 40-52.

The present disclosure further contemplates additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate biological properties of the antibody, including but not limited to, specificity, thermostability, expression level, effector function (s) , glycosylation, immunogenicity, and/or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

Variations can be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide sequence. In some embodiments, amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions can be in the range of about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. In some embodiments, variations in the amino acid sequence that are biologically useful and/or relevant can be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parent protein.

It is known in the art that the constant region (s) of an antibody mediates several effector function and these effector functions can vary depending on the isotype of the antibody. For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR) . There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors) , IgE (epsilon receptors) , IgA (alpha receptors) and IgM (mu receptors) . Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell cytotoxicity or ADCC) , antibody-dependent cellular phagocytosis (ADCP) , release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgA antibody. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgD antibody. In some embodiments, anti-CD40 antibody or antigen- binding fragment described herein comprise at least one constant region of a human IgE antibody. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG antibody. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgM antibody. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG1 antibody. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG2 antibody. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG3 antibody. In some embodiments, anti-CD40 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG4 antibody.

Also provided are engineered and modified antibodies that can be prepared using an antibody having one or more of the VH and/or VL sequences disclosed herein as starting material to engineer a modified antibody, which modified antibody can have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL) , for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region (s) , for example to alter the effector function (s) of the antibody. One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six CDRs. Because CDR sequences are responsible for most antibody -antigen interactions, recombinant antibodies that mimic the properties of specific naturally -occurring antibodies can be expressed by constructing expression vectors that include CDR sequences from the specific naturally-occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. el al. (1986) Nature 321 : 522-525; Queen, C. et al (1989) Proc. Natl. Acad Sci. U.S.A. 86: 10029-10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al. ) .

Accordingly, some embodiments described herein pertain to an isolated monoclonal antibody, or antigen binding portion thereof, comprising VL comprising CDR1, CDR2, and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; respectively, and a VH comprising CDR1, CDR2, and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48; respectively. Thus, such antibodies contain the VH and VL CDR sequences of monoclonal antibodies 40-18, 40-37, 40-38, 40-45, 40-47, or 40-52, yet can contain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “Vbase” human germline sequence database (available on the Internet at www. mrc-cpe. cam. ac. uk/vbase) , as well as in Kabat, E.A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M, et al. (1992) Mol. Biol. 227 : 776-798; and Cox, J.P.L. et al. (1994) , Eur. J. Immunol. 24: 827-836; the contents of each of which are expressly incorporated herein by reference.

In some embodiments, the framework sequences for use in the anti-CD40 antibodies or antigen-binding fragments described herein are those that are structurally similar to the framework sequences used by the anti-CD40 antibodies described herein. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al. ) .

Engineered anti-CD40 antibodies or antigen-binding fragments described herein include those in which modifications have been made to framework residues within VH and/or VL, e.g., to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U S. Patent Publication No. 20030153043 by Carr et al.

Another type of variable region modification is to mutate amino acid residues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation (s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. In some embodiments, conservative modifications (as discussed above) are introduced. The mutations can be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

Anti-CD40 variable regions described herein can be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3 or IgG4 Fc, which can be of any allotype or isoallotype, e.g., for IgG1 : G1m, G1m1 (a) , G1m2 (x) , G1m3 (f) , G1m17 (z) ; for IgG2: G2m, G2m23 (n) ; for IgG3: G3m, G3m21 (g1) , G3m28 (g5) , G3m11 (b0) , G3m5 (b1) , G3m13 (b3) , G3m14 (b4) , G3m10 (b5) , G3m15 (s) , G3m16 (t) , G3m6 (c3) , G3m24 (c5) , G3m26 (u) , G3m27 (v) ; and for K: Km, Km1, Km2, Km3 (see, e.g., Jefferies et al. (2009) mAbs 1 : 1) .

In some embodiments, anti-CD40 variable regions described herein are linked to an effectorless or mostly effectorless Fc, e.g., IgG4.

Generally, variable regions described herein can be linked to an Fc comprising one or more modification, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody described herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

The Fc region encompasses domains derived from the constant region of an immunoglobulin, including a fragment, analog, variant, mutant or derivative of the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE and IgM. The constant region of an immunoglobulin is defined as a naturally-occurring or synthetically-produced polypeptide homologous to the immunoglobulin C-terminal region, and can include a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in combination. In some embodiments, at least one or more of the constant regions has been modified or deleted in the anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, the antibodies comprise modifications to one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and/or to the light chain constant region (CL) . In some embodiments, the heavy chain constant region of the modified antibodies comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibodies comprises more than one human constant region. In some embodiments, modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the entire CH2 domain has been removed from an antibody (ΔCH2 constructs) . In some embodiments, a deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent constant region. In some embodiments, a modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, a modified antibody comprises a peptide spacer inserted between the hinge region and modified CH2 and/or CH3 domains.

Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specific for the IgG class of antibody, namely FcγRI, FcγRII, and FcγRIII. The important sequences for the binding of IgG to the FcγR receptors have been reported to be located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by the ability of that antibody to bind to an Fc receptor (FcR) . In some embodiments, an anti-CD40 antibody or antigen-binding fragment comprises a Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge can be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc region of human IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the modified antibodies (e.g., modified Fc region) provide for altered effector functions that, in turn, affect the biological profile of the antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region reduces Fc receptor binding of the modified antibody as it circulates. In some embodiments, the constant region modifications reduce the immunogenicity of the antibody. In some embodiments, the constant region modifications increase the serum half-life of the antibody. In some embodiments, the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region modifications decrease or remove ADCC, ADCP, and/or complement dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in a human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce effector functions (e.g., ADCC, ADCP and CDC) in the modified antibody. In some embodiments, an antibody does not have one or more effector functions (e.g., “effectorless” antibodies) . In some embodiments, the antibody has no ADCC activity and/or no CDC activity. In some embodiments, the antibody does not bind an Fc receptor and/or complement factors. In some embodiments, the antibody has no effector function (s) . In some embodiments, the constant region modifications increase or enhance ADCC, ADCP, and/or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites. In some embodiments, an anti-CD40 antibody or antigen-binding fragment comprises a variant Fc region that is engineered with substitutions at specific amino acid positions as compared to a native Fc region.

In some embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant) , to provide desirable structural features and/or biological activity. Generally, variants of the constant region or portions thereof, e.g., CH1, CL, hinge, CH2 or CH3 domains can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and/or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation, or 1-10 or 1-5 mutations, or comprise an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to that of the corresponding wild-type region or domain (CH1, CL, hinge, CH2, or CH3 domain, respectively) , provided that the heavy chain constant region comprising the specific variant retains the necessary biological activity.

For example, one can make modifications in the Fc region in order to generate an Fc variant that (a) has increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC) , (b) increased or decreased antibody-dependent cell-mediated phagocytosis (ACDP) , (c) increased or decreased complement mediated cytotoxicity (CDC) , (d) has increased or decreased affinity for C1q and/or (e) has increased or decreased affinity for a Fc receptor relative to the parent Fc. Such Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable. For example, the variant Fc region can include two, three, four, five, etc. substitutions therein, e.g., of the specific Fc region positions identified herein.

A variant Fc region can also comprise a sequence alteration wherein amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal can avoid reaction with other cysteine-containing proteins present in the host cell used to produce the anti-CD40 antibodies or antigen-binding fragments described herein. Even when cysteine residues are removed, single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently. In some embodiments, the Fc region can be modified to make it more compatible with a selected host cell. For example, one can remove the PA sequence near the N-terminus of a typical native Fc region, which can be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. In some embodiments, one or more glycosylation sites within the Fc domain can be removed. Residues that are typically glycosylated (e.g., asparagine) can confer cytolytic response. Such residues can be deleted or substituted with unglycosylated residues (e.g., alanine) . In some embodiments, sites involved in interaction with complement, such as the C1q binding site, can be removed from the Fc region. For example, one can delete or substitute the EKK sequence of human IgG1. In some embodiments, sites that affect binding to Fc receptors can be removed, preferably sites other than salvage receptor binding sites. In some embodiments, an Fc region can be modified to remove an ADCC site. In some embodiments, an Fc region can be modified to remove an ADCP site. ADCC and ADCP sites are known in the art; see, for example, Molec. Immunol. 29 (5) : 633-9 (1992) with regard to ADCC sites in IgG1, Herbrand, U. (2016) . BioProcessing, 15 (1) , 1538-8786 with regard to ADCP sites in IgG1. Specific examples of variant Fc domains are disclosed for example, in WO 97/34631 and WO 96/32478.

In some embodiments, the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of Fc is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In some embodiments, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function (s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and/or 331 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC) . This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

In some embodiments, hybrid IgG isotypes with particular biological characteristics can be used. For example, an IgG1/IgG3 hybrid variant can be constructed by substituting IgG1 positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody can be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In some embodiments described herein, an IgG1/IgG2 hybrid variant can be constructed by substituting IgG2 positions in the CH2 and/or CH3 region with amino acids from IgG1 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody can be constructed that comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to an insertion of a glycine at position 236) , and 327 A.

In some embodiments, the Fc region can be modified to decrease ADCC, ADCP, and/or to decrease the affinity for an Fcγ receptor by modifying one or more amino acids at the following positions: 220, 226, 228, 229, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 318, 320, 322, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 220S, 226S, 228P, 229S, 233P, 234A, 234V, 235A, 235E, 236A, 236E, 236R, 237A, 238S, 239D, 267R, 239E, 268D, 267E, 268E, 268Q, 268F, 269R, 297A, 309L, 318A, 324T, 325L, 328R, 330S, 331S, 332D, 332E, and any combination thereof. Exemplary variants include 234A/235A, 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F7324T. Other modifications for reducing FcγR and complement interactions include removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20: 685-691.

In some embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region for FcRn. For example, one or more of more of following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and/or T256F. Alternatively, to increase the biological half-life, the antibody can be altered within the CH or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 259I, 308F, 428L, 428M, 434S, 434I, 434F, 434Y, and 434X1. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428 L, 428F, 250Q/428L (Hinton et al. 2004, J. Biol. Chem. 279 (8) : 6213-6216, Hinton et al. 2006 Journal of Immunology 176: 346-356) , 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276 (9) : 6591-6604) , 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311 S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dali Acqua et al., Journal of Immunology, 2002, 169: 5171-5180, Dall Acqua et al., 2006, Journal of Biological Chemistry 281: 23514-23524) . Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182: 7663-7671.

Fc modifications that increase binding to an Fc receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in abat (WO00/42072) . Modifications for altering binding to FcγRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332, according to the EU index. Exemplary substitutions for enhancing FcγRIIb affinity include but are not limited to 234A, 234D, 234E, 234F, 234W, 235A, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328 Y, 330S, 33 IS, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcγRIIb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.

Other modifications for enhancing FcγR and complement interactions include but are not limited to substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20: 685-691.

Moreover, the binding sites on human IgG1 for FcγRI. FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al. (2001) J. Biol. Chem. 276: 6591-6604) . Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A, which has been shown to exhibit enhanced FcγRIIIa binding and ADCC activity (Shields et al., 2001) . Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with S239D/I332E and S239D/I332E/A330L mutations which showed the greatest increase in affinity for FcγRIIIa, a decrease in FcγRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006) . Introduction of the triple mutations into antibodies such as alemtuzumab (CD52-specific) , trastuzumab (HER2/neu-specific) , rituximab (CD20-specific) , and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, and the S239D/I332E variant showed an enhanced capacity to deplete B cells in monkeys (Lazar et al., 2006) . In addition, IgG1 mutants containing L235 V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcγRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcγRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al. 2007; Nordstrom et al. 2011) . Other Fc mutants that can be used include: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L, L235V/F243L/R292P/Y300L/P396L, and M428L/N434S.

In some embodiments, an Fc is chosen that has reduced binding to FcγRs. An exemplary Fc, e.g., IgG1 Fc, with reduced FcγR binding comprises the following three amino acid substitutions: L234A, L235E and G237A. In some embodiments, an Fc is chosen that has reduced complement fixation. An exemplary Fc, e.g., IgG1 Fc, with reduced complement fixation has the following two amino acid substitutions: A330S and P331S. In some embodiments, an Fc is chosen that has essentially no effector function, i.e., it has reduced binding to FcγRs and reduced complement fixation. An exemplary Fc, e.g., IgG1 Fc, that is effectorless comprises the following five mutations: L234A, L235E, G237A, A330S and P331S. When using an IgG4 constant domain, it can include the substitution S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes IgG4 molecules. In some embodiments, the IgG4 constant domain includes the substitutions S228P and L235E.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region that comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L234F, L235A, L235E, G236R, G237A, D265A, N297A, N297Q, N297G, E318A, L328R, P329G, A330S, P331S, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises an amino acid substitution selected from the group consisting of N297A, N297Q and N297G, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises the amino acid substitutions of L234A and L235A, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises the amino acid substitutions of G236R and L328R, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises the amino acid substitutions of L234F, L235E, and P331S per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises the amino acid substitutions of L234A, L235A, and P329G, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises the amino acid substitutions of L234F, L235E, and D265A per EU numbering.

Optionally, the Fc region can comprise a non-naturally-occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCX Patent Publications WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/0201 14) .

The affinities and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) , biolayer interferometry (BLI) , or radioimmunoassay (RIA) ) , or kinetics (e.g., BIACORE analysis) , and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET) , gel electrophoresis and chromatography (e.g., gel filtration) . These and other methods can utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W.E., ed., FUNDAMENTAL IMMUNOLOGY, 4th Ed., Lippincott-Raven, Philadelphia (1999) , which focuses on antibody-immunogen interactions.

In some embodiments, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation) . Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

Glycosylation of the constant region on N297 can be prevented by mutating the N297 residue to another residue, e.g., N297A, and/or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC and/or ADCP ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant anti-CD40 antibodies or antigen-binding fragments described herein to thereby produce an antibody with altered glycosylation. For example, EP 1, 176, 195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J. Biol. Chem. 277: 26733-26740) . PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta (l, 4) -N-acetylglucosaminyltransferase III (GnTIII) ) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180) .

Another modification of the anti-CD40 antibodies or antigen-binding fragments described herein is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG) , such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. In some embodiments, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer) . As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI-CIO) alkoxy-or arylox -polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the anti-CD40 antibodies or antigen-binding fragments described herein. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

In some embodiments, variants can include addition of amino acid residues at the amino-and/or carboxyl-terminal end of the antibody or polypeptide. The length of additional amino acids residues can range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, the variant comprises an additional polypeptide/protein (e.g., Fc region) to create a fusion protein. In some embodiments, a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent tag or an enzyme) .

The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment disclosed herein can retain the ability to bind CD40 to a similar extent, the same extent, or to a higher extent, as the parent antibody or antigen-binding fragment. In some embodiments, the variant can be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%or more identical in amino acid sequence to the parent antibody or antigen-binding fragment. In certain embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises the amino acid sequence of the parent anti-CD40 antibody or antigen-binding fragment with one or more conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.

In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises the amino acid sequence of the parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-CD40 antibody or antigen-binding fragment comprises the amino acid sequence of the parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitution, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., CD40 binding) . In certain embodiments, the one or more conservative amino acid substitutions and/or the one or more non-conservative amino acid substitutions can enhance a biological activity of the variant, such that the biological activity of the functional variant is increased as compared to the parent antibody or antigen-binding fragment.

In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) of the antibody or antigen-binding fragment.

Described herein are antibodies, e.g., fully human antibodies, which are characterized by particular functional features or properties. For example, the antibodies specifically bind human CD40, and more specifically, a particular domain (e.g., a functional domain) within the extracellular domain of human CD40. In some embodiments, the antibodies are antagonist antibodies, i.e., they inhibit or suppress the T cell inhibitory activity of CD40 on cells, e.g., T cells. In some embodiments, anti-CD40 antibodies cross-react with CD40 from one or more non-human primates, such as cynomolgus CD40. In some embodiments, the antibodies specifically bind to the extracellular region of human CD40 and the extracellular region of cynomolgus CD40. In some embodiments, the antibodies bind to human CD40 with high affinity.

Epitope mapping is a method of identifying the binding site, region, or epitope on a target protein where an antibody binds. A variety of methods are known in the art for mapping epitopes on target proteins. These methods include mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology) ; display methods (e.g., phage display, microbial display, and ribosome/mRNA display) ; methods involving proteolysis and mass spectroscopy; and structural determination (e.g., X-ray crystallography and NMR) . In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein bind to human CD40 with high affinity, for example, with a K _D of 10 ^-7 M or less, 10 ^-8 M or less, 5×10 ^-9 M or less, 10 ^-9 M or less, 5×10 ^-10 M or less, 10 ^-10 M or less, 5×10 ^-11 M or less, 10 ^-11 M or less, 5×10 ^-12 M or less, 10 ^-12 M or less, 10 ^-12 M to 10 ^-7 M, 10 ^-11 M to 10 ^-7 M, 10 ^-10 M to 10 ^-7 M, 10 ^-9 M to 10 ^-7 M, 10 ^-8 M to 10 ^-7 M, 10 ^-10 M to 10 ^-8 M, 10 ^-9 M to 10 ^-8 M, 10 ^-11 M to 10 ^-9 M, or 10 ^-10 M to 10 ^- ⁹ M. In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein bind to human CD40 with a K _D of 10 ^-11 M to 5×10 ^-9 M. In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein bind to soluble human CD40 with high affinity, e.g., as determined by BLI, with a K _D of 10 ^-7 M or less, 10 ^-8 M or less, 5×10 ^-9 M or less, 10 ^-9 M or less, 5×10 ^-10 M or less, 10 ^-10 M or less, 5×10 ^-11 M or less, 10 ^-11 M or less, 5×10 ^-12 M or less, 10 ^-12 M or less, 10 ^-12 M to 10 ^-7 M, 10 ^-11 M to 10 ^-7 M, 10 ^-10 M to 10 ^-7 M, 10 ^-9 M to 10 ^-7 M, 10 ^-8 M to 10 ^-7, 10 ^-10 M to 10 ^-8 M, 10 ^-9 M to 10 ^-8 M, 10 ^-11 M to 10 ^-9 M, or 10 ^-10 M to 10 ^-9 M. In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein bind to soluble human CD40 with a K _D of 10 ^-11 M to 5×10 ^-9 M. In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein bind to bound (e.g., cell membrane bound) human CD40, such as on activated human T cells, e.g., as determined by flow cytometry and Scatchard plot, with a K _D of 10 ^- ⁷ M or less, 10 ^-8 M or less, 5×10 ^-9 M or less, 10 ^-9 M or less, 5×10 ^-10 M or less, 10 ^-10 M or less, 5×10 ^- ¹¹ M or less, 10 ^-11 M or less, 5×10 ^-12 M or less, 10 ^-12 M or less, 10 ^-12 M to 10 ^-7 M, 10 ^-11 M to 10 ^-7 M, 10 ^-10 M to 10 ^-7 M, 10 ^-9 M to 10 ^-7 M, 10 ^-8 M to 10 ^-7, 10 ^-10 M to 10 ^-8 M, 10 ^-9 M to 10 ^-8 M, 10 ^-11 M to 10 ^-9 M, or 10 ^-10 M to 10 ^-9 M. In some embodiments, an anti-CD40 antibody or antigen-binding fragment binds to bound (e.g., cell membrane bound) human CD40, such as on activated human T cells, e.g., as determined by flow cytometry, with an EC ₅₀ of 10 μg/mL or less, 5 μg/mL or less, 1 μg/mL or less, 0.9 μg/mL or less, 0.8 μg/mL or less, 0.7 μg/mL or less, 0.6 μg/mL or less, 0.5 μg/mL or less, 0.4 μg/mL or less, 0.3 μg/mL or less, 0.2 μg/mL or less, 0.1 μg/mL or less, 0.05 μg/mL or less, or 0.01 μg /mL or less.

Accordingly, an anti-CD40 antibody or antigen-binding fragment that exhibits one or more of these functional properties (e.g., biochemical, immunochemical, cellular, physiological or other biological activities, or the like) as determined according to methodologies known to the art and described herein, will be understood to exhibit a statistically significant difference in the particular activity relative to that seen in the absence of the antibody (e.g., or when a control antibody of irrelevant specificity is present) . In some embodiments, anti-CD40 antibody or antigen-binding fragment-induced increases in a measured parameter (e.g., T cell proliferation, cytokine production) in a given assay effects a statistically significant increase by at least 10%of the measured parameter, e.g., by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% (i.e., 2 fold) , 3 fold, 5 fold or 10 fold, and in some embodiments, an antibody described herein can increase the measured parameter, e.g., by greater than 92%, 94%, 95%, 97%, 98%, 99%, 100% (i.e., 2 fold) , 3 fold, 5 fold or 10 fold, relative to the same assay conducted in the absence of the antibody. Conversely, anti-CD40 antibody-induced decreases in a measured parameter (e.g., tumor volume, CD40 ligand binding to human CD40) in a given assay effects a statistically significant decrease by at least 10%of the measured parameter, e.g., by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%or 90%, and in some embodiments, an antibody described herein can decrease the measured parameter, e.g., by greater than 92%, 94%, 95%, 97%, 98%or 99%, relative to the same assay conducted in the absence of the antibody.

The anti-CD40 antibodies or antigen-binding fragments can be analyzed for their physical, chemical and/or biological properties by various methods known in the art. In some embodiments, an anti-CD40 antibody is tested for its ability to bind CD40 (e.g., human CD40) . Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore) , ELISA, FACS, Western blots, and RIAs. In addition, antibodies can be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency. Assays to evaluate the effects of the antibodies on functional properties of CD40 (e.g., ligand binding, T cell proliferation, cytokine production) are described in further detail below and in the Examples.

In some embodiments, anti-CD40 antibodies are not native antibodies or are not naturally-occurring antibodies. For example, anti-CD40 antibodies have post-translational modifications that are different from those of antibodies that are naturally-occurring, such as by having more, less or a different type of post-translational modification.

In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein are chemically modified naturally or by intervention. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques. The anti-CD40 antibodies or antigen-binding fragments can comprise one or more analogs of an amino acid (including, for example, unnatural amino acids) , as well as other modifications known in the art.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment is conjugated to a cytotoxic agent or moiety. In some embodiments, an anti-CD40 antibody or antigen-binding fragment is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate) . In some embodiments, the cytotoxic moiety is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs) , or other intercalating agents. In some embodiments, the cytotoxic moiety is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4) , and tubulysins. In some embodiments, the cytotoxic moiety is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, an antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment described herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. A detectable substance can include, but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine (s) ; fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC) , rhodamine, tetramethylrhodamine isothiocyanate (TRITC) , dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3) , and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as ²¹²Bi, ¹⁴C, ⁵⁷Co, ⁵¹Cr, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ¹⁵³Gd, ¹⁵⁹Gd, ⁶⁸Ge, ³H, ¹⁶⁶Ho, ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I, ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In, ¹⁴⁰La, ¹⁷⁷Lu, ⁵⁴Mn, ⁹⁹Mo, ³²P, ¹⁰³Pd, ¹⁴⁹Pm, ¹⁴²Pr, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁰⁵Rh, ⁹⁷Ru, ³⁵S, ⁴⁷Sc, ⁷⁵Se, ¹⁵³Sm, ¹¹³Sn, ¹¹⁷Sn, ⁸⁵Sr, ^99mTc, ²⁰¹Ti, ¹³³Xe, ⁹⁰Y, ⁶⁹Yb, ¹⁷⁵Yb, ⁶⁵Zn; positron emitting metals; and magnetic metal ions positron emitting metals; and magnetic metal ions.

In some embodiments, anti-CD40 antibodies or antigen-binding fragments described herein can be bispecific molecules or multi-specific molecules such as bispecific antibodies or multi-specific antibodies. A monospecific anti-CD40 antibody, or antigen binding portion thereof, can be derivatized or linked to another binding moiety, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. For example, an anti-CD40 antibody or antigen-binding fragment can be linked to an antibody or scFv that binds specifically to any protein that can be used as potential targets for combination treatments, such as the proteins described herein. The second target can be PD-1, PD-L1, CEACAM1, CEACAM5, GITR, or LAG-3. The antibodies and antigen-binding fragments described herein can in fact be derived or linked to more than one other functional molecule to generate multispecific molecules that have more than two different binding sites and bind to more than two target molecules. To create a bispecific molecule (e.g., a bispecific antibody) described herein, an antibody described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antigen-binding fragment, peptide or binding mimetic, such that a bispecific molecule results.

Accordingly, provided herein are bispecific molecules (e.g., bispecific antibodies) comprising at least a first binding specificity for CD40 and a second binding specificity for a second target antigen. In some embodiments, provided herein are multispecific molecules (e.g., multispecific antibodies) , which can further include a third binding specificity.

In some embodiments, the bispecific molecules described herein comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F (ab') 2, Fv, or a single chain Fv (scFv) . The antibody can also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in U.S. Patent No. 4,946,778.

While human or humanized monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific molecules described herein are murine and chimeric monoclonal antibodies.

The bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA) , 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) , o-phenylenedimaleimide (oPDM) , N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) , and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648) . Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83) , and Glennie et al. (1987) J. Immunol. 139: 2367-2375) . Some conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL) .

When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In some embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, mAb x (scFv) ₂, Fab x F (ab') ₂ or ligand x Fab fusion protein. A bispecific antibody can comprise an antibody comprising an scFv at the C-terminus of each heavy chain. A bispecific molecule described herein can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules can comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858.

Binding of the bispecific molecules to their specific targets can be confirmed using art-recognized methods, such as enzyme-linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , FACS analysis, bioassay (e.g., growth inhibition) , or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

An anti-CD40 antibody or antigen-binding fragment described herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, an immobilized anti-CD40 antibody or antigen-binding fragment is used in an immunoassay. In some embodiments, an immobilized anti-CD40 antibody or antigen-binding fragment is used in purification of the target antigen (e.g., human CD40) .

5.3 LACO-Stim fusion proteins

Provided herein are also fusion proteins comprising a first domain that activates an antigen-presenting cell ( “APC” ; e.g., a dendritic cell) and a second domain that activates an immune effector cell (e.g., a T cell) , wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof disclosed herein, and the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. Such fusion proteins are also referred to as Lymphocytes- APCs Co-stimulators ( “LACO-Stim” molecules or “LACO” molecules) .

In some embodiments, the fusion protein is a membrane protein. In some embodiments, the fusion protein is a soluble protein. In some embodiments, the fusion protein is a bispecific antibody. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, the first domain and the second domain are linked via a linker. In some embodiments, the linker is a trimerization motif. In some embodiments, the linker is a trimerization motif selected from the group consisting of a T4 fibritin trimerization motif, an isoleucine zipper, a GCN4II motif, a Matrilin-1 motif, and a collagen XV trimerization motif.

The first domain can have any anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, the anti-CD40 antibody or antigen-binding fragment can be one of the followings scFv clones: 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52. In some embodiments, the anti-CD40 antibody or antigen-binding fragment has a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 1, 7, 13, 19, 25, and 31, respectively; (2) SEQ ID NOs: 2, 8, 14, 20, 26, and 32, respectively; (3) SEQ ID NOs: 3, 9, 15, 21, 27, and 33, respectively; (4) SEQ ID NOs: 4, 10, 16, 22, 28, and 34, respectively; (5) SEQ ID NOs: 5, 11, 17, 23, 29, and 35, respectively; or (6) SEQ ID NOs: 6, 12, 18, 24, 30, and 36, respectively; or a variant thereof

“Immune effector cells” as used herein and understood in the art refer to cells that are of hematopoietic origin and play a direct role in the immune response against a target, such as a pathogen, a cancer cell, or a foreign substance. Immune effector cells include T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils. In some embodiments, the second domain of the fusion proteins provided herein that activates an immune effector cell comprises a co-stimulatory receptor of the immune effector cell. In some embodiments, the immune effector cell is a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, or a granulocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a macrophage.

“Stimulation” of an immune effector cell means a primary response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event in the immune effector cell which can alter expression of certain genes and/or reorganization of cytoskeletal structures, and the like. A “stimulatory molecule” of an immune effector cell refers to a molecule on the immune effector cell that, upon binding with its cognate ligand, which is commonly present on an APC, can mediate signal transduction to promote the maturation, differentiation, proliferation, and/or activation of the immune effector cell. For example, a stimulatory molecule of the T cells, the TCR/CD3 complex triggers the activation of the T cells. The ligand for a stimulatory molecule, or “stimulatory ligand, ” means a ligand that is commonly present on an APC and can bind with a stimulatory molecule on the immune effector cell to mediate a primary response by the immune effector cell, including, but not limited to, maturation, differentiation, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, for example, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A “co-stimulatory signal, ” as used herein and understood in the art, refers to a signal from a co-stimulatory receptor (e.g., CD28 or 4-1BB) , which in combination with a primary signal (e.g., TCR/CD3) promotes optimal clonal expansion, differentiation and effector functions of immune effector cells (e.g., T cells) . A “co-stimulatory receptor” of an immune effector cell, s used herein and understood in the art, refers to a molecule on the immune effector cell that specifically binds with a “co-stimulatory ligand” to mediate a co-stimulatory response by the immune effector cell, such as heightened activation or proliferation of the immune effector cell. Co-stimulatory receptors for immune effector cells include, but are not limited to, CD28, 4-1BB, ICOS, CD27, OX40, DAP10, CD30, 2B4, CD2, LIGHT, GITR, TLR, DR3, and CD43. A “functional fragment” of a co-stimulatory receptor is a fragment of the co-stimulatory receptor that retains its function to mediate a co-stimulatory signal and stimulate the immune effector cell. In some embodiments, a functional fragment of a co-stimulatory receptor retains the co-stimulatory domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory domain is the cytoplasmic domain of the co-stimulatory receptor. In some embodiments, signals from co-stimulatory receptors of immune effector cells (e.g., T cells) lower the activation threshold for the immune effector cells. In some embodiments, signals from co-stimulatory receptors of T cells lead to the augmentation of TCR signaling events necessary for efficient cytokine production (via augmented transcriptional activity and messenger RNA stabilization) , cell cycle progression, survival, regulation of metabolism and T cell responses.

A “co-stimulatory ligand, ” as used herein and understood in the art, refers to a molecule that specifically binds a cognate co-stimulatory receptor on an immune effector cell, thereby providing a signal which, in addition to the primary signal provided by the stimulatory molecule, mediates a response in the immune effector cell, including, but not limited to, proliferation, activation, differentiation, and the like. The co-stimulatory ligand can be present on an APC (e.g., a dendritic cell) . Co-stimulatory ligands include, but are not limited to, CD58, CD70, CD83, CD80, CD86, CD137L (4-1BBL) , CD252 (OX40L) , CD275 (ICOS-L) , CD54 (ICAM-1) , CD49a, CD112 (PVRL2) , CD150 (SLAM) , CD155 (PVR) , CD265 (RANK) , CD270 (HVEM) , TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153 (CD30L) , CD48, CD160, CD200R (OX2R) , and CD44. A “receptor-binding fragment” of a co-stimulatory ligand refers to a fragment of the ligand that retains its capacity to bind its receptor.

Some co-stimulatory receptors and co-stimulatory ligands are exemplified below. It is understood that any co-stimulatory receptors and/or co-stimulatory ligands provided herein or otherwise known in the art can be used as part of the fusion proteins provided herein.

CD28: Cluster of Differentiation 28 (CD28) is a protein expressed on T cells that provides co-stimulatory signals for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. CD28 is a co-stimulatory receptor for optimal T cell clonal expansion, differentiation and effector functions. CD28 engagement lowers the T cell activation threshold and leads to the augmentation of TCR signaling events necessary for efficient cytokine production (via augmented transcriptional activity and messenger RNA stabilization) , cell cycle progression, survival, regulation of metabolism and T cell responses. CD28 is a crucial player for immunological synapse (IS) organization, where it enhances close contact between T cells and APCs.

In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises a CD28 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of CD28. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD28. In some embodiments, the ligand of CD28 is CD80. In some embodiments, the second domain comprises an antibody that binds CD28, or an antigen-binding fragment thereof. In some embodiments, the second domain of fusion proteins provided herein comprises a functional fragment of CD28, which comprises a portion of an intracellular/cytoplasmic domain of CD28 that can function as a co-stimulatory signaling domain. A CD28 can have an amino acid sequence corresponding to the sequence having GenBank No. P10747 (P10747.1, GI: 115973) or NP_006130 (NP_006130.1, GI: 5453611) , as provided below, or functional fragments thereof. In one embodiment, a fusion protein disclosed herein can have an amino acid sequence comprising the cytoplasmic domain of CD28 corresponding to amino acids 180 to 220 of CD28 or a fragment thereof. In another embodiment, a fusion protein disclosed herein can have an amino acid sequence further comprising the transmembrane domain of CD28 corresponding to amino acids 153 to 179, or a functional fragment thereof. It is understood that sequences of CD28 that are shorter or longer than a specific delineated domain can be included in a fusion protein disclosed herein, if desired.

4-1BB: 4-1BB, also referred to as tumor necrosis factor receptor superfamily member 9, can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity (Stephan MT et al., Nat Med (2007) 13 (12) : 1440-1449) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises a 4-1BB polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of 4-1BB. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds 4-1BB. In some embodiments, the ligand of 4-1BB is 4-1BBL. In some embodiments, the second domain comprises an antibody that binds 4-1BB, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory signaling domain derived from 4-1BB. A 4-1BB polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P41273 (P41273.1, GI: 728739) or NP_001552 (NP_001552.2, GI: 5730095) or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can have a co-stimulatory domain comprising the cytoplasmic domain of 4-1BB corresponding to amino acids 214 to 255 of the sequence below, or a functional fragment thereof. It is understood that sequences of 4-1BB that are shorter or longer than a specific delineated domain can be included in a fusion protein disclosed herein, if desired.

ICOS. Inducible T-cell co-stimulator precursor (ICOS) , also referred to as CD278, is a CD28-superfamily co-stimulatory receptor that is expressed on activated T cells. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40s antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises an ICOS polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of ICOS. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds ICOS. In some embodiments, the ligand of ICOS is CD275 (ICOS-L) . In some embodiments, the second domain comprises an antibody that binds ICOS, or an antigen-binding fragment thereof. In one embodiment, the second domain comprises a co-stimulatory signaling domain derived from ICOS. An ICOS polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_036224 (NP_036224.1, GI: 15029518) , provided below, or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can have a co-stimulatory domain comprising the cytoplasmic domain of ICOS corresponding to amino acids of the sequence below, or a functional fragment thereof. It is understood that sequences of ICOS that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD27: CD27 (TNFRSF7) is a transmembrane receptor expressed on subsets of human CD8+ and CD4+ T-cells, NKT cells, NK cell subsets and hematopoietic progenitors and induced in FOXP3+ CD4 T-cells and B cell subsets. Previous studies have found that CD27 can provide costimulatory signals that improve human T-cell survival and anti-tumor activity in vivo. (See Song and Powell; Oncoimmunology 1 (4) : 547-549 (2012) ) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises a CD27 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of CD27. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD27. In some embodiments, the ligand of CD27 is CD70. In some embodiments, the second domain comprises an antibody that binds CD27, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from CD27. A CD27 polypeptide can have an amino acid sequence corresponding to the sequence having UniProtKB/Swiss-Prot No.: P26842.2 (GI: 269849546) , provided below, or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of CD27 corresponding to amino acids 213 to 260 of the sequence below, or a functional fragment thereof. It is understood that sequences of CD27 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

OX40. OX40, also referred to as tumor necrosis factor receptor superfamily member 4 precursor or CD134, is a member of the TNFR-superfamily of receptors. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises an OX40 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of OX40. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds OX40. In some embodiments, the ligand of OX40 is CD252. In some embodiments, the second domain comprises an antibody that binds OX40, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory signaling domain derived from OX40. An OX40 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P43489 (P43489.1, GI: 1171933) or NP_003318 (NP_003318.1, GI: 4507579) , provided below, or fragments thereof. In one embodiment, fusion proteins provided herein can have a co-stimulatory domain comprising the cytoplasmic domain of OX40 corresponding to amino acids 236 to 277of the sequence below, or a functional fragment thereof. It is understood that sequences of OX40 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

DAP10. DAP10, also referred to as hematopoietic cell signal transducer, is a signaling subunit that associates with a large family of receptors in hematopoietic cells. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises an DAP10 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of DAP10. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds DAP10. In some embodiments, the second domain comprises an antibody that binds DAP10, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory signaling domain derived from DAP10. A DAP10 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_055081.1 (GI: 15826850) , provided below, or fragments thereof. In one embodiment, fusion proteins provided herein can have a co-stimulatory domain comprising the cytoplasmic domain of DAP10 corresponding to amino acids 70 to 93 of the sequence below, or a functional fragment thereof. It is understood that sequences of DAP10 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD30: CD30 and its ligand (CD30L) are members of the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamilies, respectively. CD30 enhances proliferation and cytokine production induced by TCR stimulation. (Goronzy and Weyand, Arthritis research &therapy 10, no. S1 (2008) : S3. ) In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises a CD30 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of CD30. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD30. In some embodiments, the ligand of CD30 is CD153. In some embodiments, the second domain comprises an antibody that binds CD30, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from CD30. A CD30 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No.: AAA51947.1 (GI: 180096) , provided below, or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of CD30 corresponding to amino acids 407 to 595 of the sequence below, or a functional fragment thereof. It is understood that sequences of CD30 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

2B4 2B4 (CD244) is a co-stimulatory receptor expressed on both NK cells and CD8+ T cells. It targets a non-MHC like molecule (CD48) expressed on hematopoietic cells, including B and T cells, as well as on activated monocytes and granulocytes. Activation of 2B4 by binding of its ligand on target cells leads to NK (or T cell) activation, and target killing. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises a 2B4 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of 2B4. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds 2B4. In some embodiments, the second domain comprises an antibody that binds 2B4, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from 2B4. A 2B4 polypeptide can have an amino acid sequence corresponding to the sequence having Accession No: Q9BZW8.2 (GI: 47605541) , provided below, or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of 2B4 corresponding to amino acids 251 to 370 of the sequence below, or a functional fragment thereof. It is understood that sequences of 2B4 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD2 The engagement of the CD2 molecule by its ligand CD58 co-stimulates proliferation, cytokine production, and effector function in T cells, especially the CD28-deficient T cells subset. CD58 is broadly expressed on APCs including dendritic cells. (Judith Leitner J et al., Immunol, 2015, 195 (2) 477-487) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises a CD2 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of CD2. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD2. In some embodiments, the ligand of CD2 is CD58. In some embodiments, the ligand of CD2 is CD48. In some embodiments, the second domain comprises an antibody that binds CD2, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from CD2. A CD2 polypeptide can have an amino acid sequence corresponding to the sequence having Accession: NP_001758.2 GI: 156071472, provided below, or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of CD2 corresponding to amino acids 236 to 351 of the sequence below, or a functional fragment thereof. It is understood that sequences of CD2 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

LIGHT TNF superfamily member 14 (also known as LTg, CD258, HVEML, LIGHT) is a co-stimulatory receptor involved in cellular immune responses. LIGHT can function as a costimulatory factor for the activation of lymphoid cells and as a deterrent to infection by herpesvirus. LIGHT has been shown to stimulate the proliferation of T cells, and trigger apoptosis of various tumor cells. LIGHT is expressed on immature dendritic cells (DCs) generated from human PBMCs. Engagement of LIGHT co-stimulates human T cell proliferation, amplifies the NF-κB signaling pathway, and preferentially induces the production of IFN-γ. (Tamada K et al., J Immunol, 2000, 164 (8) 4105-4110) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises a LIGHT polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of LIGHT. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds LIGHT. In some embodiments, the second domain comprises an antibody that binds LIGHT, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from LIGHT. A LIGHT polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: NP_001363816.1 GI: 1777376047) , or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of LIGHT corresponding to amino acids 1 to 37 of the sequence below, or a functional fragment thereof. It is understood that sequences of LIGHT that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

GITR TNF receptor superfamily member 18 (also known as TNFRSF18, AITR, GITR; CD357; GITR-D; ENERGEN) has been shown to have increased expression upon T-cell activation. Stimulation of T cells through GITR has been shown to enhance immunity to tumors and viral pathogens, and to exacerbate autoimmune disease. The effects of stimulation through GITR are generally thought to be caused by attenuation of the effector activity of immunosuppressive CD4+CD25+ regulatory T (TReg) cells. (Shevach, E. and Stephens, G. Nat Rev Immunol 6, 613–618 (2006) ) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises a GITR polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of GITR. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds GITR. In some embodiments, the ligand of GITR is GITR-L. In some embodiments, the second domain comprises an antibody that binds GITR, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from GITR. A GITR polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: AAI52382.1 GI: 158931986) , or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of GITR corresponding to amino acids 184 to 241 of the sequence below or a functional fragment thereof. It is understood that sequences of GITR that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

DR3 TNF receptor superfamily member 25 (also known as DR3, TR3, DDR3, LARD, APO-3, TRAMP, WSL-1, GEF720, WSL-LR, PLEKHG5, or TNFRSF12) is expressed preferentially in the tissues enriched in lymphocytes, and it plays a role in regulating lymphocyte homeostasis. This receptor has been shown to stimulate NF-kappa B activity and regulate cell apoptosis. The alternative splicing of this gene in B and T cells encounters a programmed change upon T-cell activation, which predominantly produces full-length, membrane bound isoforms, and is involved in controlling lymphocyte proliferation induced by T-cell activation. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises a DR3 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of DR3. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds DR3. In some embodiments, the second domain comprises an antibody that binds DR3, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from DR3. A DR3 polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: Accession: Accession: AAI17190.1 GI: 109658976) , or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of DR3 corresponding to amino acids 221 to 417 of the sequence below, or a functional fragment thereof. It is understood that sequences of DR3 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD43 CD43 (also known as SPN sialophorin, LSN, GALGP, GPL115) is a highly sialylated glycoprotein that functions in antigen-specific activation of T cells, and is found on the surface of thymocytes, T lymphocytes, monocytes, granulocytes, and some B lymphocytes. In stimulated immune effector cells, proteolytic cleavage of the extracellular domain occurs in some cell types, releasing a soluble extracellular fragment. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises a CD43 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of CD43. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD43. In some embodiments, the second domain comprises an antibody that binds CD43, or an antigen-binding fragment thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from CD43. A CD43 polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: EAW80016.1 GI: 119600422; Accession: EAW80015.1 GI: 119600421) , or fragments thereof. In one embodiment, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of CD43 corresponding to amino acids 277 to 400 of the sequence below, or a functional fragment thereof. It is understood that sequences of CD43 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD58 (also known as AG3; LFA3; LFA-3) is a member of the immunoglobulin superfamily and a ligand of the T lymphocyte CD2 protein. CD58 is localized to the plasma membrane and functions in adhesion and activation of T lymphocytes. (See e.g Abdul Razak FR, et al. Genes Immun, 2016 Sep. PMID 27467287; Schneider M, et al. Genes Chromosomes Cancer, 2015 Oct. PMID 26194173. ) A polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession NP_001770; NP_001138294) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD58, or a receptor-binding fragment thereof. In some embodiments, the second domain of the fusion proteins provided herein comprises the extracellular domain of CD58 corresponding to amino acids 29-215 of the sequence below. It is understood that sequences of CD58 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD70 (also known as Ki-24, CD27L, TNFSF7) is known to enhance the generation of cytotoxic T-cells and contribute to T-cell activation. CD70 is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family, which is a ligand for TNFRSF27/CD27. It is a surface antigen on activated T and B lymphocytes. It induces proliferation of costimulated T cells, enhances the generation of cytolytic T cells, and contributes to T cell activation. This cytokine is also reported to play a role in regulating B-cell activation, cytotoxic function of natural killer cells, and immunoglobulin synthesis. (See e.g., Masamoto I, et al. Leuk Lymphoma, 2016; Jacobs J, et al. Pharmacol Ther, 2015 Nov) . A CD70 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession: NP_001243; NP_001317261; XP_016883012) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD70, or a receptor-binding fragment thereof. In some embodiments, the second domain of the fusion proteins provided herein comprises the extracellular domain of CD70 corresponding to amino acids 39-193 of the sequence below. It is understood that sequences of CD70 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD83 (also known as BL11, HB15) is a single-pass type I membrane protein and member of the immunoglobulin superfamily of receptors. CD83 can bind CD83L and is involved in the regulation of antigen presentation. (Li Z, et al. Haematologica, 2018 Apr.; Ju X, et al. J Immunol, 2016 Dec 15. PMID 29351987; Horvatinovich JM, et al. J Immunol, 2017 Mar 15. PMID 28193829. ) A CD83 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_001035370, NP_001238830, NP_004224) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD83, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD83 corresponding to amino acids 20-144 of the sequence below. It is understood that sequences of CD83 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD80 (also known as B7, B7-1, B7.1, BB1, CD28LG, CD28LG1, LAB7) is a single-pass type I membrane protein and member of the immunoglobulin superfamily of receptors. CD80’s function involves antigen presentation regulation and immune stimulation. CD80 binds CD28 or CTLA-4, which induces T-cell proliferation and cytokine production. (See e.g., Feng XY, et al. Future Oncol, 2019 Feb. PMID 30628844) A CD80 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., EAW79565.1; NP_005182) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD80, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD80 corresponding to amino acids 35-242 of the sequence below. It is understood that sequences of CD80 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD86 (also known as B70, B7-2, CD28LG2) is an integrin alpha X chain protein which can bind CD28 and CD152. This protein combines with the beta 2 chain (ITGB2) to form a leukocyte-specific integrin referred to as inactivated-C3b (iC3b) receptor 4 (CR4) . The alpha X beta 2 complex overlap the properties of the alpha M beta 2 integrin in the adherence of neutrophils and monocytes to stimulated endothelium cells, and in the phagocytosis of complement coated particles. (See e.g., Takács F, et al. Pathol Oncol Res, 2019 PMID 30406401; Schütz C et al. Leukemia. 2017; 31 (4) : 829-836. doi: 10.1038/leu. 2017.9. ) A CD86 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession: NP_787058.5 NP_001193853) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD86, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD86 corresponding to amino acids 24-247 of the sequence below. It is understood that sequences of CD86 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD137L (also known as 4-1BBL, TNFSF9, CDw137, ILA) is a member of the tumor necrosis factor (TNF) receptor family. This transmembrane cytokine is a bidirectional signal transducer that acts as a ligand for TNFRSF9/4-1BB, which is a costimulatory receptor molecule in T lymphocytes. This cytokine and its receptor are involved in the antigen presentation process and in the generation of cytotoxic T cells. 4-1BBL has been shown to reactivate anergic T lymphocytes in addition to promoting T lymphocyte proliferation. This cytokine has also been shown to be required for the optimal CD8 responses in CD8 T cells. This cytokine is expressed in carcinoma cell lines and is thought to be involved in T cell-tumor cell interaction. (See e.g., Shen YL, et al. J Dig Dis, 2017 Jul. PMID 28547807; Qian Y, et al. Med Oncol, 2015 Mar. PMID 25631633. ) A CD137L polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_003802.1) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD137L, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD137L corresponding to amino acids 50-254 of the sequence below. It is understood that sequences of CD137L that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD252 (also known as OX40L, gp34) an integrin beta chain, which combines with different alpha chains to form integrin heterodimers. CD252 is the ligand for receptor TNFRSF4 (OX40) . CD252 co-stimulates T-cell proliferation and cytokine production. CD252 also functions in T cell APC interactions and mediates adhesion of activated T cells to endothelial cells. (See e.g., Roszik J, et al. Cancer Immunol Immunother, 2019 Sep. PMID 31501955) . A CD252 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_001284491 XP_005245532; NP_003317) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD252, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD252 corresponding to amino acids 51-183 of the sequence below. It is understood that sequences of CD252 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD275 (also known as ICOS-L, B7-H2, B7-RP1, GL50) . CD275 is a ligand for ICOS/CD278, which is a costimulatory receptor that promotes T-cell proliferation and cytokine secretion. CD275 can also induce B-cell proliferation and differentiation. (See e.g., Han Y, et al. Front Immunol, 2018. PMID 30319662; Cao Y, et al. Int Immunopharmacol, 2018 Mar. PMID 29414642. ) A CD275 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_001269979, NP_001269980, NP_001269981, NP_056074, NP_001352688 XP_016883799) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD275, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD275 corresponding to amino acids 19-256 of the sequence below. It is understood that sequences of CD275 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD54 (also known as ICAM-1) is a cell surface glycoprotein which is typically expressed on endothelial cells and cells of the immune system. It binds to integrins of type CD11a /CD18, or CD11b /CD18. The function of CD54 includes cell adhesion, lymphocyte activation, and migration. (See e.g., Reyes‐Botella, C., et al. Journal of Periodontology 71.4 (2000) : 614-617; Schildberg, Frank A., et al. Hepatology 54.1 (2011) : 262-272. ) A CD54 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_000192) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD54, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD54 corresponding to amino acids 28-480 of the sequence below. It is understood that sequences of CD54 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD49a (also known as VLA1, or ITGA1) is an alpha 1 subunit of integrin receptor. CD49a is known to mediate memory CD8+ T cell persistence and response and NK cell activity. CD49a is found to be expressed on macrophages. (See e.g., Bromley et al., Am Assoc Immnol (2020) : 81-10; Li et al. American Journal of Reproductive Immunology 81.4 (2019) : e13101.; Sun et al. Cancer immunology research (2019) . ) A CD49a polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_852478) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD49a, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD49a corresponding to amino acids 29-1141 of the sequence below. It is understood that sequences of CD49a that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD112 (also known as PVRL2, PRR2, Nectin-2, HVEB) is a human plasma membrane glycoprotein. It can bind, for example, CD226, Nectin-3, DNAM-1, and Afadin. Among other things, CD112 is found to bind to DNAM-1 on NK cells to induce its cytolytic activity. (See e.g., Bekes I, et al.Cancer Sci, 2019 Jun. PMID 30843637; Fujimoto Y, et al. Acta Virol, 2016 Mar. PMID 26982466; J Exp Med (2003) 198 (4) : 557–567) . A CD112 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession NO: NP_001036189, NP_002847) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD112, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD112 corresponding to amino acids 32-360 of the sequence below. It is understood that sequences of CD112 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD150 (also known as SLAM, SLAMF1, IPO-3) belongs to the signaling lymphocytic activation molecule family. CD150 can bind CD45. The function of CD150 includes co-stimulation of T-cells and B-cells. (See e.g., Sidorenko and Clark. Nature immunology 4.1 (2003) : 19-24. Yusuf et al. The Journal of Immunology 185.1 (2010) : 190-202.; De Salort et al. Immunology letters 134.2 (2011) : 129-136. ) A CD150 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession NO: NP_001317683, XP_016857618, NP_003028) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD150, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD150 corresponding to amino acids 21-237 of the sequence below. It is understood that sequences of CD150 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD155 (also known as PVR, NECL-5) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. The external domain mediates cell attachment to the extracellular matrix molecule vitronectin, while its intracellular domain interacts with the dynein light chain Tctex-1/DYNLT1. CD155 serves as a cellular receptor for poliovirus in the first step of poliovirus replication. CD155 can bind poliovirus, vitronectin, CD226, CD96, αVβ3, CD111, CD112. CD155 is known to mediate NK cell adhesion and trigger their effector functions. (See e.g., Chan et al. The journal of immunology 184.2 (2010) : 902-911. ) A CD155 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_001129240; NP_001129241; NP_001129242; NP_006496) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD155, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD155 corresponding to amino acids 21-343 of the sequence below. It is understood that sequences of CD155 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD265 (also known as RANK, TRANCE-R, ODFR, TNFRSF11A) is a member of the TNF-receptor superfamily. CD265 induces the activation of NF-kappa B and MAPK8/JNK and plays important role in regulating interaction between T cells and dendritic cells. CD265 can bind TRANCE. CD265 enhances T-cell growth and dendritic cell function, and regulates in lymph node organogenesis. (See e.g., Hanada et al., Journal of Molecular Medicine 89.7 (2011) : 647-656. ) A CD265 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_001257878, NP_001257879, NP_003830) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD265, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD265 corresponding to amino acids 30-212 of the sequence below. It is understood that sequences of CD265 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD270 (also known as HVEM, HveA, TR2, TNFRSF14) is a member of the TNF receptor superfamily. CD270 can bind CD258 and CD272. It functions in signal transduction pathways that activate inflammatory and inhibitory T-cell immune response. It binds herpes simplex virus (HSV) viral envelope glycoprotein D (gD) , mediating its entry into cells. (See e.g., Meng Q, et al. J Immunol, 2019, PMID 30770415) . A CD270 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_001284534; NP_003811) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD270, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD270 corresponding to amino acids 39-202 of the sequence below. It is understood that sequences of CD270 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

TL1A (also known as TL1; TL1A; VEGI; TNFSF15, TNLG1B; VEGI192A) is a cytokine that belongs to the TNF ligand family. This cytokine is a ligand for receptor TNFRSF25 and decoy receptor TNFRSF21/DR6. TL1A can activate NF-kappaB and MAP kinases, and acts as an autocrine factor to induce apoptosis in endothelial cells. This cytokine is also found to stimulate enhance IFN-γproduction in human T cells and NK cells. (See e.g., Papadakis et al., The Journal of Immunology 172.11 (2004) : 7002-7007. ) A TL1A polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession No. NP_005109; NP_001191273) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises TL1A, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of TL1A corresponding to amino acids 57-251 of the sequence below. It is understood that sequences of TL1A that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD127 (also known as ILRA; CD127; IL7RA; CDW127; IL-7R-alpha) is an s a receptor for interleukin 7 (IL7) . This protein has been shown to play a critical role in V (D) J recombination during lymphocyte development. Defects in this gene may be associated with severe combined immunodeficiency (SCID) . (See e.g., Carrette et al. Seminars in immunology. 24 (3) Academic Press, 2012. ) A CD127 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession No: NP_002176, XP_942460) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD127, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD127 corresponding to amino acids 21-239 of the sequence below. It is understood that sequences of CD127 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

IL-4R (also known as CD124; IL4RA; IL-4RA) is a type I transmembrane protein that can bind interleukin 4 and interleukin 13 to regulate IgE production. It can promote differentiation of Th2 cells. It is also found to activate macrophage during allergy and parasitic infections. (See e.g., Maldonado et al. Journal of Experimental Medicine 206.4 (2009) : 877-892. ) An IL-4R polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession No.: NP_000409, NP_001244335, NP_001244336, NP_001244926) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises IL-4R, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of IL-4R corresponding to amino acids 26-232 of the sequence below. It is understood that sequences of IL-4R that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

GITR-L (also known as AITRL, GITRL, TL6, TNF18, TNLG2A, hGITRL) is a cytokine that belongs to the TNF ligand family. This cytokine is a ligand for receptor NFRSF18/AITR/GITR. It has been shown to modulate T lymphocyte survival in peripheral tissues. This cytokine is also found to be expressed in endothelial cells and is thought to be important for interaction between T lymphocytes and endothelial cells. (See e.g., Tang X, et al. Oncotarget, 2016 Feb 23. PMID 26657118; Placke T, et al. J Immunol, 2012 Jul 1. PMID 22649191) . A GITR-L polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., NP_005083) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises GITR-L, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of GITR-L corresponding to amino acids 72-199 of the sequence below. It is understood that sequences of GITR-L that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

TIM-4 (also known as SMUCKLER, TIMD4) TIM-4 is expressed on APC and can deliver co-stimulating signals to T cells by binding to TIM-1. It has been found to induce T cell differentiation, expansion and survival. (See e.g., Rodriguez-Manzanet et al. The Journal of Immunology 180.7 (2008) : 4706-4713.; Nurtanio and Yang. North American journal of medical sciences 3.5 (2011) : 217. ) A TIM-4 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession: NP_001140198.1; NP_612388.2; Q96H15.2) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises TIM-4, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of TIM-4 corresponding to amino acids 25-314 of the sequence below. It is understood that sequences of TIM-4 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD153 (CD30L, TNFSF8) , cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for TNFRSF8/CD30, which is a cell surface antigen and a marker for Hodgkin lymphoma and related hematologic malignancies. CD153 binds to CD30 and induces proliferation and activation of T-cells (See e.g., Shimozato, et al. Biochemical and biophysical research communications 256.3 (1999) : 519-526; Croft, Nature Reviews Immunology 3.8 (2003) : 609-620. Marín and Luis, Tuberculosis 102 (2017) : 8-15. ) A CD153 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession NO: NP_001235, NP_001239219) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD153, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD153 corresponding to amino acids 63-234 of the sequence below. It is understood that sequences of CD153 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD48 (also known as BCM1, BLAST, BLAST1, MEM-102, or SLAMF2) is a member of the CD2 subfamily of immunoglobulin-like receptors which includes SLAM (signaling lymphocyte activation molecules) proteins. CD48 can bind to CD2 and deliver a co-stimulatory signal to T cells. CD48 is found on the surface of lymphocytes and other immune cells, dendritic cells, and endothelial cells, and participates in activation and differentiation pathways in these cells. A CD48 polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: EAW52705.1 GI: 119573090; Accession: CAG33293.1 GI: 48146141) , or fragments thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD48, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the mature form of CD48 corresponding to amino acids 27-220 of the sequence below. It is understood that sequences of CD48 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD160 (also known as NK1, BY55, or NK28) is a 27 kDa glycoprotein. The expression of CD160 is tightly associated with peripheral blood NK cells and CD8 T lymphocytes with cytolytic effector activity. A CD160 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession: EAW71440.1 GI: 119591846; Accession: CAI13713.1 GI: 55959477) , or fragments thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD160, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the mature form of CD160 corresponding to amino acids 25-159 of the sequence below. It is understood that sequences of CD160 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD200R (also known as HCRTR2, MOX2R, OX2R) can bind the OX-2 membrane glycoprotein. CD200R is a cell surface glycoprotein containing two immunoglobulin-like domains. It is reported to control myeloid function in a tissue-specific manner. It is also reported to regulate activity of an immune cell by recruiting accessory molecules (e.g., DAP12) to cell surface (See e.g., Gorczynski, International Scholarly Research Notices 2012 (2012) . ) A CD200R polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession NO: NP_620161; NP_620385) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD200R, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD200R corresponding to amino acids 29-243 of the sequence below. It is understood that sequences of CD200R that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD44 (also known as H-CAM, Pgp-1, Epican, HUTCH-I, LHR, ECMR-III) is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. It is a receptor for hyaluronic acid (HA) and can also interact with other ligands, such as osteopontin, collagens, and matrix metalloproteinases (MMPs) . This protein participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis. (See e.g., Huet et al. The Journal of Immunology 143.3 (1989) : 798-801. ) A CD44 polypeptide can have an amino acid sequence corresponding to the sequence provided below (e.g., Accession No. NP_000601, NP_001001389) . In some embodiments, provided herein are fusion proteins comprising a first domain that comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and a second domain that activates an immune effector cell, wherein the second domain comprises CD44, or a receptor-binding fragment thereof. In some embodiments, the second domain comprises the extracellular domain of CD44 corresponding to amino acids 21-649 of the sequence provided below. It is understood that sequences of CD44 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

5.3.1 Exemplary LACO-Stim Fusion Proteins

Accordingly, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an anti-CD40 antibody or antigen-binding fragment described herein and wherein the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a ligand that binds a co-stimulatory receptor of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. In some embodiments, the APC is selected from the group consisting of a dendritic cell, a macrophage, a myeloid derived suppressor cell, a monocyte, a B cell, a T cell, and a Langerhans cell. In some embodiments, the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.

The first domain can comprise any anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, the first domain comprises a monoclonal antibody. In some embodiments the first domain comprises a chimeric antibody. In some embodiments the first domain comprises a humanized antibody. In some embodiments the first domain comprises a human antibody. In some embodiments, the first domain comprises a Fab, Fab’, F (ab’) 2, Fv, scFv, (scFv) 2, single chain antibody, dual variable region antibody, diabody, nanobody, or single variable region antibody. In some embodiments the first domain comprises a human antibody. In some embodiments, the first domain comprises a scFv.

In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 antibody or antigen-binding fragment thereof. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the antibody designated as 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52 as provided below in Section 5.2 above.

In some embodiments, the first domain of the fusion proteins provided herein comprises an anti-CD40 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has (a) a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and/or (b) a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. In some embodiments, the first domain of the fusion proteins provided herein comprises an anti-CD40 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has a VL and a VH having the amino acid sequences of (1) SEQ ID NOs: 37 and 43, respectively; (2) SEQ ID NOs: 38 and 44, respectively; (3) SEQ ID NOs: 39 and 45, respectively; (4) SEQ ID NOs: 40 and 46, respectively; (5) SEQ ID NOs: 41 and 47, respectively; or (6) SEQ ID NOs: 42 and 48, respectively.

In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-66. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 61. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 62. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 64. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 65. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 66.

In some embodiments, the second domain of fusion proteins provided herein comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, or (b) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. The immune effector cell can be selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte. In some embodiments, the second domain of fusion proteins provided herein comprises a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, wherein the immune cell is a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, or a granulocyte. In some embodiments, the co-stimulatory receptor of the immune effector cell is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, DR3, and CD43. In some embodiments, the second domain of fusion proteins provided herein comprises a functional fragment of a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, DR3, and CD43. In some embodiments, the functional fragment comprises the cytoplasmic domain of the co-stimulatory receptor. In some embodiments, the second domain further comprises the transmembrane domain of the co-stimulatory receptor. In some embodiments, the second domain comprises a functional fragment of CD28. In some embodiments, the second domain comprises the cytoplasmic domain of CD28. In some embodiments, the second domain comprises a functional fragment of 4-1BB. In some embodiments, the second domain comprises the cytoplasmic domain of 4-1BB. In some embodiments, the second domain comprises a functional fragment of ICOS. In some embodiments, the second domain comprises the cytoplasmic domain of ICOS. In some embodiments, the second domain comprises a functional fragment of CD27. In some embodiments, the second domain comprises the cytoplasmic domain of CD27. In some embodiments, the second domain comprises a functional fragment of OX40. In some embodiments, the second domain comprises the cytoplasmic domain of OX40. In some embodiments, the second domain comprises a functional fragment of DAP10. In some embodiments, the second domain comprises the cytoplasmic domain of DAP10. In some embodiments, the second domain comprises a functional fragment of 2B4. In some embodiments, the second domain comprises the cytoplasmic domain of 2B4. In some embodiments, the second domain comprises a functional fragment of CD30. In some embodiments, the second domain comprises the cytoplasmic domain of CD30. In some embodiments, the second domain comprises a functional fragment of CD2. In some embodiments, the second domain the cytoplasmic domain of CD2. In some embodiments, the second domain comprises a functional fragment of LIGHT. In some embodiments, the second domain comprises the cytoplasmic domain of LIGHT. In some embodiments, the second domain comprises a functional fragment of GITR. In some embodiments, the second domain comprises the cytoplasmic domain of GITR. In some embodiments, the second domain comprises a functional fragment of DR3. In some embodiments, the second domain comprises the cytoplasmic domain of DR3. In some embodiments, the second domain comprises a functional fragment of CD43. In some embodiments, the second domain comprises the cytoplasmic domain of CD43.

In some embodiments, the second domain of fusion proteins provided herein comprises an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. The immune effector cell can be selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte. In some embodiments, the co-stimulatory receptor of the immune effector cell is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, DR3, and CD43. In some embodiments, the second domain comprises an antibody that binds CD28, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds 4-1BB, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds ICOS, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD27, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds OX40, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds DAP10, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds 2B4, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD30, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD2, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds LIGHT, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds GITR, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds DR3, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD43, or an antigen-binding fragment thereof.

In some embodiments, the second domain comprises a monoclonal antibody. In some embodiments the second domain comprises a chimeric antibody. In some embodiments the second domain comprises a humanized antibody. In some embodiments the second domain comprises a human antibody. In some embodiments, the second domain comprises a Fab, Fab’, F (ab’) 2, Fv, scFv, (scFv) 2, single chain antibody, dual variable region antibody, diabody, nanobody, or single variable region antibody. In some embodiments the second domain comprises a human antibody. In some embodiments, the second domain comprises a scFv.

In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD28 scFv. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the antibody that is designated 1412.

Table 4: Exemplary Anti-CD28 Antibody

In some embodiments, the second domain of the fusion proteins provided herein comprises an anti-CD28 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has (a) a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 162; and/or (b) a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 163. In some embodiments, the second domain of the fusion proteins provided herein comprises an anti-CD28 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has (a) a VH having an amino acid sequence that is SEQ ID NO: 162; and/or (b) a VL having an amino acid sequence that is SEQ ID NO: 163. In some embodiments, the second domain of the fusion proteins provided herein comprise an anti-CD28 scFv having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 164. In some embodiments, the second domain of the fusion proteins provided herein comprise an anti-CD28 scFv having the amino acid sequence of SEQ ID NO: 164.

The fusion proteins described herein (i.e., the LACO molecules) can include any combinations of the anti-CD40 antibody or antigen binding fragment described herein and immune effector cell activators (co-stimulatory receptors or antibodies that bind co-stimulatory receptors) disclosed herein or otherwise known in the art. For illustration purposes, provided below are various forms of the anti-CD40-CD28 LACO molecules that activates APCs (e.g., the dendritic cells) via the CD40/CD40L signaling and activates immune effector cells (e.g., the T cells) via the CD28 signaling.

5.3.1.1 Exemplary LACO-Stim (1) : anti-CD40 + antibody for co-stimulatory receptor (e.g., aCD40/aCD28 bispecific Ab)

In some embodiments, provided herein are bispecific antibodies. A “bispecific antibody, ” as used herein and understood in the art, refers to an antibody having binding specificities for at least two different antigenic epitopes. The epitopes can be from the same antigen or two different antigens. In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an anti-CD40 antibody or antigen binding fragment described herein, and wherein the second domain comprises an antibody that binds a co-stimulatory receptor of the immune effector cell (e.g., a T cell) , or an antigen-binding fragment thereof. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the scFv designated as 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52 as provided below in Section 5.2 above. In some embodiments, the second domain comprise an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain.

In some embodiments, provided herein are bispecific antibodies comprising a first domain that is the anti-CD40 scFv designated as 40-18, and a second domain comprising an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is the anti-CD40 scFv designated as 40-37, and a second domain comprising an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is the anti-CD40 scFv designated as 40-38, and a second domain comprising an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is the anti-CD40 scFv designated as 40-45, and a second domain comprising an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is the anti-CD40 scFv designated as 40-47, and a second domain comprising an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is the anti-CD40 scFv designated as 40-52, and a second domain comprising an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43.

In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or antigen-binding fragment described herein, and a second domain comprising an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43. In some embodiments, the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-4-1BB antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-ICOS antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-CD27 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-OX40 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-DAP10 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-2B4 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-CD30 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-CD2 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-LIGHT antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-GITR antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-TLR antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-DR3 antibody or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an anti-CD43 antibody or an antigen-binding fragment thereof.

Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan et al. (1985) Science 229: 81. Bispecific antibodies include bispecific antigen-binding fragments. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 6444-48; Gruber et al. (1994) J. Immunol. 152: 5368. Techniques for making bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983) , WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991) ) , and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168) . Multi-specific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1) ; cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science 229: 81 (1985) ) ; using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148 (5) : 1547-1553 (1992) ) ; using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) ) ; and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368 (1994) ) ; and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991) . Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies, ” are also included herein (see, e.g., US 2006/0025576A1) . Bispecific antibodies can be constructed by linking two different antibodies, or portions thereof. For example, a bispecific antibody can comprise Fab, F (ab′) ₂, Fab′, scFv, and sdAb from two different antibodies.

In some embodiments, the anti-CD28 antibody or antigen-binding fragment can be any anti-CD28 antibody or antigen-binding fragment disclosed herein or otherwise known in the art that activate CD28 signaling. In some embodiments, the anti-CD28 antibody or antigen-binding fragment is the antibody designated 1412. In some embodiments, the anti-CD28 antibody or antigen-binding fragment thereof has (a) a VH having an amino acid sequence that is SEQ ID NO: 162; and/or (b) a VL having an amino acid sequence that is SEQ ID NO: 163. In some embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises an anti-CD28 scFv having the amino acid sequence of SEQ ID NO: 161.

As a person of ordinary skill in the art would understand, the anti-CD28 antibody or antigen-binding fragment in the fusion proteins exemplified herein can be replaced with an antibody or antigen-binding fragment that binds another co-stimulator for immune effector cells that is disclosed herein or otherwise known in the art, including, for example, an antibody or antigen-binding fragment that binds 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43.

5.3.1.2 Exemplary LACO-Stim (2) : anti-CD40 + co-stimulatory receptor (e.g., aCD40-CD28; aCD40-4-1BB)

In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an anti-CD40 antibody or antigen-binding fragment disclosed herein and wherein the second domain comprises a co-stimulatory receptor of the immune effector cell (e.g., T cell) , or a functional fragment thereof. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, provided herein are antibody-based membrane fusion protein.

In some embodiments, the first and second domains are linked via a CD8 hinge, a CD28 hinge, or an IgG Fc region. In some embodiments, the first and second domains are linked via a CD8 hinge. In some embodiments, the CD8 hinge has the amino acid sequence of SEQ ID NO: 164. In some embodiments, the first and second domains are linked via a CD28 hinge. In some embodiments, the CD28 hinge has the amino acid sequence of SEQ ID NO: 165. In some embodiments, the first and second domains are linked via an IgG Fc region. In some embodiments, the IgG Fc region has the amino acid sequence of SEQ ID NO: 166.

In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the scFv designated as 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52 as provided below in Section 5.2 above. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-18, and a second domain comprising a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-37, and a second domain comprising a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-38, and a second domain comprising a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-45, and a second domain comprising a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-47, and a second domain comprising a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-52, and a second domain comprising a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof.

The second domain of the fusion proteins can comprise a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain of the fusion proteins provided herein comprises a CD28 cytoplasmic domain (e.g., amino acids 180 to 220 of SEQ ID NO: 109) . In some embodiments, the second domain of the fusion proteins provided herein can have an amino acid sequence that is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to amino acids 180 to 220 of SEQ ID NO: 109. In some embodiments, the second domain of the fusion proteins provided herein has amino acids 180 to 220 of SEQ ID NO: 109. In some embodiments, the second domain of the fusion proteins provided herein further comprises a CD28 transmembrane domain (e.g., amino acids 153 to 179 of SEQ ID NO: 109) . In some embodiments, the second domain of the fusion proteins provided herein comprises a 4-1BB cytoplasmic domain (e.g., amino acids 214 to 255 of SEQ ID NO: 110) . In some embodiments, the second domain of the fusion proteins provided herein can have an amino acid sequence that is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to amino acids 214 to 255 of SEQ ID NO: 110. In some embodiments, the second domain of the fusion proteins provided herein has amino acids 214 to 255 of SEQ ID NO: 110. In some embodiments, the second domain of the fusion proteins provided herein further comprises a 4-1BB transmembrane domain (e.g., amino acids 187 to 213 of SEQ ID NO: 110) .

In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or antigen-binding fragment described herein, and a second domain that the second domain that comprises a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the second domain that comprises a 4-1BB cytoplasmic domain. In some embodiments, the second domain comprises an ICOS cytoplasmic domain. In some embodiments, the second domain comprises a CD27 cytoplasmic domain. In some embodiments, the second domain comprises an OX40 cytoplasmic domain. In some embodiments, the second domain comprises a DAP10 cytoplasmic domain. In some embodiments, the second domain comprises a 2B4 cytoplasmic domain. In some embodiments, the second domain comprises a CD30 cytoplasmic domain. In some embodiments, the second domain comprises a CD2 cytoplasmic domain. In some embodiments, the second domain comprises a LIGHT cytoplasmic domain. In some embodiments, the second domain comprises a GITR cytoplasmic domain. In some embodiments, the second domain comprises a TLR cytoplasmic domain. In some embodiments, the second domain comprises a DR3 cytoplasmic domain. In some embodiments, the second domain comprises a CD43 cytoplasmic domain.

In some embodiments, fusion proteins provided herein further comprise a transmembrane region. In some embodiments, the transmembrane region is derived from the same co-stimulatory receptor. In some embodiments, the transmembrane region is derived from a different co-stimulatory receptor. In some embodiments, the second domain comprises a CD28 transmembrane region and a CD28 cytoplasmic domain. In some embodiments, provided herein are fusion proteins having a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a 4-1BB transmembrane region and a 4-1BB cytoplasmic domain.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-18.28 (SEQ ID NO: 67) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%identical to SEQ ID NO: 67. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 90%identical to SEQ ID NO: 67. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 95%identical to SEQ ID NO: 67. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 98%identical to SEQ ID NO: 67. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 99%identical to SEQ ID NO: 67. In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 67.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-37.28 (SEQ ID NO: 68) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%identical to SEQ ID NO: 68. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 90%identical to SEQ ID NO: 68. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 95%identical to SEQ ID NO: 68. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 98%identical to SEQ ID NO: 68. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 99%identical to SEQ ID NO: 68. In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 68.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-38.28 (SEQ ID NO: 69) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%identical to SEQ ID NO: 69. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 90%identical to SEQ ID NO: 69. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 95%identical to SEQ ID NO: 69. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 98%identical to SEQ ID NO: 69. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 99%identical to SEQ ID NO: 69. In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 69.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-45.28 (SEQ ID NO: 70) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%identical to SEQ ID NO: 70. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 90%identical to SEQ ID NO: 70. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 95%identical to SEQ ID NO: 70. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 98%identical to SEQ ID NO: 70. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 99%identical to SEQ ID NO: 70. In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 70.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-47.28 (SEQ ID NO: 71) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%identical to SEQ ID NO: 71. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 90%identical to SEQ ID NO: 71. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 95%identical to SEQ ID NO: 71. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 98%identical to SEQ ID NO: 71. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 99%identical to SEQ ID NO: 71. In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 71.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-52.28 (SEQ ID NO: 72) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 85%identical to SEQ ID NO: 72. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 90%identical to SEQ ID NO: 72. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 95%identical to SEQ ID NO: 72. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 98%identical to SEQ ID NO: 72. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 99%identical to SEQ ID NO: 72. In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 72.

As a person of ordinary skill in the art would understand, the CD28 cytoplasmic domain in the fusion proteins exemplified herein can be replaced with the cytoplasmic domain of another co-stimulator for immune effector cells that is disclosed herein or otherwise known in the art, including, for example, the cytoplasmic domain of 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43; or a different functional fragment of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43 that retains the function of the full-length protein to activate the immune effector cell.

5.3.1.3 Exemplary LACO-Stim (3) : anti-CD40 + ligand for co-stimulatory receptor (e.g., aCD40-CD80; aCD40-CD86)

In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an anti-CD40 antibody or antigen-binding fragment described herein; and wherein the second domain comprises a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, provided herein are antibody-based soluble fusion protein. In some embodiments, provided herein are antibody-based soluble fusion protein.

In some embodiments, the fusion protein comprises a first domain that comprises an anti-CD40 antibody or antigen-binding fragment thereof disclosed herein, and a second domain comprises the ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, CD44, and receptor-binding fragments thereof. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the scFv designated as 40-18, 40-37, 40-38, 40-45, 40-47, and 40-52 as provided below in Section 5.2 above. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-18, and a second domain comprising a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44, or a receptor-binding fragments thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-37, and a second domain comprising a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44, or a receptor-binding fragments thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-38, and a second domain comprising a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44, or a receptor-binding fragments thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-45, and a second domain comprising a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44, or a receptor-binding fragments thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-47, and a second domain comprising a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44, or a receptor-binding fragments thereof. In some embodiments, provided herein are fusion proteins comprising a first domain that is the anti-CD40 scFv designated as 40-52, and a second domain comprising a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44, or a receptor-binding fragments thereof.

In some embodiments, the second domain comprises CD58 (e.g., SEQ ID NO: 122) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD70 (e.g., SEQ ID NO: 123) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD83 (e.g., SEQ ID NO: 124) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD80 (e.g., SEQ ID NO: 125) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD86 (e.g., SEQ ID NO: 126) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD137L (e.g., SEQ ID NO: 127) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD252 (e.g., SEQ ID NO: 128) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD275 (e.g., SEQ ID NO: 129) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD54 (e.g., SEQ ID NO: 130) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD49a (e.g., SEQ NO: 131) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD112 (e.g., SEQ ID NO: 132) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD150 (e.g., SEQ ID NO: 133) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD155 (e.g., SEQ ID NO: 134) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD265 (e.g., SEQ ID NO: 135) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD270 (e.g., SEQ ID NO: 136) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises TL1A (e.g., SEQ ID NO: 137) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD127 (e.g., SEQ ID NO: 138) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises IL-4R (e.g., SEQ ID NO: 139) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises GITR-L (e.g., SEQ ID NO: 140) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises TIM-4 (e.g., SEQ ID NO: 141) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD153 (e.g., SEQ ID NO: 142) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD48 (e.g., SEQ ID NO: 143) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD160 (e.g., SEQ ID NO: 144) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD200R (e.g., SEQ ID NO: 145) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD44 (e.g., SEQ ID NO: 146) or a receptor-binding fragment thereof. A person of ordinary skill in the art can readily determine a proper receptor-binding fragment of a ligand that retains its binding affinity toward its receptor and function to activate the receptor.

5.4 Polynucleotides and Vectors

Also provided herein are polynucleotides that encode a polypeptide (e.g., an anti-CD40 antibody or antigen-binding fragment, or a LACO molecule) described herein. The term “polynucleotide that encode a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single stranded DNA can be the coding strand or non-coding (anti-sense) strand. The polynucleotides of the disclosure can be mRNA.

Expressly contemplated herein are polynucleotides encode any anti-CD40 antibody or antigen-binding fragment disclosed herein. For illustrative purposes, in some embodiments, the polynucleotides provided herein encode an anti-CD40 antibody or antigen-binding fragment comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-30; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the polynucleotides provided herein encode an anti-CD40 antibody or antigen-binding fragment comprising (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and/or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-CD40 antibody or antigen-binding fragment disclosed herein comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 1, 7, 13, 19, 25, and 31, respectively; (2) SEQ ID NOs: 2, 8, 14, 20, 26, and 32, respectively; (3) SEQ ID NOs: 3, 9, 15, 21, 27, and 33, respectively; (4) SEQ ID NOs: 4, 10, 16, 22, 28, and 34, respectively; (5) SEQ ID NOs: 5, 11, 17, 23, 29, and 35, respectively; or (6) SEQ ID NOs: 6, 12, 18, 24, 30, and 36, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-CD40 antibody or antigen-binding fragment disclosed herein comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of (1) SEQ ID NOs: 37 and 43, respectively; (2) SEQ ID NOs: 38 and 44, respectively; (3) SEQ ID NOs: 39 and 45, respectively; (4) SEQ ID NOs: 40 and 46, respectively; (5) SEQ ID NOs: 41 and 47, respectively; or (6) SEQ ID NOs: 42 and 48, respectively. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-CD40 antibody or antigen-binding fragment disclosed herein comprising a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42. Also provided is a polynucleotide that hybridizes to a polynucleotide having a nucleotide sequence encoding an anti-CD40 antibody or antigen-binding fragment disclosed herein comprising a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 49-54. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 49. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 50. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 51. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 52. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 53. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 54.

In some embodiments, the polynucleotides provided herein encode an anti-CD40 antibody or antigen-binding fragment disclosed herein comprising a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. Also provided is a polynucleotide that hybridizes to a polynucleotide having a nucleotide sequence encoding an anti-CD40 antibody or antigen-binding fragment disclosed herein comprising a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55-60. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 55. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 56. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 57. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 58. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 59. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 60.

In some embodiments, the hybridization is under conditions of high stringency as is known to those skilled in the art. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

The present disclosure also provides variants of the polynucleotides described herein, wherein the variants encode, for example, fragments, analogs, and/or derivatives of an anti-CD40 antibody or antigen-binding fragment disclosed herein. In some embodiments, the present disclosure provides a polynucleotide having a nucleotide sequence at least about 80%identical, at least about 85%identical, at least about 90%identical, at least about 95%identical, at least about 96%identical, at least about 97%identical, at least about 98%identical, or at least about 99%identical to a polynucleotide sequence encoding an anti-CD40 antibody or antigen-binding fragment described herein.

In some embodiments, the polynucleotides provided herein encode the anti-CD40 scFv designated as 40-18, 40-37, 40-38, 40-45, 40-47, or 40-52. In some embodiments, the polynucleotides provided herein encode an anti-CD40 scFv having an amino acid sequence selected from the group consisting of SEQ ID NOs: 61-66. In some embodiments, the polynucleotides provided herein encode an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 61. In some embodiments, the polynucleotides provided herein encode an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 62. In some embodiments, the polynucleotides provided herein encode an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 63. In some embodiments, the polynucleotides provided herein encode an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 64. In some embodiments, the polynucleotides provided herein encode an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 65. In some embodiments, the polynucleotides provided herein encode an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 66.

Provided herein are also polynucleotides that encode the fusion proteins provided herein, which comprise a first domain that activates an APC and a second domain that activates an immune effector cell (e.g., a T cell) , wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof disclosed herein, and the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. In some embodiments, provided herein are polynucleotides that encode a . The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, provided herein are polynucleotides that encode the provided LACO molecules designated as 40-18.28, 40-37.28, 40-38.28, 40-45.28, 40-47.28, or 40-52.28. In some embodiments, the polynucleotides provided herein encode a fusion protein having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-72. In some embodiments, the polynucleotides provided herein encode a fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-72. In some embodiments, the polynucleotides provided herein encode a fusion protein having the amino acid sequence of SEQ ID NO: 67. In some embodiments, the polynucleotides provided herein encode a fusion protein having the amino acid sequence of SEQ ID NO: 68. In some embodiments, the polynucleotides provided herein encode a fusion protein having the amino acid sequence of SEQ ID NO: 69. In some embodiments, the polynucleotides provided herein encode a fusion protein having the amino acid sequence of SEQ ID NO: 70. In some embodiments, the polynucleotides provided herein encode a fusion protein having the amino acid sequence of SEQ ID NO: 71. In some embodiments, the polynucleotides provided herein encode a fusion protein having the amino acid sequence of SEQ ID NO: 72.

In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 73-78. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 73. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 74. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 75. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 76. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 77. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 78. Also provided is a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 73-78. In some embodiments, the hybridization is under conditions of high stringency as is known to those skilled in the art.

As used herein, the phrase “a polynucleotide having a nucleotide sequence at least about 95%identical to a polynucleotide sequence” means that the nucleotide sequence of the polynucleotide is identical to a reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5%of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code) . Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) . In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a polynucleotide which aids in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide) . The polypeptide can have the leader sequence cleaved by the host cell to form a “mature” form of the polypeptide.

In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a marker or tag sequence. For example, in some embodiments, a marker sequence is a hexa-histidine tag (HIS-tag) that allows for efficient purification of the polypeptide fused to the marker. In some embodiments, a marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG ^TM tag. In some embodiments, a marker can be used in conjunction with other markers or tags.

In some embodiments, a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, provided herein are vectors comprising a polynucleotide provided herein. The vectors can be expression vectors. In some embodiments, vectors provided herein comprise a polynucleotide encoding an anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of an anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a fusion protein described herein.

In some embodiments, provided herein are recombinant expression vectors that can be used to amplify and express a polynucleotide encoding an anti-CD40 antibody or antigen-binding fragment described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-CD40 antibody, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, provided herein are recombinant expression vectors that can be used to amplify and express a polynucleotide encoding a fusion protein described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of a fusion protein, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, a viral vector is used. DNA regions are “operatively linked” when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in certain expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In some embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide can include an N-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment described herein or a fusion protein is expressed from one or more vectors. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art.

Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

The present disclosure also provides host cells comprising the polypeptides described herein, polynucleotides encoding polypeptides described herein, or vectors comprising such polynucleotides. In some embodiments, provided herein are host cells comprising a vector comprising a polynucleotide disclosed herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding an anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding a polypeptide that is part of an anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, host cells provided herein comprise a polynucleotide encoding an anti-CD40 antibody or antigen-binding fragment described herein. In some embodiments, the cells produce the anti-CD40 antibodies or antigen-binding fragments described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding a fusion protein described herein. In some embodiments, host cells provided herein comprise a polynucleotide encoding a fusion protein described herein. In some embodiments, the cells produce the fusion protein described herein.

5.5 Genetically engineered immune effector cells

Provided herein are genetically engineered immune effector cells recombinantly expressing the fusion proteins disclosed herein. Provided herein are also genetically engineered cells comprising the polynucleotides disclosed herein. In some embodiments, provided herein are also genetically engineered cells comprising the vectors disclosed herein.

In some embodiments, the genetically engineered immune effector cell provided herein is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte. In some embodiments, the cell provided herein is a T cell. In some embodiments, the cell provided herein is an NK cell. In some embodiments, the cell provided herein is an NKT cell. In some embodiments, the cell provided herein is a macrophage. In some embodiments, the cell provided herein is a neutrophil. In some embodiments, the cell provided herein is a granulocyte. In some embodiments, the genetically engineered immune effector cells provided herein are isolated. In some embodiments, the genetically engineered immune effector cells provided herein are substantially pure.

In some embodiments, the immune effector cell provided herein is a T cell. The T cell can be a cytotoxic T cell, a helper T cell, or a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, an effector memory TEMRA cell, or a gamma delta T cell. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the genetically engineered T cells provided herein are isolated. In some embodiments, the genetically engineered T cells provided herein are substantially pure.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a subject. As used herein, a genetically engineered cell that is “derived from” a source cell means that the genetically engineered cell is obtained by taking the source cell and genetically manipulating the source cell. The source cell can be from a natural source. For example, the source cell can be a primary cell isolated from a subject. The subject can be an animal or a human. The source cell can also be a cell that has undergone passages or genetically manipulation in vitro.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a human. Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell lines available in the art can be used. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from bone marrow. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood mononuclear cells (PBMC) .

In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a stem or progenitor cell. In some embodiments, the stem or progenitor cell is selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a T cell progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic stem and progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic multipotent progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an embryonic stem cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an induced pluripotent cell.

In some embodiments, provided herein are a population of the genetically engineered cells disclosed herein. The population of cells can be a homogenous population of cells. The population of cells can be a heterogeneous population of cells. In some embodiments, the population of cells can be a heterogeneous population of cells comprising any combination of the cells disclosed herein. In some embodiments, the population of genetically engineered cells provided herein are derived from tumor-infiltrating lymphocytes (TIL) . In some embodiments, the population of genetically engineered cells provided herein are derived from peripheral blood mononuclear cells (PBMC) . In some embodiments, the population of genetically engineered cells provided herein are derived from peripheral blood leukocytes (PBL) . In some embodiments, the population of genetically engineered cells provided herein are derived from tumor infiltrating lymphocytes (TIL) . In some embodiments, the population of genetically engineered cells provided herein are derived from marrow infiltrate lymphocytes (MILs) . In some embodiments, the population of genetically engineered cells provided herein are derived from cytokine-induced killer cells (CIK) . In some embodiments, the population of genetically engineered cells provided herein are derived from lymphokine-activated killer cells (LAK) .

In some embodiments, the genetically engineered immune effector cells provided herein further recombinantly express a chimeric antigen receptor (CAR) , a T cell receptor (TCR) or a Bi-specific T-cell engager (BiTE) . In some embodiments, the genetically engineered cells disclosed herein further express a CAR. In some embodiments, the genetically engineered cells disclosed herein further express a TCR. In some embodiments, the genetically engineered cells disclosed herein further express a BiTE. In some embodiments, the genetically engineered immune effector cells provided herein further comprise a polynucleotide that encodes a CAR, a TCR or a BiTE (CAR/TCR/BiTE) . In some embodiments, the genetically engineered immune effector cells provided herein further comprise a polynucleotide that encodes a CAR. In some embodiments, the genetically engineered immune effector cells provided herein further comprise a polynucleotide that encodes a TCR. In some embodiments, the genetically engineered immune effector cells provided herein further comprise a polynucleotide that encodes a BiTE. In some embodiments, the CAR, TCR or BiTE binds a tumor antigen or a viral antigen.

In some embodiments, the genetically engineered immune effector cells provided herein further expresses a CAR or comprises a polynucleotide that encodes a CAR. The CAR can be any CAR disclosed herein or otherwise known in the art. In some embodiments, the CAR comprises an antigen-binding domain that specifically binds a tumor antigen. As such, in some embodiments, provided herein are also genetically engineered cells expressing a fusion protein disclosed herein and a CAR. In some embodiments, genetically engineered cells provided herein comprise a polynucleotide that comprises a first fragment encoding a fusion protein, and a second fragment encoding a CAR. In some embodiments, genetically engineered cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a CAR.

In some embodiments, the genetically engineered immune effector cells provided herein further expresses a CAR or comprises a polynucleotide that encodes a TCR. The TCR can be any TCR disclosed herein or otherwise known in the art. In some embodiments, the TCR comprises an antigen-binding domain that specifically binds a tumor antigen. As such, in some embodiments, provided herein are also genetically engineered cells expressing a fusion protein disclosed herein and a TCR. In some embodiments, genetically engineered cells provided herein comprise a polynucleotide that comprises a first fragment encoding a fusion protein, and a second fragment encoding a TCR. In some embodiments, genetically engineered cells provided herein comprise a first polynucleotide encoding a fusion protein and a second polynucleotide encoding a TCR.

In some embodiments, the genetically engineered immune effector cells provided herein further expresses a CAR or comprises a polynucleotide that encodes a BiTE. The BiTE can be any BiTE disclosed herein or otherwise known in the art. In some embodiments, the BiTE comprises an antigen-binding domain that specifically binds a tumor antigen. As such, in some embodiments, provided herein are also genetically engineered cells expressing a fusion protein disclosed herein and a BiTE. In some embodiments, genetically engineered cells provided herein comprise a polynucleotide that comprises a first fragment encoding a fusion protein, and a second fragment encoding a BiTE. In some embodiments, genetically engineered cells provided herein comprise a first polynucleotide encoding a fusion protein and a second polynucleotide encoding a BiTE.

In some embodiments, the CAR, TCR, or BiTE provided herein include a target-binding domain that binds an antigen. In some embodiments, the antigen is a viral antigen. In some embodiments, the viral antigen is EBV. In some embodiments, the viral antigen is HPV. In some embodiments, the viral antigen is HIV. It is understood that these or other viral antigens can be utilized for targeting by a CAR, TCR, or BiTE disclosed herein.

In some embodiments, the CAR, TCR, or BiTE provided herein include a target-binding domain that binds a cancer antigen or a tumor antigen. Any suitable cancer antigen or tumor antigen can be chosen based on the type of cancer exhibited by a subject (cancer patient) to be treated. It is understood that the selected cancer antigen is expressed in a manner such that the cancer antigen is accessible for binding. Generally, the cancer antigen to be targeted by a cell expressing a CAR, TCR, or BiTE is expressed on the cell surface of a cancer cell. However, it is understood that any cancer antigen that is accessible for binding is suitable for targeting.

Suitable antigens include, but are not limited to, B-cell maturation antigen (BCMA) , mesothelin (MSLN) , prostate specific membrane antigen (PSMA) , prostate stem cell antigen (PSCA) , carbonic anhydrase IX (CAIX) , carcinoembryonic antigen (CEA) , CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD123, CD133, CD138, CD33, CD200R, alpha-fetoprotein (AFP) , B7H3, B7H4, IL3Ra2, CS1, C-Met, Ber-EP4 (EpCAM-1) , ) epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM) , folate-binding protein (FBP) , fetal acetylcholine receptor (AChR) , folate receptor-α and β (FRα and β) , Ganglioside G2 (GD2) , Ganglioside G3 (GD3) , human Epidermal Growth Factor Receptor 2 (HER-2/ERB2) , Epidermal Growth Factor Receptor (EGFR) , Epidermal Growth Factor Receptor vIII (EGFRvIII) , ERB3, ERB4, GDNF family receptor alpha 4 (GFRa4) , Histone 3 variant (H3.3) , human telomerase reverse transcriptase (hTERT) , Interleukin-13 receptor subunit alpha-2 (IL13Rα2) , κ-light chain, kinase insert domain receptor (KDR) , Lewis A (CA19.9) , Lewis Y (LeY) , L1 cell adhesion molecule (LlCAM) , melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1) , MAGE-A3, Mucin 16 (Muc-16) , Mucin 1 (Muc-1) , Tn-MUC1, NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4) , tumor-associated glycoprotein 72 (TAG-72) , vascular endothelial growth factor R (VEGF-R) , Wilms tumor protein (WT-1) , type 1 tyrosine-protein kinase transmembrane receptor (ROR1) , B7-H3 (CD276) , B7-H6 (Nkp30) , Chondroitin sulfate proteoglycan-4 (CSPG4) , DNAX Accessory Molecule (DNAM-1) , Ephrin type A Receptor 2 (EpHA2) , Fibroblast Associated Protein (FAP) , Gp100/HLA-A2, Glypican 3 (GPC3) , HA-1H, HERK-V, IL-11Rα, Latent Membrane Protein 1 (LMP1) , MAG3, Neural cell-adhesion molecule (N-CAM/CD56) , NY-ESO-1, Melan-A (MART1) , PD-L1, WT1 transcription factor (WT1) , P53, KRAS, TCRB1, TCRB2, and Trail Receptor (TRAIL R) . It is understood that these or other cancer antigens can be utilized for targeting by a CAR, TCR, or BiTE disclosed herein.

Additionally, it is recognized in the art that the cell–based immune system frequently responds to the neoantigens that arise from DNA damage that can lead to malignant transformation, and recognition of neoantigens can be an important driver of the clinical activity of cell therapies, such as adoptive T cell therapies (e.g., Schumacher, Science 348.6230 (2015) : 69-74. Schumacher et al., Annual review of immunology 37 (2019) : 173-200) . Neoantigens can be identified using classical approaches focusing on common shared mutations (e.g., mutated BRAF, KRAS, and p53) , or using next-generation sequencing techniques (e.g., Lu, Yong-Chen, and Paul F. Robbins. Seminars in immunology. Vol. 28. No. 1. Academic Press, 2016) . In some embodiments, the CAR, TCR, or BiTE provided herein include a target-binding domain that binds a cancer neoantigen or a tumor neoantigen.

In some embodiments, the genetically engineered immune effector cells provided herein further comprise a polynucleotide that encodes a CAR, TCR, or BiTE that binds a cancer antigen or tumor antigen. In some embodiments, the genetically engineered immune effector cells provided herein further recombinantly express a CAR, TCR, or BiTE that binds a cancer antigen or tumor antigen. In some embodiments, the cancer antigen or tumor antigen is selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin. In some embodiments, the cancer antigen or tumor antigen is Her2. In some embodiments, the cancer antigen or tumor antigen is NY-ESO-1. In some embodiments, the cancer antigen or tumor antigen is CD19. In some embodiments, the cancer antigen or tumor antigen is CD20. In some embodiments, the cancer antigen or tumor antigen is CD22. In some embodiments, the cancer antigen or tumor antigen is PSMA. In some embodiments, the cancer antigen or tumor antigen is c-Met. In some embodiments, the cancer antigen or tumor antigen is GPC3. In some embodiments, the cancer antigen or tumor antigen is IL13ra2. In some embodiments, the cancer antigen or tumor antigen is EGFR. In some embodiments, the cancer antigen or tumor antigen is CD123. In some embodiments, the cancer antigen or tumor antigen is CD7. In some embodiments, the cancer antigen or tumor antigen is GD2. In some embodiments, the cancer antigen or tumor antigen is PSCA. In some embodiments, the cancer antigen or tumor antigen is EBV16-E7. In some embodiments, the cancer antigen or tumor antigen is H3.3. In some embodiments, the cancer antigen or tumor antigen is EGFRvIII. In some embodiments, the cancer antigen or tumor antigen is BCMA. In some embodiments, the cancer antigen or tumor antigen is mesothelin.

5.5.1 CARs

The genetically engineered immune effector cells (e.g., T cells) provided herein can be used in cancer treatment. In some embodiments, provided herein is a genetically engineered T cell that expresses the fusion protein disclosed herein. In some embodiments, provided herein is a genetically engineered T cell that comprises the polynucleotide disclosed herein. In some embodiments, provided herein is a CAR-T cell.

In some embodiments, the fusion proteins provided herein can be co-expressed with a CAR in an immune effector cell. In some embodiments, a fusion protein provided herein can be conjugated to a CAR. CARs retarget immune effector cells (e.g., T cells) to tumor surface antigens (Sadelain et al., Nat. Rev. Cancer. 3 (1) : 35-45 (2003) ; Sadelain et al., Cancer Discovery 3 (4) : 388-398 (2013) ) . CARs are engineered receptors that provide both antigen binding and immune effector cell activation functions. CARs can be used to graft the specificity of an antibody, such as a monoclonal antibody, onto an immune effector cell such as a T cell, a NK cell, or a macrophage. First-generation receptors link an antibody-derived tumor-binding element, such as an scFv, that is responsible for antigen recognition to either CD3zeta or Fc receptor signaling domains, which trigger T-cell activation. The advent of second-generation CARs, which combine activating and costimulatory signaling domains, has led to encouraging results in patients with chemorefractory B-cell malignancies (Brentjens et al., Science Translational Medicine 5 (177) : 177ra38 (2013) ; Brentjens et al., Blood 118 (18) : 4817-4828 (2011) ; Davila et al., Science Translational Medicine 6 (224) : 224ra25 (2014) ; Grupp et al., N. Engl. J. Med. 368 (16) : 1509-1518 (2013) ; Kalos et al., Science Translational Medicine 3 (95) : 95ra73 (2011) ) . The extracellular antigen-binding domain of a CAR is usually derived from a monoclonal antibody (mAb) or from receptors or their ligands. Antigen binding by the CARs triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the intracellular domain, initiating a signaling cascade required for cytolysis induction, cytokine secretion, and proliferation.

In some embodiments, a fusion protein provided herein can be conjugated to CAR that has an antigen binding domain that binds to a cancer antigen. In some embodiments, the CAR can be a “first generation, ” “second generation” or “third generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ; Jensen et al., Immunol. Rev. 257: 127-133 (2014) ; Sharpe et al., Dis. Model Mech. 8 (4) : 337-350 (2015) ; Brentjens et al., Clin. Cancer Res. 13: 5426-5435 (2007) ; Gade et al., Cancer Res. 65: 9080-9088 (2005) ; Maher et al., Nat. Biotechnol. 20: 70-75 (2002) ; Kershaw et al., J. Immunol. 173: 2143-2150 (2004) ; Sadelain et al., Curr. Opin. Immunol. 21 (2) : 215-223 (2009) ; Hollyman et al., J. Immunother. 32: 169-180 (2009) ) .

“First generation” CARs are typically composed of an extracellular antigen binding domain, for example, a single-chain variable fragment (scFv) , fused to a transmembrane domain, which is fused to a cytoplasmic/intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3ζ-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs) . “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4 ⁺ and CD8 ⁺ T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second-generation” CARs comprise a cancer antigen-binding domain fused to an intracellular signaling domain capable of activating immune effector cells such as T cells and a co-stimulatory domain designed to augment immune effector cell, such as T cell, potency and persistence (Sadelain et al., Cancer Discov. 3: 388-398 (2013) ) . CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory receptors, for example, CD28, 4-1BB, ICOS, OX40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell. “Second generation” CARs provide both co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3ζ signaling domain. Studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4-1BB domains, and activation, for example, by comprising a CD3ζ activation domain.

As described above, a CAR also contains a signaling domain that functions in the immune effector cell expressing the CAR. Such a signaling domain can be, for example, derived from CDζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b) , CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP12. In general, the signaling domain will induce persistence, trafficking and/or effector functions in the transduced immune effector cells such as T cells (Sharpe et al., Dis. Model Mech. 8: 337-350 (2015) ; Finney et al., J. Immunol. 161: 2791-2797 (1998) ; Krause et al., J. Exp. Med. 188: 619-626 (1998) ) . In the case of CDζ or Fc receptor γ, the signaling domain corresponds to the intracellular domain of the respective polypeptides, or a fragment of the intracellular domain that is sufficient for signaling. Exemplary signaling domains are described below in more detail.

In certain non-limiting embodiments, an intracellular domain of a CAR can further comprise at least one co-stimulatory signaling domain. In some embodiments, an intracellular domain of a CAR can comprise two co-stimulatory signaling domains. Such a co-stimulatory signaling domain can provide increased activation of an immune effector cell. A co-stimulatory signaling domain can be derived from a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP10 polypeptide, a 2B4 polypeptide, a CD27 polypeptide, a CD30 polypeptide, a CD40 polypeptide and the like. CARs comprising an intracellular domain that comprises a co-stimulatory signaling region comprising 4-1BB, ICOS or DAP-10 have been described previously (see U.S. 7,446,190, which is incorporated herein by reference, which also describes representative sequences for 4-1BB, ICOS and DAP-10) . In some embodiments, the intracellular domain of a CAR can comprise a co-stimulatory signaling region that comprises two co-stimulatory receptors, such as CD28 and 4-1BB (see Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ) , or CD28 and OX40, or other combinations of co-stimulatory ligands, as disclosed herein.

The extracellular domain of a CAR can be fused to a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the cell surface. It is understood that, once a polypeptide containing a signal peptide is expressed at the cell surface, the signal peptide has generally been proteolytically removed during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, a polypeptide such as a CAR is generally expressed at the cell surface as a mature protein lacking the signal peptide, whereas the precursor form of the polypeptide includes the signal peptide. A signal peptide or leader can be essential if a CAR is to be glycosylated and/or anchored in the cell membrane. The signal sequence or leader is a peptide sequence generally present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway. The signal peptide is covalently joined to the N-terminus of the extracellular antigen-binding domain of a CAR as a fusion protein. Any suitable signal peptide, as are well known in the art, can be applied to a CAR to provide cell surface expression in an immune cell (see Gierasch Biochem. 28: 923-930 (1989) ; von Heijne, J. Mol. Biol. 184 (1) : 99–105 (1985) ) . Particularly useful signal peptides can be derived from cell surface proteins naturally expressed in the immune cell provided herein, including any of the signal peptides of the polypeptides disclosed herein. Thus, any suitable signal peptide can be utilized to direct a CAR to be expressed at the cell surface of an immune effector cell provided herein.

In certain non-limiting embodiments, a CAR can also comprise a spacer region or sequence that links the domains of the CAR to each other. For example, a spacer can be included between a signal peptide and an antigen binding domain, between the antigen binding domain and the transmembrane domain, between the transmembrane domain and the intracellular domain, and/or between domains within the intracellular domain, for example, between a stimulatory domain and a co-stimulatory domain. The spacer region can be flexible enough to allow interactions of various domains with other polypeptides, for example, to allow the antigen binding domain to have flexibility in orientation in order to facilitate antigen recognition. The spacer region can be, for example, the hinge region from an IgG, the CH ₂CH ₃ (constant) region of an immunoglobulin, and/or portions of CD3 (cluster of differentiation 3) or some other sequence suitable as a spacer.

The transmembrane domain of a CAR generally comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In an embodiment, the transmembrane domain of a CAR can be derived from another polypeptide that is naturally expressed in the immune effector cell. In one embodiment, a CAR can have a transmembrane domain derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζ chain, CD28, CD3 ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or other polypeptides expressed in the immune effector cell. Alternatively, the transmembrane domain can be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. Optionally, the transmembrane domain can be derived from a polypeptide that is not naturally expressed in the immune effector cell, so long as the transmembrane domain can function in transducing signal from antigen bound to the CAR to the intracellular signaling and/or co-stimulatory domains. In some embodiments, the transmembrane domain can comprise a triplet of phenylalanine, tryptophan and valine at each end. Optionally, a short oligo-or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

CD3ζ. In a non-limiting embodiment, a CAR can comprise a signaling domain derived from a CD3ζ polypeptide, for example, a signaling domain derived from the intracellular domain of CD3ζ, which can activate or stimulate an immune effector cell, for example, a T cell. A CD3ζ polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_932170 (NP_932170.1, GI: 37595565; see below) , or fragments thereof. In one embodiment, the CD3ζpolypeptide has an amino acid sequence of amino acids 52 to 164 of the CD3ζ polypeptide sequence provided below, or a fragment thereof that is sufficient for signaling activity. An exemplary CAR has an intracellular domain comprising a CD3ζ polypeptide comprising amino acids 52 to 164 of the CD3ζ polypeptide sequence provided below. Another exemplary CAR has an intracellular domain comprising a CD3ζ polypeptide comprising amino acids 52 to 164 of the CD3ζ polypeptide provided below. Still another exemplary CAR has an intracellular domain comprising a CD3ζ polypeptide comprising amino acids 52 to 164 of the CD3ζ polypeptide provided below. See GenBank NP_932170 for reference to domains within CD3ζ, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 30; transmembrane domain, amino acids 31 to 51; intracellular domain, amino acids 52 to 164.

CD28. Cluster of Differentiation 28 (CD28) is a protein expressed on T cells that provides co-stimulatory signals for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. In one embodiment, a CAR can comprise a co-stimulatory signaling domain derived from CD28. For example, as disclosed herein, a CAR can include at least a portion of an intracellular/cytoplasmic domain of CD28, for example an intracellular/cytoplasmic domain that can function as a co-stimulatory signaling domain. A CD28 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P10747 (P10747.1, GI: 115973) or NP_006130 (NP_006130.1, GI: 5453611) , as provided below, or fragments thereof. If desired, CD28 sequences additional to the intracellular domain can be included in a CAR of the invention. For example, a CAR can comprise the transmembrane of a CD28 polypeptide. In one embodiment, a CAR can have an amino acid sequence comprising the intracellular domain of CD28 corresponding to amino acids 180 to 220 of CD28, or a fragment thereof. In another embodiment, a CAR can have an amino acid sequence comprising the transmembrane domain of CD28 corresponding to amino acids 153 to 179, or a fragment thereof. An exemplary CAR can comprise a co-stimulatory signaling domain corresponding to an intracellular domain of CD28. An exemplary CAR can also comprise a transmembrane domain derived from CD28. Thus, an exemplary CAR can comprise two domains from CD28, a co-stimulatory signaling domain and a transmembrane domain. In one embodiment, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of CD28 and comprises amino acids 153 to 220 of CD28. In another embodiment, a CAR comprises amino acids 117 to 220 of CD28. In one embodiment, a CAR can comprise a transmembrane domain derived from a CD28 polypeptide comprising amino acids 153 to 179 of the CD28 polypeptide provided below. See GenBank NP_006130 for reference to domains within CD28, for example, signal peptide, amino acids 1 to 18; extracellular domain, amino acids 19 to 152; transmembrane domain, amino acids 153 to 179; intracellular domain, amino acids 180 to 220. It is understood that sequences of CD28 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

4-1BB. 4-1BB, also referred to as tumor necrosis factor receptor superfamily member 9, can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. In one embodiment, a CAR can comprise a co-stimulatory signaling domain derived from 4-1BB. A 4-1BB polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P41273 (P41273.1, GI: 728739) or NP_001552 (NP_001552.2, GI: 5730095) or fragments thereof. In one embodiment, a CAR can have a co-stimulatory domain comprising the intracellular domain of 4-1BB corresponding to amino acids 214 to 255, or a fragment thereof. In another embodiment, a CAR can have a transmembrane domain of 4-1BB corresponding to amino acids 187 to 213, or a fragment thereof. An exemplary CAR is MBBz, which has an intracellular domain comprising a 4-1BB polypeptide (for example, amino acids 214 to 255 of NP_001552) . See GenBank NP_001552 for reference to domains within 4-1BB, for example, signal peptide, amino acids 1 to 17; extracellular domain, amino acids 18 to 186; transmembrane domain, amino acids 187 to 213; intracellular domain, amino acids 214 to 255. It is understood that sequences of 4-1BB that are shorter or longer than a specific delineated domain can be included in a CAR, if desired. It is also understood that a “4-1BB polynucleotide” refers to a polynucleotide encoding a 4-1BB polypeptide.

OX40. OX40, also referred to as tumor necrosis factor receptor superfamily member 4 precursor or CD134, is a member of the TNFR-superfamily of receptors. In one embodiment, a CAR can comprise a co-stimulatory signaling domain derived from OX40. An OX40 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P43489 (P43489.1, GI: 1171933) or NP_003318 (NP_003318.1, GI: 4507579) , provided below, or fragments thereof. In one embodiment, a CAR can have a co-stimulatory domain comprising the intracellular domain of OX40 corresponding to amino acids 236 to 277, or a fragment thereof. In another embodiment, a CAR can have an amino acid sequence comprising the transmembrane domain of OX40 corresponding to amino acids 215 to 235 of OX40, or a fragment thereof. See GenBank NP_003318 for reference to domains within OX40, for example, signal peptide, amino acids 1 to 28; extracellular domain, amino acids 29 to 214; transmembrane domain, amino acids 215 to 235; intracellular domain, amino acids 236 to 277. It is understood that sequences of OX40 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired. It is also understood that an “OX40 polynucleotide” refers to a polynucleotide encoding an OX40 polypeptide.

ICOS. Inducible T-cell co-stimulator precursor (ICOS) , also referred to as CD278, is a CD28-superfamily co-stimulatory receptor that is expressed on activated T cells. In one embodiment, a CAR can comprise a co-stimulatory signaling domain derived from ICOS. An ICOS polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_036224 (NP_036224.1, GI: 15029518) , provided below, or fragments thereof. In one embodiment, a CAR can have a co-stimulatory domain comprising the intracellular domain of ICOS corresponding to amino acids 162 to 199 of ICOS. In another embodiment, a CAR can have an amino acid sequence comprising the transmembrane domain of ICOS corresponding to amino acids 141 to 161 of ICOS, or a fragment thereof. See GenBank NP_036224 for reference to domains within ICOS, for example, signal peptide, amino acids 1 to 20; extracellular domain, amino acids 21 to 140; transmembrane domain, amino acids 141 to 161; intracellular domain, amino acids 162 to 199. It is understood that sequences of ICOS that are shorter or longer than a specific delineated domain can be included in a CAR, if desired. It is also understood that an “ICOS polynucleotide” refers to a polynucleotide encoding an ICOS polypeptide.

CD8. Cluster of differentiation 8 (CD8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR) . CD8 binds to a major histocompatibility complex (MHC) molecule and is specific for the class I MHC protein. In one embodiment, a CAR can comprise a transmembrane domain derived from CD8. A CD8 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_001139345.1 (GI: 225007536) , as provided below, or fragments thereof. In one embodiment, a CAR can have an amino acid sequence comprising the transmembrane domain of CD8 corresponding to amino acids 183 to 203, or fragments thereof. In one embodiment, an exemplary CAR has a transmembrane domain derived from a CD8 polypeptide. In one non-limiting embodiment, a CAR can comprise a transmembrane domain derived from a CD8 polypeptide comprising amino acids 183 to 203. In addition, a CAR can comprise a hinge domain comprising amino acids 137-182 of the CD8 polypeptide provided below. In another embodiment, a CAR can comprise amino acids 137-203 of the CD8 polypeptide provided below. In yet another embodiment, a CAR can comprise amino acids 137 to 209 of the CD8 polypeptide provided below. See GenBank NP_001139345.1 for reference to domains within CD8, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 182; transmembrane domain amino acids, 183 to 203; intracellular domain, amino acids 204 to 235. It is understood that additional sequence of CD8 beyond the transmembrane domain of amino acids 183 to 203 can be included in a CAR, if desired. It is further understood that sequences of CD8 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired. It also is understood that a “CD8 polynucleotide” refers to a polynucleotide encoding a CD8 polypeptide.

CD4. Cluster of differentiation 4 (CD4) , also referred to as T-cell surface glycoprotein CD4, is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. In one embodiment, a CAR can comprise a transmembrane domain derived from CD4. CD4 exists in various isoforms. It is understood that any isoform can be selected to achieve a desired function. Exemplary isoforms include isoform 1 (NP_000607.1, GI: 10835167) , isoform 2 (NP_001181943.1, GI: 303522479) , isoform 3 (NP_001181944.1, GI: 303522485; or NP_001181945.1, GI: 303522491; or NP_001181946.1, GI: 303522569) , and the like. One exemplary isoform sequence, isoform 1, is provided below. In one embodiment, a CAR can have an amino acid sequence comprising the transmembrane domain of CD4 corresponding to amino acids 397 to 418, or fragments thereof. See GenBank NP_000607.1 for reference to domains within CD4, for example, signal peptide, amino acids 1 to 25; extracellular domain, amino acids 26 to 396; transmembrane domain amino acids, 397 to 418; intracellular domain, amino acids 419 to 458. It is understood that additional sequence of CD4 beyond the transmembrane domain of amino acids 397 to 418 can be included in a CAR, if desired. It is further understood that sequences of CD4 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired. It also is understood that a “CD4 polynucleotide” refers to a polynucleotide encoding a CD4 polypeptide.

In addition to T cells, CAR can be engineered into other types of immune effector cells, such as NK cells, NKT cells, macrophages, or granulocytes. In some embodiments, the engineered cell is a NK cell. CARs provided herein can retarget NK cells to tumor surface antigens (see e.g., Hu et al. Acta Pharmacol Sin 39, 167–176 (2018) ) . CAR-NK cells can use the first generation of CAR constructs that contain CD3ζ as an intracellular signaling domain or the second generation of CAR constructs that express a second signaling domain (e.g., CD28, 4-1BB) in conjunction with CD3ζ. In general, the second generation of CARs in NK cells is more active than first-generation CARs. In some embodiments, CAR constructs are based on the activating features of NK cells. For example, DNAX-activation protein 12 (DAP12) is known to activate signaling for NK cells.

CARs provided herein can include a target-binding domain as disclosed above. In some embodiments, fusion proteins disclosed herein can be co-expressed with a CAR targeting a tumor antigen selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin in a cell. In some embodiments, fusion proteins disclosed herein is conjugated to a CAR targeting a tumor antigen selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin. In some embodiments, genetically engineered immune effector cells provided herein further comprise a polynucleotide encoding a CAR targeting a tumor antigen selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin. In some embodiments, genetically engineered immune effector cells provided herein further recombinantly express a CAR targeting a tumor antigen selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin.

In some embodiments, the CAR targets Her2. In some embodiments, the CAR targeting Her2 has the amino acid sequence of SEQ ID NO: 79. In some embodiments, the CAR targets CD19. In some embodiments, the CAR targeting CD19 has the amino acid sequence of SEQ ID NO: 80. In some embodiments, the CAR targets mesothelin. In some embodiments, the CAR targeting Mesothelin has the amino acid sequence of SEQ ID NO: 81. In some embodiments, the CAR targets PSMA. In some embodiments, the CAR targeting PSMA has the amino acid sequence of SEQ ID NO: 82. In some embodiments, the CAR targets c-Met. In some embodiments, the CAR targeting c-Met has the amino acid sequence of SEQ ID NO: 83. In some embodiments, the CAR targets BCMA. In some embodiments, the CAR targeting BCMA has the amino acid sequence of SEQ ID NO: 84. In some embodiments, the CAR targeting BCMA has the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CAR targeting BCMA has the amino acid sequence of SEQ ID NO: 86. In some embodiments, the CAR targets GPC3. In some embodiments, the CAR targeting GPC3 has the amino acid sequence of SEQ ID NO: 87. In some embodiments, the CAR targets IL13ra2. In some embodiments, the CAR targeting IL13ra2 has the amino acid sequence of SEQ ID NO: 88. In some embodiments, the CAR targets EGFR. In some embodiments, the CAR targeting EGFR has the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CAR targets CD123. In some embodiments, the CAR targeting CD123 has the amino acid sequence of SEQ ID NO: 90. In some embodiments, the CAR targets CD7. In some embodiments, the CAR targeting CD7 has the amino acid sequence of SEQ ID NO: 91. In some embodiments, the CAR targets GD2. In some embodiments, the CAR targeting GD2 has the amino acid sequence of SEQ ID NO: 92. In some embodiments, the CAR targets PSCA. In some embodiments, the CAR targeting PSCA has the amino acid sequence of SEQ ID NO: 93. In some embodiments, the CAR targets CD70. In some embodiments, the CAR targeting CD70 has the amino acid sequence of SEQ ID NO: 169.

In some embodiments, the CAR provided herein include a target-binding domain that binds a viral antigen. In some embodiments, the viral antigen is EBV. In some embodiments, the viral antigen is HPV. In some embodiments, the viral antigen is HIV.

5.5.2 TCRs

Fusion proteins provided herein can be co-expressed with a TCR in a genetically engineered cells provided herein or conjugated to a TCR. In some embodiments, provided herein are genetically engineered immune effector cells recombinantly expressing a fusion protein disclosed herein, further recombinantly expressing a TCR. In some embodiments, provided herein are genetically engineered immune effector cells comprising a polynucleotide encoding a fusion protein disclosed herein, further comprising a polynucleotide encoding a TCR.

T cell receptors (TCRs) are antigen-specific molecules that are responsible for recognizing antigenic peptides presented in the context of a product of the MHC on the surface of APCs or any nucleated cells. This system endows T cells, via their TCRs, with the potential ability to recognize the entire array of intracellular antigens expressed by a cell (including virus proteins) that are processed into short peptides, bound to an intracellular MHC molecule, and delivered to the surface as a peptide-MHC complex. This system allows foreign protein (e.g., mutated cancer antigen or virus protein) or aberrantly expressed protein to serve a target for T cells (e.g., Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544) .

The interaction of a TCR and a peptide-MHC complex can drive the T cell into various states of activation, depending on the affinity (or dissociation rate) of binding. The TCR recognition process allows a T cell to discriminate between a normal, healthy cell and, for example, one that has become transformed via a virus or malignancy, by providing a diverse repertoire of TCRs, wherein there is a high probability that one or more TCRs will be present with a binding affinity for the foreign peptide bound to an MHC molecule that is above the threshold for stimulating T cell activity (Manning and Kranz (1999) Immunology Today, 20, 417-422) .

Wild type TCRs isolated from either human or mouse T cell clones that were identified by in vitro culturing have been shown to have relatively low binding affinities (K _D = 1 -300 μΜ) (Davis et al. (1998) Annu Rev Immunol, 16, 523-544) . This is partly because that T cells that develop in the thymus are negatively selected (tolerance induction) on self-peptide-MHC ligands, such that T cells with too high of an affinity are deleted (Starr et al. (2003) Annu Rev Immunol, 21, 139-76) . To compensate for these relatively low affinities, T cells have evolved a co-receptor system in which the cell surface molecules CD4 and CD8 bind to the MHC molecules (class II and class I, respectively) and synergize with the TCR in mediating signaling activity. CD8 is particularly effective in this process, allowing TCRs with very low affinity (e.g., K _D =300 μΜ) to mediate potent antigen-specific activity.

Directed evolution can be used to generate TCRs with higher affinity for a specific peptide-MHC complex. Methods that can be used include yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92) , phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54) , and T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84) . All three approaches involve engineering, or modifying, a TCR that exhibits the normal, low affinity of the wild-type TCR, to increase the affinity for the cognate peptide-MHC complex (the original antigen that the T cells were specific for) .

As such, in some embodiments, the fusion proteins provided herein can be co-expressed with a TCR in a cell. In some embodiments, a fusion protein provided herein can be conjugated to a TCR. In some embodiments, the TCR comprises an alpha (α) chain and a beta (β) chain. In some embodiments, the TCR comprises a gamma chain (γ) and a delta (δ) chain. The extracellular regions of the αβ chains (or the γδ chains) are responsible for antigen recognition and engagement. Antigen binding stimulates downstream signaling through the multimeric CD3 complex that associates with the intracellular domains of the αβ (or γδ) chains as three dimers (εγ, εδ, ζζ) .

TCRs provided herein can be genetically engineered to bind specific antigens. In some embodiments, fusion protein disclosed herein can be co-expressed with a TCR targeting a tumor antigen in a cell. In some embodiments, fusion protein disclosed herein can be conjugated with a TCR targeting a tumor antigen. In some embodiments, provided herein are genetically engineered cells recombinantly expressing a fusion protein and a TCR targeting a tumor antigen. In some embodiments, provided herein are genetically engineered cells comprising a polynucleotide encoding a fusion protein and a polynucleotide encoding a TCR targeting a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin.

In some embodiments, the TCR comprises a TCR α chain targeting NY-ESO-1. The TCR αchain targeting NY-ESO-1 can have the amino acid sequence of SEQ ID NO: 94. In some embodiments, the TCR comprises a TCR β chain targeting NY-ESO-1. The TCR β chain targeting NY-ESO-1 can have the amino acid sequence of SEQ ID NO: 95. In some embodiments, the TCR targeting NY-ESO-1 comprises a TCR α chain and a TCR β chain.

In some embodiments, the TCR comprises a TCR α chain targeting EBV16-E7. The TCR αchain targeting EBV16-E7 can have the amino acid sequence of SEQ ID NO: 97. In some embodiments, the TCR comprises a TCR β chain targeting EBV16-E7. The TCR β chain targeting EBV16-E7 can have the amino acid sequence of SEQ ID NO: 98. In some embodiments, the TCR targeting EBV16-E7 comprises a TCR α chain and a TCR β chain. The TCR targeting EBV16-E7 can have the amino acid sequence of SEQ ID NO: 96, which can be encoded by, for example, the nucleotide sequence of SEQ ID NO: 162.

In some embodiments, the TCR comprises a TCR α chain targeting H3.3. The TCR α chain targeting H3.3 can have the amino acid sequence of SEQ ID NO: 100. In some embodiments, the TCR comprises a TCR β chain targeting H3.3. The TCR β chain targeting H3.3 can have the amino acid sequence of SEQ ID NO: 101. In some embodiments, the TCR targeting H3.3 comprises a TCR α chain and a TCR β chain. The TCR targeting H3.3 can have the amino acid sequence of SEQ ID NO: 99.

In some embodiments, the TCR provided herein include a target-binding domain that binds a viral antigen. In some embodiments, the viral antigen is EBV. In some embodiments, the viral antigen is HPV. In some embodiments, the viral antigen is HIV.

5.5.3 BiTEs

Bispecific T-cell engagers (BiTEs) are bispecific antibodies that bind to a T cell antigen (e.g., CD3) and a tumor antigen. BiTEs have been shown to induce directed lysis of target tumor cells and thus provide great potential therapies for cancers and other disorders. Fusion proteins provided herein can be co-expressed with a BiTE in a genetically engineered cells provided herein or conjugated to a BiTE. In some embodiments, provided herein are genetically engineered immune effector cells recombinantly expressing a fusion protein disclosed herein, further recombinantly expressing a BiTE. In some embodiments, provided herein are genetically engineered immune effector cells comprising a polynucleotide encoding a fusion protein disclosed herein, further comprising a polynucleotide encoding a BiTE.

BiTEs are bispecific antibodies that bind to a T cell antigen (e.g., CD3) and a tumor antigen. In some embodiments, the BiTEs bind CD3. In some embodiments, the tumor antigen is selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin.

In some embodiments, the BiTEs comprise a bispecific antibody that binds CD3 and CD19. The BiTEs that bind CD3 and CD19 can have the amino acid sequence of SEQ ID NO: 102. In some embodiments, the BiTEs comprise a bispecific antibody that binds CD3 and Her2. The BiTEs that bind CD3 and Her2 can have the amino acid sequence of SEQ ID NO: 167. In some embodiments, the BiTEs comprise a bispecific antibody that binds CD3 and EGFRvIII. The BiTEs that bind CD3 and EGFRvIII can have the amino acid sequence of SEQ ID NO: 103. In some embodiments, the BiTEs comprise a bispecific antibody that binds CD3 and Mesothelin. In some embodiments, the BiTEs comprise a bispecific antibody that binds CD3 and BCMA.

In some embodiments, the BiTE provided herein include a target-binding domain that binds a viral antigen. In some embodiments, the viral antigen is EBV. In some embodiments, the viral antigen is HPV. In some embodiments, the viral antigen is HIV.

5.6 Pharmaceutical compositions

Provided herein are also pharmaceutical compositions comprising anti-CD40 antibodies or antigen-binding fragments disclosed herein. Provided herein are also pharmaceutical compositions comprising soluble fusion proteins disclosed herein. Provided herein are also pharmaceutical compositions comprising the genetically engineered immune effector cells disclosed herein. In some embodiments, the pharmaceutical composition comprises an effective amount of the fusion proteins disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an effective amount of genetically engineered cells disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are useful in immunotherapy. In some embodiments, the pharmaceutical compositions are useful in immuno-oncology. In some embodiments, the pharmaceutical compositions are useful in inhibiting tumor growth in a subject (e.g., a human patient) . In some embodiments, the pharmaceutical compositions are useful in treating cancer in a subject (e.g., a human patient) . In some embodiments, the pharmaceutical compositions are useful in treating viral infection.

In some embodiments, the pharmaceutical compositions provided herein comprise anti-CD40 antibodies or antigen-binding fragments provided herein. The anti-CD40 antibodies or antigen-binding fragments can be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise soluble anti-CD40 antibodies or antigen-binding fragments provided herein at 1-1000 mg/ml. In some embodiments, the pharmaceutical compositions comprise soluble anti-CD40 antibodies or antigen-binding fragments provided herein at 10-500 mg/ml, 10-400 mg/ml, 10-300 mg/ml, 10-200 mg/ml, 10-100 mg/ml, 20-100 mg/ml, or 50-100 mg/ml. In some embodiments, the pharmaceutical compositions provided herein comprise anti- CD40 antibodies or antigen-binding fragments provided herein at about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 150 mg/ml, about 180 mg/ml, about 200 mg/ml, about 300 mg/ml, about 500 mg/ml, about 800 mg/ml, or about 1000 mg/ml.

In some embodiments, the pharmaceutical compositions provided herein comprise soluble fusion proteins provided herein. The fusion protein can be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise soluble fusion proteins provided herein at 1-1000 mg/ml. In some embodiments, the pharmaceutical compositions comprise soluble fusion proteins provided herein at 10-500 mg/ml, 10-400 mg/ml, 10-300 mg/ml, 10-200 mg/ml, 10-100 mg/ml, 20-100 mg/ml, or 50-100 mg/ml. In some embodiments, the pharmaceutical compositions comprise soluble fusion proteins provided herein compositions provided herein comprise soluble fusion proteins provided herein at 1-1000 mg/ml. In some embodiments, the pharmaceutical compositions provided herein comprise soluble fusion proteins provided herein at about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 150 mg/ml, about 180 mg/ml, about 200 mg/ml, about 300 mg/ml, about 500 mg/ml, about 800 mg/ml, or about 1000 mg/ml.

The pharmaceutical compositions comprising genetically engineered cells disclosed herein can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of cells in a cell population using various well-known methods, as described herein. The ranges of purity in cell populations comprising genetically modified cells provided herein can be from about 20%to about 25%, from about 25%to about 30%, from about 30%to about 35%, from about 35%to about 40%, from about 40%to about 45%, from about 45%to about 50%, from about 55%to about 60%, from about 65%to about 70%, from about 70%to about 75%, from about 75%to about 80%, from about 80%to about 85%; from about 85%to about 90%, from about 90%to about 95%, or from about 95 to about 100%. In some embodiments, the ranges of purity in cell populations comprising genetically modified cells provided herein can be from about 20%to about 30%, from about 20%to about 50%, from about 20%to about 80%, from about 20%to about 100%, from about 50%to about 80%, or from about 50%to about 100%. Dosages can be readily adjusted by those skilled in the art; for example, a decrease in purity may require an increase in dosage.

Provided herein are also kits for preparation of pharmaceutical compositions having an anti-CD40 antibody or antigen-binding fragment disclosed herein. In some embodiments, the kit further comprises a pharmaceutically acceptable excipient in one or more containers. In another embodiment, the kits can comprise an anti-CD40 antibody or antigen-binding fragment disclosed herein for administration to a subject. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of an anti-CD40 antibody or antigen-binding fragment.

Provided herein are also kits for preparation of pharmaceutical compositions having the fusion protein disclosed herein. In some embodiments, the kit further comprises a pharmaceutically acceptable excipient in one or more containers. In another embodiment, the kits can comprise fusion proteins disclosed herein for administration to a subject. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the fusion protein.

Provided herein are also kits for preparation of cells disclosed herein. In one embodiment, the kit comprises one or more vectors for generating a genetically engineered cell, such as a T cell, that expresses a fusion protein disclosed herein. The kits can be used to generate genetically engineered cells from autologous or non-autologous cells to be administered to a compatible subject. In another embodiment, the kits can comprise cells disclosed herein for administration to a subject. In specific embodiments, the kits comprise the cells disclosed herein in one or more containers. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the genetically engineered cells.

In some embodiments, provided herein is a pharmaceutical composition comprising antibodies, fusion proteins or cells provided herein wherein the composition is suitable for local administration. In some embodiments, local administration comprises intratumoral injection, peritumoral injection, juxtatumoral injection, intralesional injection and/or injection into a tumor draining lymph node, or essentially any tumor-targeted injection where the antitumor agent is expected to leak into primary lymph nodes adjacent to targeted solid tumor.

Pharmaceutically acceptable carriers that can be used in compositions or formulations provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) . Depending on the route of administration, the active ingredient, i.e., the antibodies, fusion proteins or cells, can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

Provided herein are also pharmaceutical compositions or formulations that improve the stability of the antibodies, fusion proteins or cells to allows for their long-term storage. In some embodiments, the pharmaceutical composition or formulation disclosed herein comprises: (a) the antibodies, fusion proteins or cells disclosed herein; (b) a buffering agent; (c) a stabilizing agent; (d) a salt; (e) a bulking agent; and/or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years or more. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4℃, 25℃, or 40℃.

Buffering agents useful in the pharmaceutical compositions or formulations disclosed herein can be a weak acid or base used to maintain the acidity (pH) of a solution near a chosen value after the addition of another acid or base. Suitable buffering agents can maximize the stability of the pharmaceutical formulations by maintaining pH control of the formulation. Suitable buffering agents can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties can also dependent on the pH of the formulation. Common buffering agents include, but are not limited to, histidine, citrate, succinate, acetate and phosphate. In some embodiments, a buffering agent comprises histidine (e.g., L-histidine) with isotonicity agents and potentially pH adjustment with an acid or a base known in the art. In certain embodiments, the buffering agent is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

Stabilizing agents are added to a pharmaceutical product in order to stabilize that product. Such agents can stabilize proteins in a number of different ways. Common stabilizing agents include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine, carbohydrates such as glucose, sucrose, trehalose, rafftnose, or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrins or destrans of any kind and molecular weight, or PEG. In one aspect of the invention, the stabilizing agent is chosen in order to maximize the stability of FIX polypeptide in lyophilized preparations. In certain embodiments, the stabilizing agent is sucrose and/or arginine.

Bulking agents can be added to a pharmaceutical composition or formulation in order to add volume and mass to the product, thereby facilitating precise metering and handling thereof. Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

Surfactants are amphipathic substances with lyophilic and lyophobic groups. A surfactant can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

The pharmaceutical compositions or formulations disclosed herein can further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions or formulations is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19 ^th edition, 1995.

In some embodiments, the pharmaceutical composition or formulation is an aqueous formulation. Such a formulation is typically a solution or a suspension, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term “aqueous formulation” is defined as a formulation comprising at least 50%w/w water. Likewise, the term “aqueous solution” is defined as a solution comprising at least 50 %w/w water, and the term “aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.

In some embodiments, the pharmaceutical composition or formulation disclosed herein is freeze-dried, to which the physician or the patient adds solvents and/or diluents prior to use.

Pharmaceutical compositions or formulations disclosed herein can also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA) , butylated hydroxytoluene (BHT) , lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA) , sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) , and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions described herein is contemplated. A pharmaceutical composition or formulation can comprise a preservative or can be devoid of a preservative. Supplementary active compounds can be incorporated into the compositions.

Pharmaceutical compositions or formulations typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, the compositions can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of active ingredient which can be combined with a carrier material is the amount that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutical composition or formulation disclosed herein can be prepared with carriers that protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poly lactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See. e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In some embodiments, the antibodies, fusion proteins or cells described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the activate ingredient described herein cross the BBB (if desired, e.g., for brain cancers) , they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes can comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685) . Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al) mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153: 1038) ; antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180) ; surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134) ; pl20 (Schreier et al. (1994) J. Biol. Chem. 269: 9090) ; see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4: 273.

5.7 Methods and Uses

Provided herein are also uses of the antibodies or antigen-binding fragments, the fusion proteins, and the immune effector cells disclosed herein.

5.7.1 Uses of the anti-CD40 antibodies and antigen-binding fragments

The anti-CD40 antibodies or antigen-binding fragments and pharmaceutical compositions described herein have numerous in vitro and in vivo utilities involving, for example, enhancement of immune response. For example, anti-CD40 antibodies or fusion proteins disclosed herein can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of diseases.

Accordingly, provided herein are methods of modifying an immune response in a subject comprising administering to the subject the antibody or antigen-binding fragment, fusion protein, or cells described herein such that the immune response in the subject is enhanced, stimulated or up-regulated.

Preferred subjects include human patients in whom enhancement of an immune response would be desirable. The methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., the T-cell mediated immune response) .

Also encompassed are methods for detecting the presence of CD40 (e.g., human CD40) in a sample, or measuring the amount of CD40, comprising contacting the sample, and a control sample, with an anti-CD40 antibody or antigen-binding fragment described herein, under conditions that allow for formation of a complex between the antibody or fragment thereof and CD40. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative of the presence of the CD40 molecules in the sample. Moreover, the anti-CD40 antibodies and antigen-binding fragments described herein can be used to purify CD40 via immunoaffinity purification.

Given the ability of anti-CD40 antibodies or antigen-binding fragments described herein to enhance co-stimulation of T cell responses, e.g., antigen-specific T cell responses, provided herein are in vitro and in vivo methods of using the antibodies described herein to stimulate, enhance or upregulate antigen-specific T cell responses, e.g., anti-tumor T cell responses. CD4 ⁺ and CD8 ⁺ T cell responses can be enhanced using anti-CD40 antibodies or antigen-binding fragments. The T cells can be CD4+ T cells, CD8+ T cells, T helper (T _h) cells and T cytotoxic (T _c) cells.

Further encompassed are methods of enhancing an immune response (e.g., an antigen-specific T cell response) in a subject comprising administering an anti-CD40 antibody or antigen- binding fragment described herein to the subject such that an immune response (e.g., an antigen-specific T cell response) in the subject is enhanced. In a preferred embodiment, the subject is a tumor-bearing subject and an immune response against the tumor is enhanced. A tumor can be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In some embodiments, a tumor is an immunogenic tumor. In some embodiments, a tumor is non-immunogenic. In some embodiments, a tumor is PD-L1 positive. In some embodiments a tumor is PD-L1 negative. A subject can also be a virus-bearing subject and an immune response against the virus is enhanced.

Further provided are methods for inhibiting growth of tumor cells in a subject comprising administering to the subject an anti-CD40 antibody or antigen-binding fragment described herein such that growth of the tumor is inhibited in the subject. Also provided are methods of treating chronic viral infection in a subject comprising administering to the subject an anti-CD40 antibody or antigen-binding fragment described herein such that the chronic viral infection is treated in the subject.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment is given to a subject as an adjunctive therapy. Treatments of subjects having cancer with an anti-CD40 antibody or antigen-binding fragment can lead to a long-term durable response relative to the current standard of care; long term survival of at least 1, 2, 3, 4, 5, 10 or more years, recurrence free survival of at least 1, 2, 3, 4, 5, or 10 or more years. In some embodiments, treatment of a subject having cancer with an anti-CD40 antibody or antigen-binding fragment described herein prevents recurrence of cancer or delays recurrence of cancer by, e.g., 1, 2, 3, 4, 5, or 10 or more years. An anti-CD40 treatment can be used as a primary or secondary line of treatment.

Provided herein are methods for treating a subject having cancer, comprising administering to the subject an anti-CD40 antibody or antigen-binding fragment described herein, such that the subject is treated, e.g., such that growth of cancerous tumors is inhibited or reduced and/or that the tumors regress. An anti-CD40 antibody or antigen-binding fragment can be used alone to inhibit the growth of cancerous tumors. Alternatively, an anti-CD40 antibody or antigen-binding fragment can be used in conjunction with another agent, e.g., other immunogenic agents, standard cancer treatments, or other antibodies, as described below. Combination with an inhibitor of PD-1, such as an anti-PD-l or anti-PD-Ll antibody, is also provided. See, e.g., Ellmark et al. (2015) Oncolmmunology 4: 7 elOH484.

Accordingly, provided herein are methods of treating cancer, e.g., by inhibiting growth of tumor cells, in a subject, comprising administering to the subject a therapeutically effective amount of an anti-CD40 antibody or antigen-binding fragment described herein, e.g., 40-18, 40-37, 40-38, 40-45, 40-47, or 40-52.

Cancers whose growth can be inhibited using the antibodies disclosed herein include cancers typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC) , non NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma) , ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC) ) , prostate cancer (e.g., hormone refractory prostate adenocarcinoma) , thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme) , cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma) , gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma) , bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally-induced cancers including those induced by asbestos, virus-related cancers (e.g., human papilloma virus (HPV) -related tumor) , and hematologic malignancies derived from either of the two major blood cell lineages, i.e., the myeloid cell line (which produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cell line (which produces B, T, NK and plasma cells) , such as all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL) , acute myelogenous leukemia (AML) , chronic lymphocytic leukemia (CLL) , and chronic myelogenous leukemia (CML) , undifferentiated AML (MO) , myeloblastic leukemia (Ml) , myeloblastic leukemia (M2; with cell maturation) , promyelocytic leukemia (M3 or M3 variant [M3 V] ) , myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E] ) , monocytic leukemia (M5) , erythroleukemia (M6) , megakaryoblastic leukemia (M7) , isolated granulocytic sarcoma, and chloroma; lymphomas, such as Hodgkin's lymphoma (HL) , non-Hodgkin's lymphoma (NHL) , B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL) , peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplantation lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL) , hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitf s lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL) , immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome) , and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma) , solitary plasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL) , hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL) , including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes) ; angiocentric (nasal) T-cell lymphoma; cancer of the head or neck, renal cancer, rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma, as well as any combinations of said cancers. The methods described herein may also be used for treatment of metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., with a blocking CTLA-4 or PD-l antibody) , and recurrent cancers.

Anti-CD40 antibodies or antigen-binding fragments disclosed herein can also be combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy) . Anti-CD40 antibodies or antigen-binding fragments disclosed herein can be effectively combined with chemotherapeutic regimes. In these instances, it can reduce the dose of chemotherapeutic reagent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304) . An example of such a combination is an anti-CD40 antibody or antigen-binding fragment in combination with decarbazine for the treatment of melanoma. Another example of such a combination is an anti-CD40 antibody or antigen-binding fragment in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale behind the combined use of CD40 agonists and chemotherapy is that cell death, which is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that can result in synergy with CD40 agonism through cell death are radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigen in the host.

Angiogenesis inhibitors can also be combined with CD40 agonists. Inhibition of angiogenesis leads to tumor cell death which feed tumor antigen into host antigen presentation pathways. Activating antibodies to T cell costimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097) , OX-40 (Weinberg et al. (2000) Immunol. 164: 2160-2169) , CD137/4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997) , and ICOS (Hutloff et al. (1999) Nature 397: 262-266) can also provide for increased levels of T cell activation. Inhibitors of PD1 or PD-L1 may also be used in conjunction with anti-huCD40 antibodies.

In some embodiments, anti-CD40 antibodies or antigen-binding fragments disclosed herein can be used to treat an infectious disease in a subject in need thereof. Examples of pathogens for which this therapeutic approach can be particularly useful, include, but are not limited to COVID-19, HIV, Hepatitis (A, B, &C) , Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. CD40 agonism is particularly useful against established infections by agents such as HIV that present altered antigens over the course of the infections. These novel epitopes are recognized as foreign at the time of anti-human CD40 antibody administration, thus provoking a strong T cell response.

Some examples of pathogenic viruses causing infections treatable by methods described herein include coronavirus (e.g., COVID-19) , HIV, hepatitis (A, B, or C) , herpes virus (e.g., VZV, HSV-l, HAV-6, HSV-II, and CMV, Epstein Barr virus) , adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable by methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable by methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc. ) , Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc. ) , Genus Mucorales (mucor, absidia, rhizopus) , Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable by methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.

In the above methods, the anti-CD40 antibodies or antigen-binding fragments disclosed herein can be combined with other forms of immunotherapy such as cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2) , or bispecific antibody therapy. See, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123.

The anti-CD40 antibodies or antigen-binding fragments or pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. In some embodiments, subcutaneous administration is adopted. In some embodiments, intravenous administration is adopted. In some embodiments, oral administration is adopted. In one embodiment, the antibodies or antigen-binding fragments provided herein can be delivered regionally to a tumor using well known methods, including but not limited to, hepatic or aortic pump; limb, lung or liver perfusion; in the portal vein; through a venous shunt; in a cavity or in a vein that is nearby a tumor, and the like. In another embodiment, the antibodies or antigen-binding fragments provided herein can be administered systemically. In a preferred embodiment, the antibodies or antigen-binding fragments are administered regionally at the site of a tumor. The antibodies or antigen-binding fragments can also be administered intratumorally, for example, by direct injection of the cells at the site of a tumor and/or into the tumor vasculature. For example, in the case of malignant pleural disease, mesothelioma or lung cancer, administration is preferably by intrapleural administration (see Adusumilli et al., Science Translational Medicine 6 (261) : 261ra151 (2014) ) . One skilled in the art can select a suitable mode of administration based on the type of cancer and/or location of a tumor to be treated. The antibodies or antigen-binding fragments can be introduced by injection or catheter. In one embodiment, the antibodies or antigen-binding fragments are pleurally administered to the subject in need, for example, using an intrapleural catheter.

Actual dosage levels of the active ingredients (i.e., the anti-CD40 antibodies or antigen-binding fragments) in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

As described, the anti-CD40 antibodies or antigen-binding fragments described herein can be co-administered with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody can be linked to the agent (as an immuno-complex) or can be administered separate from the agent. In the latter case (separate administration) , the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin) , cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient. Cisplatin is intravenously administered as a 100 mg/ml dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days. Coadministration of anti-CD40 antibodies, or antigen-binding fragments thereof, described herein with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells that would render them unreactive with the antibody.

5.7.2 Uses of the fusion proteins and immune effector cells

The fusion proteins provided herein can overcome immunosuppressive microenvironment in, for example, tumor or cancer tissues and potentiate a cell-mediated immune response. As disclosed herein, fusion proteins provided herein can be administered to a subject to illicit or enhance an immune response against cancer tissue or viral infection.

In some embodiments, the fusion proteins provided herein can be administered as a single therapy. In some embodiments, the fusion proteins provided herein can be administered in combination with a second therapy to enhance to efficacy of the therapy. The second therapy can be an immune therapy, wherein the administration of the fusion proteins provided herein enhance the efficacy of the immune therapy. In some embodiments, the second therapy is a cell therapy wherein an immune effector cell or cell population is administered into a subject to activate the immune system in the subject against a pathogen (e.g. a virus) or a disease (e.g. a cancer) , and the administration of the fusion proteins provided herein enhance the efficacy of the cell therapy. In some embodiments, fusion proteins provided herein can be administered to enhance the proliferation and activation of immune effector cells (e.g. T cells) . In some embodiments, fusion proteins provided herein can be administered to stimulate the maturation and epitope spreading activities of antigen-presenting cells. In some embodiments, fusion proteins provided herein can be administered to enable immune effector cells to overcome immunosuppression in tumor microenvironment. The immunosuppression in tumor microenvironment can be mediated by such as the PD1/PD-L1 signaling, regulatory T cells (Tregs) or TGF-beta signaling.

For example, in some embodiments, the fusion proteins can be administered in combination with activated immune effector cells. The activated immune effector cells can be, for example, activated T cells, activated NK cells, activated NKT cells, activated macrophages, activated neutrophils, or activated granulocytes. In some embodiments, the fusion proteins provided herein can be administered with peripheral blood leukocytes (PBL) , infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) . In some other embodiments, the fusion proteins provided herein can be administered with CART cells, TCRT cells, or BiTE. As a person of ordinary skill in the art would understand, the immune therapy (e.g. cell therapy) to be administered in combination with the fusion protein disclosed herein can be any immune therapy disclosed herein or otherwise known in the art. When the fusion protein is administered in combination with a second therapy, it can be administered prior to, concurrently with, or subsequence to the second therapy. A person of ordinary skill in the art would be able to determine the actual timing of administration to ensure that a synergistic therapeutic effect is achieved.

Additionally, as disclosed herein, immune effector cells can be genetically engineered to express the fusion proteins provided herein to acquire the capacity to overcome immunosuppressive microenvironment in tumor or cancer tissues, and to generate an enhanced immune response in a subject against a disease or a pathogen. Accordingly, the present disclosure also provides methods of using the fusion proteins, genetically engineered cells or cell populations, or pharmaceutical compositions disclosed herein in the treatment of cancer or tumor, or of viral infection.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion proteins disclosed herein. In some embodiments, provided herein are uses of the fusion proteins disclosed herein in treatment of tumor or cancer. In some embodiments, provided herein are uses of the fusion proteins provided herein for the preparation of a medicament for the treatment of tumor or cancer.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the genetically engineered cells disclosed herein. In some embodiments, provided herein are uses of the genetically engineered cells disclosed herein in treatment of tumor or cancer. In some embodiments, provided herein are uses of the genetically engineered cells provided herein for the preparation of a medicament for the treatment of tumor or cancer. In some embodiments, a population of cells comprising the genetically engineered cells is used in the treatment. The population of cells can be homogenous. The population of cells can be heterogenous.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, provided herein are uses of the pharmaceutical composition disclosed herein in treatment of tumor or cancer. In some embodiments, provided herein are uses of the pharmaceutical composition provided herein for the preparation of a medicament for the treatment of tumor or cancer.

In another embodiment, the methods and uses provided herein include administering cancer antigen-specific immune effector cells to a subject in need thereof, wherein the cells recombinantly express a CAR/TCR/BiTE comprising an antigen binding domain that specifically binds the cancer antigen. In some embodiments, a fusion protein provided herein is administered in combination with the cancer antigen-specific immune effector cell. In some embodiments, the cancer antigen-specific immune effector cell also expresses a fusion protein provided herein. The cancer antigen can be any cancer antigen disclosed herein or otherwise known in the art. In some embodiments, the cancer antigen is selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin.

The present disclosure also provides methods of using the fusion proteins, the genetically engineered cells or pharmaceutical compositions disclosed herein in treating viral infection. In some embodiments, provided herein are methods of treating viral infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion proteins, the genetically engineered cells or the pharmaceutical compositions disclosed herein. In some embodiments, provided herein are uses of the fusion proteins, the genetically engineered cells, or the pharmaceutical compositions disclosed herein in treatment of viral infection. In some embodiments, provided herein are uses of the fusion proteins, the genetically engineered cells, or the pharmaceutical compositions provided herein for the preparation of a medicament for the treatment of viral infection.

Actual dosage levels of the active ingredients (i.e., the fusion proteins or the genetically engineered immune effector cells provided herein) in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

In some embodiments, the fusion protein disclosed herein is administered to a subject in need thereof. The fusion protein can be administered at a flat dose (flat dose regimen) . In certain embodiments, the fusion protein disclosed herein is administered at a dose based on body weight. For administration of a fusion protein disclosed herein, the dosage can range from about 0.0001 to 100 mg/kg, 0.01 to 50 mg/kg, 0.01 to 10 mg/kg, 0.01 to 5 mg/kg, 1-10 mg/kg, or 1-5 mg/kg of the host body weight. For example, dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, or 10 mg/kg body weight.

Fusion proteins provided herein can be administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months, every six months, or yearly. Intervals can also be irregular as indicated by measuring blood levels of fusion protein in the subject. In some methods, dosage is adjusted to achieve a plasma concentration of about 1-1000 pg/ml and in some methods about 25-300 pg/ml. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Exemplary dosage regimens for a fusion protein described herein include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the fusion protein being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

A fusion protein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the fusion protein in the patient. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease.

As disclosed herein, the fusion proteins disclosed herein can be used in combination with a cell therapy that involves activated immune effector cells to enhance the efficacy of the cell therapy. In some embodiments, immune effector cells genetically engineered to express the fusion protein disclosed herein can be used in the therapeutic methods disclosed herein. When a cell therapy is adopted, the cells provided herein can be administered as a dose based on cells per kilogram (cells/kg) of body weight of the subject to which the cells are administered. Generally, the cell doses are in the range of about 10 ⁴ to about 10 ¹⁰ cells/kg of body weight, for example, about 10 ⁵ to about 10 ⁹, about 10 ⁵ to about 10 ⁸, about 10 ⁵ to about 10 ⁷, or about 10 ⁵ to 10 ⁶, depending on the mode and location of administration. In general, in the case of systemic administration, a higher dose is used than in regional administration, where the immune effector cells are administered in the region of a tumor. Exemplary dose ranges include, but are not limited to, 1x10 ⁴ to 1x10 ⁸, 2x10 ⁴ to 1x10 ⁸, 3x10 ⁴ to 1x10 ⁸, 4x10 ⁴ to 1x10 ⁸, 5x10 ⁴ to 1x10 ⁸, 6x10 ⁴, to 1x10 ⁸, 7x10 ⁴ to 1x10 ⁸, 8x10 ⁴ to 1x10 ⁸, 9x10 ⁴ to 1x10 ⁸, 1x10 ⁵ to 1x10 ⁸, for example, 1x10 ⁵ to 9x10 ⁷, 1x10 ⁵ to 8x10 ⁷, 1x10 ⁵ to 7x10 ⁷, 1x10 ⁵ to 6x10 ⁷, 1x10 ⁵ to 5x10 ⁷, 1x10 ⁵ to 4x10 ⁷, 1x10 ⁵ to 3x10 ⁷, 1x10 ⁵ to 2x10 ⁷, 1x10 ⁵ to 1x10 ⁷, 1x10 ⁵ to 9x10 ⁶, 1x10 ⁵ to 8x10 ⁶, 1x10 ⁵ to 7x10 ⁶, 1x10 ⁵ to 6x10 ⁶, 1x10 ⁵ to 5x10 ⁶, 1x10 ⁵ to 4x10 ⁶, 1x10 ⁵ to 3x10 ⁶, 1x10 ⁵ to 2x10 ⁶, 1x10 ⁵ to 1x10 ⁶, 2x10 ⁵ to 9x10 ⁷, 2x10 ⁵ to 8x10 ⁷, 2x10 ⁵ to 7x10 ⁷, 2x10 ⁵ to 6x10 ⁷, 2x10 ⁵ to 5x10 ⁷, 2x10 ⁵ to 4x10 ⁷, 2x10 ⁵ to 3x10 ⁷, 2x10 ⁵ to 2x10 ⁷, 2x10 ⁵ to 1x10 ⁷, 2x10 ⁵ to 9x10 ⁶, 2x10 ⁵ to 8x10 ⁶, 2x10 ⁵ to 7x10 ⁶, 2x10 ⁵ to 6x10 ⁶, 2x10 ⁵ to 5x10 ⁶, 2x10 ⁵ to 4x10 ⁶, 3x10 ⁵ to 3x10 ⁶ cells/kg, and the like. Such dose ranges can be particularly useful for regional administration. In a particular embodiment, cells are provided in a dose of 1x10 ⁵ to 1x10 ⁸, for example 1x10 ⁵ to 1x10 ⁷, 1x10 ⁵ to 1x10 ⁶, 1x10 ⁶ to 1x10 ⁸, 1x10 ⁶ to 1x10 ⁷, 1x10 ⁷ to 1x10 ⁸, 1x10 ⁵ to 5x10 ⁶, in particular 1x10 ⁵ to 3x10 ⁶ or 3x10 ⁵ to 3x10 ⁶ cells/kg for regional administration, for example, intrapleural administration. Exemplary dose ranges also can include, but are not limited to, 5x10 ⁵ to 1x10 ⁸, for example, 6x10 ⁵ to 1x10 ⁸, 7x10 ⁵ to 1x10 ⁸, 8x10 ⁵ to 1x10 ⁸, 9x10 ⁵ to 1x10 ⁸, 1x10 ⁶ to 1x10 ⁸, 1x10 ⁶ to 9x10 ⁷, 1x10 ⁶ to 8x10 ⁷, 1x10 ⁶ to 7x10 ⁷, 1x10 ⁶ to 6x10 ⁷, 1x10 ⁶ to 5x10 ⁷, 1x10 ⁶ to 4x10 ⁷, 1x10 ⁶ to 3x10 ⁷ cells/kg, and the like. Such does can be particularly useful for systemic administration. In a particular embodiment, cells are provided in a dose of 1x10 ⁶ to 3x10 ⁷ cells/kg for systemic administration. Exemplary cell doses include, but are not limited to, a dose of 1x10 ⁴, 2x10 ⁴, 3x10 ⁴, 4x10 ⁴, 5x10 ⁴, 6x10 ⁴, 7x10 ⁴, 8x10 ⁴, 9x10 ⁴, 1x10 ⁵, 2x10 ⁵, 3x10 ⁵, 4x10 ⁵, 5x10 ⁵, 6x10 ⁵, 7x10 ⁵, 8x10 ⁵, 9x10 ⁵, 1x10 ⁶, 2x10 ⁶, 3x10 ⁶, 4x10 ⁶, 5x10 ⁶, 6x10 ⁶, 7x10 ⁶, 8x10 ⁶, 9x10 ⁶, 1x10 ⁷, 2x10 ⁷, 3x10 ⁷, 4x10 ⁷, 5x10 ⁷, 6x10 ⁷, 7x10 ⁷, 8x10 ⁷, 9x10 ⁷, 1x10 ⁸, 2x10 ⁸, 3x10 ⁸, 4x10 ⁸, 5x10 ⁸, 6x10 ⁸, 7x10 ⁸, 8x10 ⁸, 9x10 ⁸, 1x10 ⁹ and so forth in the range of about 10 ⁴ to about 10 ¹⁰ cells/kg. In addition, the dose can also be adjusted to account for whether a single dose is being administered or whether multiple doses are being administered. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject, as described above. Dosages can be readily determined by those skilled in the art based on the disclosure herein and knowledge in the art.

The fusion proteins, immune effector cells, and pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. In some embodiments, subcutaneous administration is adopted. In some embodiments, intravenous administration is adopted. In some embodiments, oral administration is adopted. In one embodiment, the cells provided herein can be delivered regionally to a tumor using well known methods, including but not limited to, hepatic or aortic pump; limb, lung or liver perfusion; in the portal vein; through a venous shunt; in a cavity or in a vein that is nearby a tumor, and the like. In another embodiment, the cells provided herein can be administered systemically. In a preferred embodiment, the cells are administered regionally at the site of a tumor. The cells can also be administered intratumorally, for example, by direct injection of the cells at the site of a tumor and/or into the tumor vasculature. For example, in the case of malignant pleural disease, mesothelioma or lung cancer, administration is preferably by intrapleural administration (see Adusumilli et al., Science Translational Medicine 6 (261) : 261ra151 (2014) ) . One skilled in the art can select a suitable mode of administration based on the type of cancer and/or location of a tumor to be treated. The cells can be introduced by injection or catheter. In one embodiment, the cells are pleurally administered to the subject in need, for example, using an intrapleural catheter. Optionally, expansion and/or differentiation agents can be administered to the subject prior to, during or after administration of cells to increase production of the cells provided herein in vivo.

Proliferation of the cells provided herein is generally done ex vivo, prior to administration to a subject, and can be desirable in vivo after administration to a subject (see Kaiser et al., Cancer Gene Therapy 22: 72-78 (2015) ) . Cell proliferation should be accompanied by cell survival to permit cell expansion and persistence, such as with T cells.

In some embodiments, cancers or tumors that can be treated with the fusion proteins, cells, or pharmaceutical compositions disclosed herein are solid tumors. Cancers or tumors to be treated using the fusion proteins, cells, or pharmaceutical compositions provided herein comprise cancers typically responsive to immunotherapy. In some embodiments, the cancer or tumor can be carcinomas, sarcoma, melanoma (e.g. cutaneous or intraocular malignant melanoma) , glioma, glioblastoma, brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors, carcinoid tumors, gastric cancer, esophageal cancer, liver cancer, lung cancer (e.g. small cell lung cancer, or non-small cell lung cancer) , head and neck cancer, skin cancer, nasopharyngeal cancer, kidney cancer, colorectal cancer, breast cancer, pancreatic cancer, testicular cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer (for example, hormone refractory prostate adenocarcinoma) , bladder cancer, colon cancer, endocrine cancer, basal cell cancer, squamous cell cancer, dermatofibrosarcoma protuberans, mesothelioma, Merkel cell carcinoma, bone cancer, intestinal cancer, renal cancer (for example, clear cell carcinoma) , throat cancer, rectal cancer, cancer of the anal region, brain cancer, stomach cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the small intestine, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, synovial sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, neuroblastoma, or retinoblastoma.

In some embodiments, cancers or tumors that can be treated with the fusion proteins, cells, or pharmaceutical compositions disclosed herein are hematological cancers. In some embodiments, the hematological cancer can be lymphoma, leukemia, multiple myeloma (MM) , or myelodysplastic syndrome (MDS) . In some embodiments, the hematological cancer can be polycythemia vera, acute leukemia, acute myeloid leukemia (AML) , acute lymphocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia (CML) , chronic myelocytic leukemia, chronic lymphocytic leukemia, chronic myelomonocytic leukemia (CMML) , natural killer cell leukemia (NK leukemia) , Hodgkin’s disease, non-Hodgkin’s disease, Waldenstrom’s macroglobulinemia, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, natural killer cell lymphoma (NK lymphoma) , cutaneous T-Cell lymphoma (CTCL) , or peripheral T-cell lymphoma (PTCL) .

In cancer treatment, eliminating cancer or tumor cells in a subject can occur, but any clinical improvement constitutes a benefit. An anti-tumor effect can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An anti-tumor effect can also be manifested by the ability of the cells or pharmaceutical compositions provided herein in prevention of the occurrence of tumor in the first place. In some embodiments, an “anti-tumor effect” can be manifested by the reduction in cancer-induced immunosuppression. Clinical improvement comprises decreased risk or rate of progression or reduction in pathological consequences of the cancer or tumor. It is also understood that a method of treating cancer can include any effect that ameliorates a sign or symptom associated with cancer. Such signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting growth of a tumor, slowing the growth rate of a tumor, reducing the size of a tumor, reducing the number of tumors, eliminating a tumor, all of which can be measured using routine tumor imaging techniques well known in the art. Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.

In some embodiments, the methods or uses provided herein can reduce tumor burden. Thus, administration of the fusion proteins, cells or pharmaceutical compositions disclosed herein can reduce the number of tumor cells, reduce tumor size, and/or eradicate the tumor in the subject. Methods for monitoring patient response to administration of a pharmaceutical composition disclosed herein are known in the art and can be employed in accordance with methods disclosed herein. In some embodiments, methods known in the art can be employed to monitor the patient for response to administration of therapeutic methods disclosed herein. In some embodiments, methods known in the art can be used to monitor size of lesions, and/or size of lymph nodes. As a non-limiting example, in some embodiments, contrast-enhanced CT scans can detect and/or monitor lesions and/or lymph nodes in a patient. In some embodiments, administration of a pharmaceutical composition disclosed herein can reduce the size of lesions detected by CT scans in a patient. In some embodiments, administration of a pharmaceutical composition disclosed herein can cause shrinkage of abnormal lymph nodes. In some embodiments, the methods or uses provided herein can provide for increased or lengthened survival of a subject having cancer. In some embodiments, the methods or uses provided herein can provide for an increased immune response in the subject against the cancer.

In the methods disclosed herein, a therapeutically effective amount of the fusion proteins, cells or pharmaceutical compositions disclosed herein is administered to a subject in need of cancer treatment. The subject can be a mammal. In some embodiments, the subject is a human. Another group of suitable subjects can be a subject who has a history of cancer, but has been responsive to another mode of therapy. The prior therapy can have included, but is not restricted to, surgical resection, radiotherapy, and chemotherapy. In some embodiments, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts and are suitably defined for different types of cancers. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.

The subject can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective can be to decrease or delay the risk of recurrence. Additionally, refractory or recurrent malignancies can be treated using the fusion proteins, genetically engineered cells or pharmaceutical compositions disclosed herein.

For treatment, the amount administered is an amount effective for producing the desired effect. An effective amount or therapeutically effective amount is an amount sufficient to provide a beneficial or desired clinical result upon treatment. An effective amount can be provided in a single administration or a series of administrations (one or more doses) . An effective amount can be provided in a bolus or by continuous perfusion. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount can be determined by the physician for a particular subject. Several factors are typically considered when determining an appropriate dosage to achieve an effective amount, including for example, age, sex and weight of the subject, the condition being treated, and the severity of the condition.

Fusion proteins, cells, or pharmaceutical compositions provided herein can be administered with medical devices known in the art. For example, in some embodiments, a a needleless hypodermic injection device can be used, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use described herein include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

Combination therapy using agents with different mechanisms of action can result in additive or synergetic effects. Combination therapy can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent disclosed herein. Combination therapy can decrease the likelihood that resistant cancer cells will develop. In some embodiments, the additional therapy results in an increase in the therapeutic index of the cells or pharmaceutical compositions described herein. In some embodiments, the additional therapy results in a decrease in the toxicity and/or side effects of cells or pharmaceutical compositions described herein. In some embodiments, the fusion proteins, cells, or pharmaceutical compositions described herein can be administered in combination with an additional therapy. In some embodiments, the additional therapy can be surgical resection, radiotherapy, or chemotherapy.

The additional therapy can be administered prior to, concurrently with, or subsequent to administration of the fusion proteins, cells, or pharmaceutical compositions described herein. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. A person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.

5.8 Methods of production

5.8.1 Polynucleotides and fusion proteins

Polynucleotides provided herein can be prepared, manipulated, and/or expressed using any of a variety of well-established techniques known and available in the art. Many vectors can be used. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Exemplary transposon systems such as Sleeping Beauty and PiggyBac can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91 (4) : 501–510 (1997) ;

et al., (2007) Nucleic Acids Research. 35 (12) : e87) . Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST ^TM, pLenti6/V5-DEST ^TM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.

In some embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally. The vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotrophic herpes virus may be Epstein Barr virus (EBV) , Kaposi's sarcoma herpes virus (KSHV) , Herpes virus saimiri (HS) , or Marek's disease virus (MDV) . Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus. Typically, the host cell comprises the viral replication transactivator protein that activates the replication.

“Expression control sequences, ” “control elements, ” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5’ and 3’ untranslated regions-which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters can be used.

Illustrative ubiquitous expression control sequences that can be used in present disclosure include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) promoter (e.g., early or late) , a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1) , ferritin H (FerH) , ferritin L (FerL) , Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , eukaryotic translation initiation factor 4A1 (EIF4A1) , heat shock 70kDa protein 5 (HSPA5) , heat shock protein 90kDa beta, member 1 (HSP90B1) , heat shock protein 70kDa (HSP70) , β-kinesin (β-KIN) , the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 -1482 (2007) ) , a Ubiquitin C promoter (UBC) , a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, and a β-actin promoter.

Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone) , metallothionine promoter (inducible by treatment with various heavy metals) , MX-1 promoter (inducible by interferon) , the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323: 67) , the cumate inducible gene switch (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.

The fusion proteins described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques. The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) , MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons (1987 and annual updates) ; CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley &Sons (1987 and annual updates) Gait (ed. ) (1984) OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press; Eckstein (ed. ) (1991) OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, IRL Press; Birren et al. (eds. ) (1999) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Borrebaeck (ed. ) (1995) ANTIBODY ENGINEERING, Second Edition, Oxford University Press; Lo (ed. ) (2006) ANTIBODY ENGINEERING: METHODS AND PROTOCOLS (METHODS IN MOLECULAR BIOLOGY) ; Vol. 248, Humana Press, Inc; each of which is incorporated herein by reference in its entirety.

The fusion proteins described herein can be produced and isolated using methods known in the art. Peptides can be synthesized, in whole or in part, using chemical methods (see, e.g., Caruthers (1980) . Nucleic Acids Res. Symp. Ser. 215; Horn (1980) ; and Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA) . Peptide synthesis can be performed using various solid phase techniques (see, e.g., Roberge Science 269: 202 (1995) ; Merrifield, Methods. Enzymol. 289: 3 (1997) ) and automated synthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the manufacturer’s instructions. Peptides can also be synthesized using combinatorial methodologies. Synthetic residues and polypeptides can be synthesized using a variety of procedures and methodologies known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley &Sons, Inc., NY) . Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25: 3440 (1997) ; Frenkel, Free Radic. Biol. Med. 19: 373 (1995) ; and Blommers, Biochemistry 33: 7886 (1994) ) . Peptide sequence variations, derivatives, substitutions and modifications can also be made using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR based mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13: 4331 (1986) ; Zoller et al., Nucl. Acids Res. 10: 6487 (1987) ) , cassette mutagenesis (Wells et al., Gene 34: 315 (1985) ) , restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317: 415 (1986) ) and other techniques can be performed on cloned DNA to produce invention peptide sequences, variants, fusions and chimeras, and variations, derivatives, substitutions and modifications thereof.

The fusion proteins described herein can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant technologies, or a combination thereof. In some embodiments, a recombinant expression vector is used to express a polynucleotide encoding a fusion protein described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding a fusion protein operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, coding sequences of fusion proteins disclosed herein can be ligated into such expression vectors for their expression in mammalian cells. In some embodiments, a viral vector is used. DNA regions are “operatively linked” when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In some embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide can include an N-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

5.8.2 Antibodies and antigen-binding fragments

Provided herein are antibodies and antigen-binding fragments thereof that include but are not limited to monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, and antigen-binding fragments thereof.

Methods of antibody production are well-known in the art. See for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) ; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981) , each of which is incorporated herein by reference in its entirety. For in vivo use of antibodies in humans, it may be preferable to use human antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. A human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. For example, anti-CD19 antibodies directed against the human CD19 antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies, including, but not limited to, IgG1 (gamma 1) and IgG3. For an overview of this technology for producing human antibodies, see, Lonberg and Huszar (Int. Rev. Immunol., 13: 65-93 (1995) ) . For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif. ) and Genpharm (San Jose, Calif. ) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. For a specific discussion of transfer of a human germ-line immunoglobulin gene array in germ-line mutant mice that will result in the production of human antibodies upon antigen challenge see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993) ; Jakobovits et al., Nature, 362: 255-258 (1993) ; Bruggermann et al., Year in Immunol., 7: 33 (1993) ; and Duchosal et al., Nature, 355: 258 (1992) .

Human antibodies can also be derived from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581-597 (1991) ; Vaughan et al., Nature Biotech., 14: 309 (1996) ) . Phage display technology (McCafferty et al., Nature, 348: 552-553 (1990) ) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3: 564-571 (1993) . Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of unimmunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol., 222: 581-597 (1991) , or Griffith et al., EMBO J., 12: 725-734 (1993) . See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905, each of which is incorporated herein by reference in its entirety.

Human antibodies can also be generated by in vitro activated B cells (see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety) . Human antibodies can also be generated in vitro using hybridoma techniques such as, but not limited to, that described by Roder et al. (Methods Enzymol., 121: 140-167 (1986) ) .

Alternatively, in some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human. In some embodiment, the antigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference) , veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28 (4/5) : 489-498; Studnicka et al., 1994, Protein Engineering, 7 (6) : 805-814; and Roguska et al.,1994, PNAS, 91: 969-973, each of which is incorporated herein by its entirety by reference) , chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference) , and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002) , Caldas et al., Protein Eng., 13 (5) : 353-60 (2000) , Morea et al., Methods, 20 (3) : 267-79 (2000) , Baca et al., J. Biol. Chem., 272 (16) : 10678-84 (1997) , Roguska et al., Protein Eng., 9 (10) : 895-904 (1996) , Couto et al., Cancer Res., 55 (23 Supp) : 5973s-5977s (1995) , Couto et al., Cancer Res., 55 (8) : 1717-22 (1995) , Sandhu J S, Gene, 150 (2) : 409-10 (1994) , and Pedersen et al., J. Mol. Biol., 235 (3) : 959-73 (1994) , each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585, 089; and Riechmann et al., 1988, Nature, 332: 323, which are incorporated herein by reference in their entireties. )

A humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human. Humanization of antibodies is well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ) , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239, 400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference herein in their entirety) . In such humanized chimeric antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28 (4/5) : 489-498; Studnicka et al., Protein Engineering, 7 (6) : 805-814 (1994) ; and Roguska et al., PNAS, 91: 969-973 (1994) ) or chain shuffling (U.S. Pat. No. 5,565,332) , the contents of which are incorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) ; Chothia et al., J. Mol. Biol., 196: 901 (1987) , the contents of which are incorporated herein by reference herein in their entirety) . Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992) ; Presta et al., J. Immunol., 151: 2623 (1993) , the contents of which are incorporated herein by reference herein in their entirety) .

Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties. For example, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

A “humanized” antibody retains a similar antigenic specificity as the original antibody, for example, the ability to bind human CD40 antigen. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody for a particular antigen can be increased using methods of “directed evolution, ” as described by Wu et al., J. Mol. Biol., 294: 151 (1999) , the contents of which are incorporated herein by reference herein in their entirety.

5.8.3 Genetically engineered immune effector cells

In some embodiments, provided herein is a genetically engineered immune effector cell that recombinantly expresses a fusion protein disclosed herein. In some embodiments, provided herein is a genetically engineered immune effector cell that comprises a polynucleotide encoding a fusion protein disclosed herein. In some embodiments, provided herein is a genetically engineered immune effector cell that comprises a vector comprising a polynucleotide encoding a fusion protein disclosed herein. In some embodiments, provided herein is a genetically engineered immune effector cell that recombinantly expresses a fusion protein disclosed herein and a CAR, TCR, or BiTE (CAR/TCR/BiTE) . In some embodiments, provided herein is a genetically engineered immune effector cell that comprises a polynucleotide encoding a fusion protein disclosed herein and a CAR/TCR/BiTE.

5.8.3.1 Methods of genetic engineering

With respect to generating cells recombinantly expressing a fusion protein disclosed herein, one or more polynucleotides encoding the fusion protein is introduced into the target cell using a suitable expression vector. The target immune effector cells (e.g., T cells) are transferred with one or more polynucleotides encoding a fusion protein, or a CAR/TCR/BiTE and a fusion protein. The CAR/TCR/BiTE and fusion protein encoding polynucleotides can be on separate vectors or on the same vector, as desired. For example, a polynucleotide encoding a CAR or a fusion protein disclosed herein can be cloned into a suitable vector, such as a viral vector, and introduced into the target cell using well known molecular biology techniques (see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999) ) . Any vector suitable for expression in a cell, particularly a human cell, can be used. The vectors contain suitable expression elements such as promoters that provide for expression of the encoded nucleic acids in the target cell. In the case of a retroviral vector, cells can optionally be activated to increase transduction efficiency (see Parente-Pereira et al., J. Biol. Methods 1 (2) e7 (doi 10.14440/jbm. 2014.30) (2014) ; Movassagh et al., Hum. Gene Ther. 11: 1189-1200 (2000) ; Rettig et al., Mol. Ther. 8: 29-41 (2003) ; Agarwal et al., J. Virol. 72:3720-3728 (1998) ; Pollok et al., Hum. Gene Ther. 10: 2221-2236 (1998) ; Quinn et al., Hum. Gene Ther. 9: 1457-1467 (1998) ; see also commercially available methods such as Dynabeads ^TM human T cell activator products, Thermo Fisher Scientific, Waltham, MA) .

In one embodiment, the vector is a retroviral vector, for example, a gamma retroviral or lentiviral vector, which is employed for the introduction of a fusion protein and/or a CAR, TCR, or BiTE into the target cell. For genetic modification of the cells to express a fusion protein and/or a CAR, TCR, or BiTE, a retroviral vector can be employed for transduction. However, it is understood that any suitable viral vector or non-viral delivery system can be used. Combinations of a retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., Mol. Cell. Biol. 5: 431-437 (1985) ) ; PA317 (Miller et al., Mol. Cell. Biol. 6: 2895-2902 (1986) ) ; and CRIP (Danos et al., Proc. Natl. Acad. Sci. USA 85: 6460-6464 (1988) ) . Non-amphotropic particles are suitable too, for example, particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art (Relander et al., Mol. Therap. 11: 452-459 (2005) ) . Possible methods of transduction also include direct co-culture of the cells with producer cells (for example, Bregni et al., Blood 80: 1418-1422 (1992) ) , or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (see, for example, Xu et al., Exp. Hemat. 22: 223-230 (1994) ; Hughes, et al. J. Clin. Invest. 89: 1817-1824 (1992) ) .

Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus derived vector, or a herpes virus, such as Epstein-Barr Virus (see, for example, Miller, Hum. Gene Ther. 1 (1) : 5-14 (1990) ; Friedman, Science 244: 1275-1281 (1989) ; Eglitis et al., BioTechniques 6: 608-614 (1988) ; Tolstoshev et al., Current Opin. Biotechnol. 1: 55-61 (1990) ; Sharp, Lancet 337: 1277-1278 (1991) ; Cornetta et al., Prog. Nucleic Acid Res. Mol. Biol. 36: 311-322 (1989) ; Anderson, Science 226: 401-409 (1984) ; Moen, Blood Cells 17: 407-416 (1991) ; Miller et al., Biotechnology 7: 980-990 (1989) ; Le Gal La Salle et al., Science 259: 988-990 (1993) ; and Johnson, Chest 107: 77S-83S (1995) ) . Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med. 323: 370 (1990) ; Anderson et al., U.S. Pat. No. 5,399,346) . Generally, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8: 423-430 (1997) ; Kido et al., Current Eye Research 15: 833-844 (1996) ; Bloomer et al., J. Virol. 71: 6641-6649 (1997) ; Naldini et al., Science 272: 263-267 (1996) ; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319-10323 (1997) ) .

Particularly useful vectors for expressing a fusion protein disclosed herein and/or CAR/TCR/BiTE include vectors that have been used in human gene therapy. In one non-limiting embodiment, a vector is a retroviral vector. The use of retroviral vectors for expression in T cells or other immune effector cells, including engineered T cells, has been described (see Scholler et al., Sci. Transl. Med. 4: 132-153 (2012; Parente-Pereira et al., J. Biol. Methods 1 (2) : e7 (1-9) (2014) ; Lamers et al., Blood 117 (1) : 72-82 (2011) ; Reviere et al., Proc. Natl. Acad. Sci. USA 92: 6733-6737 (1995) ) . In one embodiment, the vector is an SGF retroviral vector such as an SGF γ-retroviral vector, which is Moloney murine leukemia-based retroviral vector. SGF vectors have been described previously (see, for example, Wang et al., Gene Therapy 15: 1454-1459 (2008) ) .

The vectors used herein employ suitable promoters for expression in a particular host cell. The promoter can be an inducible promoter or a constitutive promoter. In some embodiments, the promoter of an expression vector provides expression in a stem cell, such as a hematopoietic stem cell. In some embodiments, the promoter of an expression vector provides expression in an immune effector cell, such as a T cell. Non-viral vectors can be used as well, so long as the vector contains suitable expression elements for expression in the target cell. Some vectors, such as retroviral vectors, can integrate into the host genome.

In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide provided herein into the cell using a non-viral delivery system. For example, physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. In some embodiments, RNA electroporation can be used (Van Driessche et al. Folia histochemica et cytobiologica 43: 4 213-216 (2005) ) . In some embodiments, DNA transfection and transposon can be used. In some embodiments, the Sleeping Beauty system or PiggyBac system is used (e.g., Ivics et al., Cell, 91 (4) : 501-510 (1997) ;

et al. (2007) Nucleic Acids Research. 35 (12) : e87) . Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle) .

In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide provided herein into the cell using gene-editing. If desired, targeted integration can be implemented using technologies such as a nuclease, transcription activator-like effector nucleases (TALENs) , Zinc-finger nucleases (ZFNs) , clustered regularly interspaced short palindromic repeats (CRISPRs) , homologous recombination, non-homologous end joining, microhomology-mediated end joining, homology-mediated end joining and the like (Gersbach et al., Nucl. Acids Res. 39: 7868-7878 (2011) ; Vasileva, et al. Cell Death Dis. 6: e1831. (Jul 23 2015) ; Sontheimer, Hum. Gene Ther. 26 (7) : 413-424 (2015) ; Yao et al. Cell Research volume 27, 801-814 (2017) ) . In some embodiments, methods provided herein use a ZFN system. A zinc-finger nuclease consists of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain consists of a series of Cys2-His2 zinc-finger proteins linked in series, and each zinc-finger unit includes about 30 amino acids for specifically binding to DNA. The non-specific endonuclease is a FokI endonuclease which forms a dimer to cleave the DNA. In some embodiments, methods provided herein use a TALEN system. TALEN is a transcription activator-like effector nuclease. The TALE protein is a core component of a DNA binding domain, and generally consists of a plurality of basic repeat units linked in series. The designed and combined series of units can specifically recognize a DNA sequence and cleave a specific DNA sequence by coupling the FokI endonuclease.

In some embodiments, methods provided herein use a CRISPR-Cas system. The CRISPR-Cas system can be a CRISPR-Cas9 system. CRISPR/Cas system is a nuclease system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR binding proteins (i.e., Cas proteins) , which can cleave nearly all genomic sequences adjacent to protospacer-adjacent motifs (PAM) in eukaryocytes (Cong et al. Science 2013.339: 819-823) . The “CRISPR/Cas system” is used to refer collectively to transcripts involving CRISPR-related ( “Cas” ) genes, as well as other elements involving the expression thereof or directing the activity thereof, including sequences encoding a Cas gene, tracr (trans-activated CRISPR) sequences (for example, tracrRNA or active partial tracrRNA) , tracr pairing sequences (in the background of an endogenous CRISPR system, cover “direct repeats” and processed partial direct repeats) , guide sequences, or other sequences from the CRISPR locus and transcripts. In general, the CRISPR system is characterized as an element that facilitates the formation of a CRISPR complex at a site of a target sequence (also called a protospacer in the endogenous CRISPR system) . Unrestricted examples of the Cas protein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12) , Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 homologues, or modified forms thereof. In some embodiments, the Cas protein is a Cas9 protein (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Deltcheva, Chylinski et al. 2011; Makarova, Grishin et al. (2006) ) . Amino acid sequences of the Cas9 protein are known in the art. Exemplary sequences can be found, for example, in the SwissProt database under the accession number Q99ZW2, in the UniProt database under the number A1IQ68, Q03LF7, or J7RUA5.

The vectors and constructs can optionally be designed to include a reporter. For example, the vector can be designed to express a reporter protein, which can be useful to identify cells comprising the vector or polynucleotides provided on the vector, such as polynucleotides that have integrated into the host chromosome. In one embodiment, the reporter can be expressed as a bicistronic or multicistronic expression construct with the fusion protein or the CAR/TCR/BiTE. Exemplary reporter proteins include, but are not limited to, fluorescent proteins, such as mCherry, green fluorescent protein (GFP) , blue fluorescent protein, for example, EBFP, EBFP2, Azurite, and mKalama1, cyan fluorescent protein, for example, ECFP, Cerulean, and CyPet, and yellow fluorescent protein, for example, YFP, Citrine, Venus, and YPet.

Assays can be used to determine the transduction efficiency of a fusion protein disclosed herein or a CAR/TCR/BiTE using routine molecular biology techniques. If a marker has been included in the construct, such as a fluorescent protein, gene transfer efficiency can be monitored by FACS analysis to quantify the fraction of transduced (for example, GFP ⁺) immune effector cells, such as T cells, and/or by quantitative PCR. Using a well-established cocultivation system (Gade et al., Cancer Res. 65: 9080-9088 (2005) ; Gong et al., Neoplasia 1: 123-127 (1999) ; Latouche et al., Nat. Biotechnol. 18: 405-409 (2000) ) it can be determined whether fibroblast AAPCs expressing cancer antigen (vs. controls) direct cytokine release from transduced immune effector cells, such as T cells, expressing a CAR (cell supernatant LUMINEX (Austin TX) assay for IL-2, IL-4, IL-10, IFN-γ, TNF-α, and GM-CSF) , T cell proliferation (by carboxyfluorescein succinimidyl ester (CFSE) labeling) , and T cell survival (by Annexin V staining) . The influence of CD80 and/or 4-1BBL on T cell survival, proliferation, and efficacy can be evaluated. T cells can be exposed to repeated stimulation by cancer antigen positive target cells, and it can be determined whether T cell proliferation and cytokine response remain similar or diminished with repeated stimulation. The cancer antigen CAR constructs can be compared side by side under equivalent assay conditions. Cytotoxicity assays with multiple E: T ratios can be conducted using chromium-release assays.

Combinations and permutations of various methods described herein or otherwise known in the art are expressly contemplated to prepare the genetically engineered cells disclosed herein.

5.8.3.2 Sources of immune effector cells

Immune effector cells provided herein can be obtained from a subject. Sources for the immune effector cells provided herein include, but are not limited to, peripheral blood, umbilical cord blood, bone marrow, or other sources of hematopoietic cells. Immune effector cells (e.g., T cells) can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, cell lines available in the art can be used. Immune effector cells provided herein can be isolated by methods well known in the art, including commercially available isolation methods (see, for example, Rowland-Jones et al., LYMPHOCYTES: A PRACTICAL APPROACH, Oxford University Press, New York (1999) ) . Various methods for isolating immune effector cells have been described previously, and can be used, including but not limited to, using peripheral donor lymphocytes (Sadelain et al., Nat. Rev. Cancer 3 : 35-45 (2003) ; Morgan et al., Science 314: 126-129 (2006) , and using selectively in v/Yro-expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or dendritic cells (Dupont et al., Cancer Res. 65: 5417-5427 (2005) ; Papanicolaou et al., Blood 102: 2498-2505 (2003) ) .

In certain embodiments, immune effector cells (e.g., T cells) disclosed herein can be obtained from a unit of blood collected from a subject using any techniques known to the skilled artisan, such as Ficoll ^TM separation. In some embodiments, cells from the circulating blood of an individual are obtained 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 some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS) . In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step can be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca ²⁺-free, Mg ²⁺-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed, and the cells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ^TM gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3 ⁺, CD28 ⁺, CD4 ⁺, CD8 ⁺, CD45RA ⁺, and CD45RO ⁺T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3×28) -conjugated beads, such as

M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein) , subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention.

Various techniques can be employed to separate the cells to enrich for desired immune effector cells. For instance, negative selection methods can be used to remove cells that are not the desired immune effector cells. Additionally, positive selection methods can be used to isolate or enrich for desired immune effector cells or precursor cells thereof, or a combination of positive and negative selection methods can be employed. Monoclonal antibodies (MAbs) are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation for both positive and negative selections. If a particular type of cell is to be isolated, for example, a particular type of T cell, various cell surface markers or combinations of markers, including but not limited to, CD3, CD4, CD8, CD34 (for hematopoietic stem and progenitor cells) and the like, can be used to separate the cells, as is well known in the art (see Kearse, T CELL PROTOCOLS: DEVELOPMENT AND ACTIVATION, Humana Press, Totowa NJ (2000) ; De Libero, T CELL PROTOCOLS, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009) ) . In some embodiments, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4 ⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4 ⁺, CD25 ⁺, CD62L ^hi, GITR ⁺, and FoxP3 ⁺. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

Procedures for separation of immune effector cells include, but are not limited to, density gradient centrifugation, coupling to particles that modify cell density, magnetic separation with antibody-coated magnetic beads, affinity chromatography; cytotoxic agents joined to or used in conjunction with a monoclonal antibody (mAb) , including, but not limited to, complement and cytotoxins, and panning with an antibody attached to a solid matrix, for example, a plate or chip, elutriation, flow cytometry, or any other convenient technique (see, for example, Recktenwald et al., CELL SEPARATION METHODS AND APPLICATIONS, Marcel Dekker, Inc., New York (1998) ) . It is understood that the immune effector cells used in methods provided herein can be substantially pure cells or can be a polyclonal population. In some embodiments, a polyclonal population can be enriched for a desired immune effector cell. Such an enrichment can take place prior to or after genetically engineering the cells to express a fusion protein provided herein, as desired.

The immune effector cells can be autologous or non-autologous to the subject to which they are administered in the methods of treatment disclosed herein. Autologous cells are isolated from the subject to which the engineered cells are to be administered. Optionally, the cells can be obtained by leukapheresis, where leukocytes are selectively removed from withdrawn blood, made recombinant, and then retransfused into the donor. Alternatively, allogeneic cells from a non-autologous donor that is not the subject can be used. In the case of a non-autologous donor, the cells are typed and matched for human leukocyte antigen (HLA) to determine an appropriate level of compatibility, as is well known in the art. The cells can optionally be cryopreserved after isolation and/or genetic engineering, and/or expansion of genetically engineered cells (see Kaiser et al., supra, 2015) ) . Methods for cyropreserving cells are well known in the art (see, for example, Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, 4th ed., Wiley-Liss, New York (2000) ; Harrison and Rae, GENERAL TECHNIQUES OF CELL CULTURE, Cambridge University Press (1997) ) .

In some embodiments, isolated immune effector cells are genetically engineered ex vivo for recombinant expression of a fusion protein. In some embodiments, isolated immune effector cells are genetically engineered ex vivo for recombinant expression of a fusion protein and a CAR/TCR/BiTE. In some embodiments, immune effector cells provided herein are obtained by in vitro sensitization, wherein the sensitization can occur before or after the immune effector cells are genetically engineered to recombinantly express the fusion protein disclosed herein. In an embodiment where the sensitized immune effector cells, such T cells, are isolated from in vivo sources, it will be self-evident that genetic engineering occurs of the already-sensitized immune effector cells.

Also contemplated in the present disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the genetically engineered cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993) . In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT) , cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

In a further embodiment, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained can be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated to collect blood cells, including T cells, NK cells, or other immune effector cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

The immune effector cells disclosed herein can be subjected to conditions that favor maintenance or expansion of cells as well known in the art. (De Libero, T Cell Protocols, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009) ; Parente-Pereira et al., J. Biol. Methods 1 (2) e7 (doi 10.14440/jbm. 2014.30) (2014) ; Movassagh et al., Hum. Gene Ther. 11: 1189-1200 (2000) ; Rettig et al., Mol. Ther. 8: 29-41 (2003) ; Agarwal et al., J. Virol. 72: 3720-3728 (1998) ; Pollok et al., Hum. Gene Ther. 10: 2221-2236 (1999) ; Quinn et al., Hum. Gene Ther. 9: 1457-1467 (1998) ; see also commercially available methods such as Dynabeads ^TM human T cell activator products, Thermo Fisher Scientific, Waltham, MA) ) . The immune effector cells disclosed herein (e.g. T cells) can optionally be expanded prior to or after ex vivo genetic engineering. Expansion of the cells is particularly useful to increase the number of cells for administration to a subject. Such methods for expansion of cells are well known in the art (see e.g. Kaiser et al., Cancer Gene Therapy 22: 72-78 (2015) ; Wolfl et al., Nat. Protocols 9: 950-966 (2014) ) . Furthermore, the cells can optionally be cryopreserved after isolation and/or genetic engineering, and/or expansion of genetically engineered cells (see Kaiser et al., supra, 2015) ) . Methods for cyropreserving cells are well known in the art (see, for example, Freshney, Culture of Animal Cells: A Manual of Basic Techniques, 4th ed., Wiley-Liss, New York (2000) ; Harrison and Rae, General Techniques of Cell Culture, Cambridge University Press (1997) ) .

In some embodiments, provided herein are immune effector cells, such as T cells, that recognize and are sensitized to a viral antigen or a tumor antigen, and also recombinantly express a fusion protein provided herein. Such immune effector cells, such as T cells, can but need not express a CAR that binds to a viral antigen or a tumor antigen, since the cells already are antigen-specific so that their immune response (for example, cytotoxicity) is stimulated specifically by such antigen. Such immune effector cells, such as T cells, that recognize and are sensitized to a viral antigen or a tumor antigen can be obtained by known methods, by way of example, in vitro sensitization methods using naive T cells (see, for example, Wolfl et al., Nat. Protocols 9: 950-966 (2014) ) or hematopoietic progenitor cells (see van Lent et al., J. Immunol. 179: 4959-4968 (2007) ) ; or obtained from a subject that has been exposed to and is mounting an immune response against the antigen, such as a subject having a viral infection or a tumor antigen (i.e., in vivo sensitized immune effector cells) . Methods for isolating an antigen-specific T cell from a subject are well known in the art. Such methods include, but are not limited to, a cytokine capture system or cytokine secretion assay, which is based on the secretion of cytokines from antigen stimulated T cells that can be used to identify and isolate antigen-specific, and expansion of cells in vitro (see Assenmacher et al., Cytometric Cytokine Secretion Assay, in Analyzing T Cell Responses: How to Analyze Cellular Immune Responses Against Tumor Associated Antigens, Nagorsen et al., eds., Chapter 10, pp. 183-195, Springer, The Netherlands (2005) ; Haney et al., J. Immunol. Methods 369: 33-41 (2011) ; Bunos et al., Vox Sanguinis DOI: 10.1 I l l/vox. 12291 (2015) ; Montes et al., Clin. Exp. Immunol. 1 42: 292-302 (2005) ; Adusumilli et al., Sci TranslMed. 6: 261ral51 (2014) ) . Such cytokines include, but are not limited to interferon-γ and tumor necrosis factor-a. The antigen-specific T cells can be isolated using well known techniques as described above for isolating immune effector cells, which include, but are not limited to, flow cytometry, magnetic beads, panning on a solid phase, and so forth. Antigen-specific T cell isolation techniques are also commercially available, which can be used or adapted for clinical applications (see, for example, Miltenyi Biotec, Cambridge, MA; Proimmune, Oxford, UK; and the like) . Methods for T cell activation and expansion are described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041.

Generally, the T cells provided herein can be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory receptor on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8) : 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9) : 13191328, 1999; Garland et al., J. Immunol Meth. 227 (1-2) : 53-63, 1999) .

5.9 Experimental

The examples provided below are for purposes of illustration only, which are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

In this study, we selected some scFvs against CD40 from fully human antibody phage display library, and generated fusion protein of the CD40 scFv with the intracellular domain of CD28 (CD40 scFv-CD28 fusion, or LACO) . When co-introduced into human T lymphocytes with Her2 CARs, some of the CD40 scFv-CD28 fusion molecules strongly increased T cell anti-tumor activities compared to Her2 CART cells alone (namely, without the fusion protein) .

5.9.1 Example 1: Preparation of anti-human CD40 monoclonal antibodies

Anti-CD40 antibodies were prepared using fully human antibody phage display library following the steps below:

(1) Expression and purification of phage display library: the log phase TG1 library culture was infected with freshly thawed M13K07 helper phage with a multiplicity of infection of 20: 1 (phage-to-cell-ratio) and overnight induction by IPTG; the phage library was purified by PEG/NaCl precipitated method and phage titer was determined. The phage was stored at 4℃ and the scFv selection was performed shortly after.

(2) Selection of CD40-specific scFv-phages: for the first round of selection, Maxisorp plate was coated with 20 μg/ml CD40-6His protein dissolved in 1×PBS and incubated overnight at 4℃. (For subsequent rounds of selection, lower protein concentration was used for more stringent selection, including 2 μg/ml in the 2nd round bio-panning, and 0.5 μg/ml in the 3rd round bio-panning. ) The plates were then washed with PBS, and blocking buffer (5%milk+1%BSA in 1×PBS) was added to each well. After 2-hour incubation at room temperature, the blocking buffer was discarded, phage solution added, and the plate was sealed with parafilm, and incubated for 2 hours with gently shaking. In the first selection round, the plate was then washed 10 times with PBST. (For following rounds, increase stringency of washing was adopted by adding more wash cycles: 20 cycles in the 2nd round, 30 cycles in the 3rd round) . The antigen-bound scFv-phages were then eluted by incubating in 1 ml acid elution buffer (pH 2.2) for about 8 minutes. The eluted pages were pipetted to about 100 μl neutralization buffer in a novel PP tube, inoculated in 15 ml of log-phase TG1 culture (OD600=0.5) , cultured at 37℃ by 30 min standing and 30 min shaking, plated onto 2xYT-GA agar plate, and cultured overnight at 30℃ for subsequent selection.

(3) mpELISA screening: after three round selection, positive colonies were selected for monoclonal phage ELISA (mpELISA) screening. Phage supernatant was generated from individual bacterial clones and tested for the binding to CD40-6His protein. The supernatant was incubated with pre-blocked Maxisorp plate coated with 2 μg/ml CD40-6His protein. After three washes, 100 μl/well of HRP-conjugated anti-M13 antibody diluted 1: 5000 in blocking buffer (5%milk+1%BSA in 1×PBS) was added and incubate for 60 min at RT. After washing plate 5 times with PBST, 100 μl/well TMB substrate solution was added and incubated for 10-30 min until blue color had appeared. Reaction was stopped by adding 50 μl/well of stop solution (2N H2SO4) . Absorbance was read at 450 nm in a microplate reader. FIG. 1 shows five representative 96-well plates of anti-human CD40-Fc monoclonal phage ELISA.

(4) Cloning and sequence analysis: A total of 56 positive clones were selected according to the ELISA results, and used as templates for PCR cloning of the scFv sequence. The CDR regions of scFv were analyzed through abysis website (http: //abysis. org/) , and are provided above in Tables 1 and 2.

5.9.2 Example 2: Preparation of CD40 scFv-CD28 fusion (LACO)

The CD40 scFv-CD28 fusion was synthesized by Sangon Biotech (Shanghai, China) . Next, the pUC57-CAR plasmid was linearized by digestion with Spe1 enzyme. The completeness of the digestion was checked by running agarose DNA gel. The linearized vector was purified using PCR Cleanup kit (#28106, QIAGEN) and eluted with EB from the kit water. The concentration of DNA was measured by nanodrop. Then, in vitro transcription (IVT) was performed following the protocol of manufacturer (Thermofisher, Cat No: AM13455) . For one reaction, 1 μg template DNA, NTP/ARCA buffer, T7 buffer, GTP, T7 enzyme and RNase free H2O were added to 0.2 ml PCR tube and incubated at 37 ℃ for 3 hours. 3 hours later, 2 μl DNase was added per reaction, and incubated at 37 ℃ for 15 min. The tailing procedure was performed according to the manufacturer’s suggestion. The IVT mRNA was purified using the RNeasy Mini kit (#74106, QIAGEN) , and eluted with RNase-free water. The concentration of RNA was measured by nanodrop. RNA integrity and size were examined by agarose gel electrophoresis.

Table 5: RNA Used in this study.

ID	RNA (10 μg each)
C4	40-18.28+4D5. BBZ
C5	40-37.28+4D5. BBZ
C6	40-38.28+4D5. BBZ
C7	40-45.28+4D5. BBZ
C8	40-47.28+4D5. BBZ
C9	40-52.28+4D5. BBZ
C11	A40C28+4D5. BBZ
C13	4D5. BBZ
C14	NO EP

4D5: anti-Her2 scFv; 4D5. BBZ: anti-Her2 CAR having 4D5, 4-1BB costimulatory domain and CD3ζsignaling domain; 40-18.28: the LACO molecule having the anti-CD40 scFv 40-18 fused with the intracellular domain of CD28 (same for the other listed LACO molecules 40-37.28, 40-37.28, 40-37.28, 40-37.28, 40-37.28, 40-37.28) ; A40C28: the LACO molecule having anti-CD40 scFv A40C fused with the intracellular domain of CD28; NO EP: T cells without CAR.

Binding of the anti-CD40 scFv expressed on CARTs cells to CD40-Fc protein was measured by FACS staining. As shown in FIG. 2, C5, C7, C8, and C9 showed strong binding to CD40-Fc recombinant protein.

5.9.3 Example 3: Tumor cell lines and primary human lymphocytes

A549-ESO-CBG cell line was generated by using lentiviral transduction of A549 cells with Click beetle green (CBG) and EGFP, followed by lentiviral transduction of HLA-A2. Primary lymphocytes from normal donors were stimulated with anti-CD3/CD28 Dynabeads (Life Technologies) and cultured in R10 medium (RPMI-1640 supplemented with 10%FCS; Invitrogen) . T cells were cryopreserved at day 10 after stimulation in a solution of 90 %FCS and 10%DMSO at 1e8 cells/vial.

5.9.4 Example 4: Preparation and characterization of LACO-expressing CART cells

CART cells expressing LACO provided herein were prepared by electroporation with the following procedures: T cells were collected and washed with Opti-MEM medium for 3 times. The cell pellets were resuspended with Opti-MEM medium, and the cell concentration was adjusted to 5 ×10 ⁷/ml. Certain amount of RNA was aliquoted to 1.5 ml EP tube, added with 100 μl T cells (≥ 5×10 ⁶ cells) , and mixed gently to avoid bubbles. Electroporation was performed using BTX machine at the following parameters for T cells: 500 voltage, 0.7 ms, for one pulse. The cells were then transferred to pre-warmed culture medium and cultured at 37℃.

The cytotoxicity of the LACO-expressing CART cells against tumor cells was measured in in vitro cytotoxicity assay. A549-ESO-CGB cells were adjusted to 30,000/ml and seeded to flat-bottomed 96-well plate at 3000 cells/100 μl/well. CART cells were diluted to appropriate concentration, seeded at 100 μl/well with tumor cells at different E/T ratios, such as 10: 1, 3: 1, 1: 1, or 0.3: 1. Care was taken to avoid bubbles. The co-culture plates were placed into IncuCyte S3 machine, and scanning parameters were set. After 3 days of scanning, the Total Green Object Integrated Intensity (GCU x μm ²/well) was analyzed to calculate the killing efficiency.

As shown in FIG. 3, T cells C7, C9 or C11 showed significantly enhanced killing effect against tumor cells compared to T cells expressing Her2 CAR alone, confirming that co-expression of respective LACO molecules enhanced tumor killing effect of the CART cells.

5.9.5 Example 5: Specific activation of LACO-expressing CART cells by cancer cells

CD107a is an early phase-activating marker for T cells. Activation of CART cells by tumor cells was measured by CD107a staining with the following procedures: 20μl PE-CD107a mAb was added to each well of a 96-well plate; tumor cells were diluted to 2×10 ⁶/ml and seeded on 96-well round plates (100 μl/well) ; CART cells were diluted to 1×10 ⁶/ml and seeded in 96-well round plates (100 μl/well) ; the plates were centrifuged at 500 rpm×5 min to attach cells and cultured at 37℃ for 1 hour; Golgi stop was diluted by 1500× with medium and added to each well (20 μl/well) ; cells were cultured at 37℃ for another 2.5 hours, stained with anti-CD3-APC and anti-CD8-FITC antibodies at 37℃ for 30 min, washed and analyze by flow cytometry.

FIGs. 4A-4C show CD107a staining of CAR-T cells in the coculture and killing assay with A549 (FIG. 4A) , PC-3 (FIG. 4B) , and SK-OV3 (FIG. 4C) . As shown, higher percentages of T cells C7, C9 and C11 were activated by the coculture with tumor cells, confirming that co-expression of the respectively LACO molecules enhanced tumor-induced activation of CAR T cells.

5.10 The instant application disclosed the following specific embodiments:

Embodiment 1: An antibody or antigen-binding fragment thereof that specifically binds human CD40, comprising:

(a) a light chain variable region (VL) comprising

(1) a light chain CDR1 (VL CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6;

(2) a light chain CDR2 (VL CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12; and

(3) a light chain CDR3 (VL CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18;

or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or

(b) a heavy chain variable region (VH) comprising

(1) a heavy chain CDR1 (VH CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24;

(2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-30; and

(3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36;

or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VH CDRs.

Embodiment 2: The antibody or antigen-binding fragment of embodiment 1, wherein

(a) the VL CDR1, CDR2 and CDR3 have

(1) the amino acid sequences of SEQ ID NOs: 1, 7, and 13, respectively;

(2) the amino acid sequences of SEQ ID NOs: 2, 8, and 14, respectively;

(3) the amino acid sequences of SEQ ID NOs: 3, 9, and 15, respectively;

(4) the amino acid sequences of SEQ ID NOs: 4, 10, and 16, respectively;

(5) the amino acid sequences of SEQ ID NOs: 5, 11, and 17, respectively; or

(6) the amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively;

(b) the VH CDR1, CDR2 and CDR3 have

(1) the amino acid sequences of SEQ ID NOs: 19, 25, and 31, respectively;

(2) the amino acid sequences of SEQ ID NOs: 20, 26, and 32, respectively;

(3) the amino acid sequences of SEQ ID NOs: 21, 27, and 33, respectively;

(4) the amino acid sequences of SEQ ID NOs: 22, 28, and 34, respectively;

(5) the amino acid sequences of SEQ ID NOs: 23, 29, and 35, respectively; or

(6) the amino acid sequences of SEQ ID NOs: 24, 30, and 36, respectively;

Embodiment 3: The antibody or antigen-binding fragment of embodiment 1, wherein

(1) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 7, and 13, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 19, 25, and 31, respectively;

(2) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 8, and 14, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 20, 26, and 32, respectively;

(3) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 3, 9, and 15, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 21, 27, and 33, respectively;

(4) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 10, and 16, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 22, 28, and 34, respectively;

(5) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 5, 11, and 17, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 23, 29, and 35, respectively; or

(6) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 24, 30, and 36, respectively.

Embodiment 4: The antibody or antigen-binding fragment of embodiment 1, comprising a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3 having the amino acid sequences of SEQ ID NOs: 4, 10, 16, 22, 28, and 34, respectively.

Embodiment 5: The antibody or antigen-binding fragment of embodiment 1, comprising a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3 having the amino acid sequences of SEQ ID NOs: 6, 12, 18, 24, 30, and 36, respectively

Embodiment 6: An antibody or antigen-binding fragment thereof that specifically binds human CD40, comprising:

(a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and/or

(b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-48.

Embodiment 7: The antibody or antigen-binding fragment of embodiment 6 comprising a VL and a VH, wherein the VL and VH each have at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequences of (1) SEQ ID NOs: 37 and 43, respectively; (2) SEQ ID NOs: 38 and 44, respectively; (3) SEQ ID NOs: 39 and 45, respectively; (4) SEQ ID NOs: 40 and 46, respectively; (5) SEQ ID NOs: 41 and 47, respectively; or (6) SEQ ID NOs: 42 and 48, respectively.

Embodiment 8: The antibody or antigen-binding fragment of embodiment 6 comprising a VL and a VH, wherein the VL and VH each have at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequences of SEQ ID NOs: 40 and 46, respectively.

Embodiment 9: The antibody or antigen-binding fragment of embodiment 6 comprising a VL and a VH, wherein the VL and VH each have at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to the amino acid sequences of SEQ ID NOs: 42 and 48, respectively.

Embodiment 10: An antibody or antigen-binding fragment thereof that specifically binds human CD40, comprising

(a) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-42; and/or

(b) a VH comprising VH CDR1, CDR2, and CDR3 from a VH having an amino acid sequence selected from group consisting of SEQ ID NOs: 43-48.

Embodiment 11: The antibody or antigen-binding fragment thereof embodiment 10, comprising

(1) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 37, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 43;

(2) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 38, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 44;

(3) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 39, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 45;

(4) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 40, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 46;

(5) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 41, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 47; or

(6) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 42, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 48.

Embodiment 12: The antibody or antigen-binding fragment thereof embodiment 10 comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 40, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 46.

Embodiment 13: The antibody or antigen-binding fragment thereof embodiment 10 comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 42, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 48.

Embodiment 14: An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of embodiments 1 to 13 for binding to human CD40.

Embodiment 15: The antibody or antigen-binding fragment of any one of embodiments 1 to 14 that is a monoclonal antibody or antigen-binding fragment.

Embodiment 16: The antibody or antigen-binding fragment of any one of embodiments 1 to 15 that is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

Embodiment 17: The antibody or antigen-binding fragment of any one of embodiments 1 to 15 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .

Embodiment 18: The antibody or antigen-binding fragment of embodiment 17 that is a scFv.

Embodiment 19: The antibody or antigen-binding fragment of any one of embodiments 1 to 18 that is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

Embodiment 20: The antibody or antigen-binding fragment of embodiment 19 that is a human antibody or antigen-binding fragment.

Embodiment 21: The antibody or antigen-binding fragment of any one of embodiments 1 to 20 that is a bispecific antibody or a multispecific antibody.

Embodiment 22: A polynucleotide encoding the antibody or antigen-binding fragment of any one of embodiments 1 to 21.

Embodiment 23: A vector comprising the polynucleotide of embodiment 22.

Embodiment 24: A fusion protein comprising a first domain and a second domain, wherein (i) the first domain comprises the antibody or antigen-binding fragment of any one of embodiments 1 to 21; and (ii) the second domain activates an immune effector cell and comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

Embodiment 25: The fusion protein of embodiment 24, wherein the second domain comprises a cytoplasmic domain of the co-stimulatory receptor.

Embodiment 26: The fusion protein of embodiment 25, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.

Embodiment 27: The fusion protein of embodiment 25, wherein the co-stimulatory receptor is CD28.

Embodiment 28: The fusion protein of embodiment 25, wherein the co-stimulatory receptor is 4-1BB.

Embodiment 29: The fusion protein of any one of embodiments 25 to 28, wherein the second domain further comprises the transmembrane domain of the co-stimulatory receptor.

Embodiment 30: The fusion protein of embodiment 24, wherein the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof.

Embodiment 31: The fusion protein of embodiment 30, wherein the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.

Embodiment 32: The fusion protein of embodiment 24, wherein the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof.

Embodiment 33: The fusion protein of embodiment 32, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.

Embodiment 34: The fusion protein of embodiment 32, wherein the co-stimulatory receptor is CD28.

Embodiment 35: The fusion protein of embodiment 34, wherein the anti-CD28 antibody or an antigen-binding fragment thereof is a scFv having the amino acid sequence of SEQ ID NO: 161.

Embodiment 36: The fusion protein of any one of embodiments 24 to 35 wherein the N-terminus of the first domain is linked to the C-terminus of the second domain.

Embodiment 37: The fusion protein of any one of embodiments 24 to 35 wherein the N-terminus of the second domain is linked to the C-terminus of the first domain.

Embodiment 38: The fusion protein of any one of embodiments 24 to 37, wherein the first domain and the second domain are linked via a linker.

Embodiment 39: The fusion protein of embodiment 24 having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 67.

Embodiment 40: The fusion protein of embodiment 24 having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 68.

Embodiment 41: The fusion protein of embodiment 24 having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 69.

Embodiment 42: The fusion protein of embodiment 24 having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 70.

Embodiment 43: The fusion protein of embodiment 24 having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 71.

Embodiment 44: The fusion protein of embodiment 24 having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 72.

Embodiment 45: A polynucleotide that encodes the fusion protein of any one of embodiments 24 to 44.

Embodiment 46: A vector that comprises the polynucleotide of embodiment 45.

Embodiment 47: The vector of embodiment 46 that is a viral vector.

Embodiment 48: A genetically engineered immune effector cell that recombinantly expresses the fusion protein of any one of embodiments 24 to 44, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.

Embodiment 49: A genetically engineered immune effector cell comprising the polynucleotide of embodiment 45 or the vector of embodiment 46 or 47, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.

Embodiment 50: The cell of embodiment 48 that further recombinantly expresses a chimeric antigen receptor (CAR) , a T cell receptor (TCR) or a Bi-specific T-cell engager (BiTE) , wherein the CAR, TCR or BiTE binds a tumor antigen or a viral antigen.

Embodiment 51: The cell of embodiment 49, further comprising a polynucleotide that encodes a CAR, a TCR, or BiTE, wherein the CAR, TCR or BiTE binds a tumor antigen or a viral antigen.

Embodiment 52: The cell of embodiment 50 or 51, wherein the CAR, TCR or BiTE binds a viral antigen selected from the group consisting of HPV, EBV, and HIV.

Embodiment 53: The cell of embodiment 50 or 51, wherein the CAR, TCR or BiTE binds a tumor antigen selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin.

Embodiment 54: The cell of embodiment 53, wherein the CAR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-93 and 169.

Embodiment 55: The cell of embodiment 53, wherein the TCR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-101.

Embodiment 56: The cell of embodiment 53, wherein the BiTE has an amino acid sequence selected from the group consisting of SEQ ID NO: 102, 103, and 167.

Embodiment 57: The cell of any one of embodiments 48 to 56, that is derived from a cell isolated from peripheral blood or bone marrow.

Embodiment 58: The cell of any one of embodiments 48 to 56, that is derived from a cell differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.

Embodiment 59: The cell of any of embodiments 48 to 58 that is a T cell.

Embodiment 60: The T cell of embodiment 59 that is a cytotoxic T cell, a helper T cell, or a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, an effector memory TEMRA cell, or a gamma delta T cell.

Embodiment 61: A population of the genetically engineered immune effector cell of any one of embodiments 48 to 60 that are derived from cells isolated from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .

Embodiment 62: A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 21, and a pharmaceutically acceptable excipient

Embodiment 63: A pharmaceutical composition comprising the fusion protein of any one of embodiments 24 to 44, and a pharmaceutically acceptable excipient.

Embodiment 64: A pharmaceutical composition comprising the cell or population of cells of any one of embodiments 48 to 61, and a pharmaceutically acceptable excipient.

Embodiment 65: Use of the antibody or antigen-binding fragment of any one of embodiments 1 to 21 or the fusion protein of any one of embodiments 24 to 44 in cancer treatment.

Embodiment 66: Use of the antibody or antigen-binding fragment of any one of embodiments 1 to 21 or the fusion protein of any one of embodiments 24 to 44 for the preparation of a medicament for the treatment of cancer.

Embodiment 67: The use of embodiment 65 or 66, wherein the fusion protein is used in combination with an immune effector cell.

Embodiment 68: The use of embodiment 67, wherein the immune effector cell is selected from the group consisting of a CAR T cell, a TCRT cell, a TIL, a CIK, a LAK, and a MIL.

Embodiment 69: Use of the cell or population of cells of any one of embodiments 48 to 61 in cancer treatment.

Embodiment 70: Use of the cell or population of cells of any one of embodiments 48 to 61 for the preparation of a medicament for the treatment of cancer.

Embodiment 71: The use of any one of embodiments 65 to 70, wherein the antibody or antigen-binding fragment, the fusion protein, the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.

Embodiment 72: A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment of any one of embodiments 1 to 21 or the fusion protein of any one of embodiments 24 to 44 to the subject.

Embodiment 73: The method of embodiment 72, further comprising administering a cell therapy to the subject.

Embodiment 74: The method of embodiment 73, wherein the cell therapy is selected from the group consisting of a CAR T therapy, a TCRT therapy, a TIL therapy, a CIK therapy, a LAK therapy, and a MIL therapy.

Embodiment 75: A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the cell or population of cells of any one of embodiments 48 to 61 to the subject.

Embodiment 76: The method of any one of embodiments 72 to 75, further comprising administering an additional therapy to the subject.

Embodiment 77: The method of any one of embodiments 72 to 76, wherein the subject is a human.

Embodiment 78: The use or method of any one of embodiments 65 to 77, wherein the fusion protein, the cell, population of cells, or pharmaceutical composition reduces cancer-induced immunosuppression.

Embodiment 79: The use or method of any one of embodiments 65 to 78, wherein the cancer is a hematological cancer.

Embodiment 80: The use or method of any one of embodiments 65 to 78, wherein the cancer is a solid tumor.

Embodiment 81: A method of genetically engineering an immune effector cell comprising transferring the polynucleotide of embodiment 45 into the cell.

Embodiment 82: The method of embodiment 81, wherein the polynucleotide is transferred via electroporation.

Embodiment 83: The method of embodiment 81, wherein the polynucleotide is transferred via viral transduction.

Embodiment 84: The method of embodiment 81, wherein the polynucleotide is transferred using a transposon system.

Embodiment 85: The method of embodiment 81, wherein the polynucleotide is transferred using gene-editing.

Embodiment 86: The method of embodiment 85, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.

Embodiment 87: The method of any one of embodiments 81 to 86, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.

Claims

An antibody or antigen-binding fragment thereof that specifically binds human CD40, comprising:

(a) a light chain variable region (VL) comprising

(1) a light chain CDR1 (VL CDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 6;

(2) a light chain CDR2 (VL CDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 12; and

(3) a light chain CDR3 (VL CDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 18;

or a variant thereof having up to about 3 amino acid substitutions, additions, and/or

deletions in the VL CDRs; and/or

(b) a heavy chain variable region (VH) comprising

(1) a heavy chain CDR1 (VH CDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 24;

(2) a heavy chain CDR2 (VH CDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 30; and

(3) a heavy chain CDR3 (VH CDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 36;

or a variant thereof having up to about 3 amino acid substitutions, additions, and/or

deletions in the VH CDRs.
The antibody or antigen-binding fragment of claim 1, wherein

(a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or

(b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 24, 30, and 36, respectively; or a variant thereof having up to about 3 amino acid substitutions, additions, and/or deletions in the VH CDRs.
The antibody or antigen-binding fragment of claim 1, wherein the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 6, 12, and 18, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 24, 30, and 36, respectively.
An antibody or antigen-binding fragment thereof that specifically binds human CD40, comprising:

(a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence as set forth in SEQ ID NO: 42; and/or

(b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence as set forth in SEQ ID NO: 48.
An antibody or antigen-binding fragment thereof that specifically binds human CD40, comprising

(a) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having an amino acid sequence as set forth in SEQ ID NO: 42; and/or

(b) a VH comprising VH CDR1, CDR2, and CDR3 from a VH having an amino acid sequence s as set forth in SEQ ID NO: 48.
An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of claims 1 to 5 for binding to human CD40.
The antibody or antigen-binding fragment of any one of claims 1 to 6 that is a monoclonal antibody or antigen-binding fragment.
The antibody or antigen-binding fragment of any one of claims 1 to 7 that is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.
The antibody or antigen-binding fragment of any one of claims 1 to 7 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .
The antibody or antigen-binding fragment of claim 9 that is a scFv.
The antibody or antigen-binding fragment of any one of claims 1 to 10 that is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.
The antibody or antigen-binding fragment of claim 11 that is a human antibody or antigen-binding fragment.
The antibody or antigen-binding fragment of any one of claims 1 to 12 that is a bispecific antibody or a multispecific antibody.
A polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1 to 13.
A vector comprising the polynucleotide of claim 14.
A fusion protein comprising a first domain and a second domain, wherein (i) the first domain comprises the antibody or antigen-binding fragment of any one of claims 1 to 15; and (ii) the second domain activates an immune effector cell and comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.
The fusion protein of claim 16 wherein the second domain comprises a cytoplasmic domain of the co-stimulatory receptor.
The fusion protein of claim 17, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The fusion protein of claim 17, wherein the co-stimulatory receptor is CD28.
The fusion protein of claim 17, wherein the co-stimulatory receptor is 4-1BB.
The fusion protein of any one of claims 17 to 20, wherein the second domain further comprises the transmembrane domain of the co-stimulatory receptor.
The fusion protein of claim 16, wherein the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof.
The fusion protein of claim 22, wherein the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.
The fusion protein of claim 16, wherein the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof.
The fusion protein of claim 24, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The fusion protein of claim 24, wherein the co-stimulatory receptor is CD28.
The fusion protein of claim 26, wherein the anti-CD28 antibody or an antigen-binding fragment thereof is a scFv having the amino acid sequence of SEQ ID NO: 161.
The fusion protein of any one of claims 16 to 27 wherein the N-terminus of the first domain is linked to the C-terminus of the second domain.
The fusion protein of any one of claims 16 to 27 wherein the N-terminus of the second domain is linked to the C-terminus of the first domain.
The fusion protein of any one of claims 16 to 29, wherein the first domain and the second domain are linked via a linker.
The fusion protein of claim 16 having an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 72.
A polynucleotide that encodes the fusion protein of any one of claims 16 to 31.
A vector that comprises the polynucleotide of claim 32.
The vector of claim 33 that is a viral vector.
A genetically engineered immune effector cell that recombinantly expresses the fusion protein of any one of claims 16 to 31, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.
A genetically engineered immune effector cell comprising the polynucleotide of claim 32 or the vector of claim 33 or 34, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.
The cell of claim 35 that further recombinantly expresses a chimeric antigen receptor (CAR) , a T cell receptor (TCR) or a Bi-specific T-cell engager (BiTE) , wherein the CAR, TCR or BiTE binds a tumor antigen or a viral antigen.
The cell of claim 36, further comprising a polynucleotide that encodes a CAR, a TCR, or BiTE, wherein the CAR, TCR or BiTE binds a tumor antigen or a viral antigen.
The cell of claim 37 or 38, wherein the CAR, TCR or BiTE binds a viral antigen selected from the group consisting of HPV, EBV, and HIV.
The cell of claim 37 or 38, wherein the CAR, TCR or BiTE binds a tumor antigen selected from the group consisting of Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA, and Mesothelin.
The cell of claim 40, wherein the CAR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 79-93 and 169.
The cell of claim 40, wherein the TCR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-101.
The cell of claim 40, wherein the BiTE has an amino acid sequence selected from the group consisting of SEQ ID NO: 102, 103, and 167.
The cell of any one of claims 35 to 43, that is derived from a cell isolated from peripheral blood or bone marrow.
The cell of any one of claims 35 to 43, that is derived from a cell differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.
The cell of any of claims 35 to 45 that is a T cell.
The T cell of claim 46 that is a cytotoxic T cell, a helper T cell, or a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, an effector memory TEMRA cell, or a gamma delta T cell.
A population of the genetically engineered immune effector cell of any one of claims 35 to 47 that are derived from cells isolated from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .
A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 13, and a pharmaceutically acceptable excipient
A pharmaceutical composition comprising the fusion protein of any one of claims 16 to 31, and a pharmaceutically acceptable excipient.
A pharmaceutical composition comprising the cell or population of cells of any one of claims 35 to 48, and a pharmaceutically acceptable excipient.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 13 or the fusion protein of any one of claims 16 to 31 in cancer treatment.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 13 or the fusion protein of any one of claims 16 to 31 for the preparation of a medicament for the treatment of cancer.
The use of claim 52 or 53, wherein the fusion protein is used in combination with an immune effector cell.
The use of claim 54, wherein the immune effector cell is selected from the group consisting of a CAR T cell, a TCRT cell, a TIL, a CIK, a LAK, and a MIL.
Use of the cell or population of cells of any one of claims 35 to 48 in cancer treatment.
Use of the cell or population of cells of any one of claims 35 to 48 for the preparation of a medicament for the treatment of cancer.
The use of any one of claims 52 to 57, wherein the antibody or antigen-binding fragment, the fusion protein, the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.
A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 13 or the fusion protein of any one of claims 16 to 31 to the subject.
The method of claim 59, further comprising administering a cell therapy to the subject.
The method of claim 60, wherein the cell therapy is selected from the group consisting of a CAR T therapy, a TCRT therapy, a TIL therapy, a CIK therapy, a LAK therapy, and a MIL therapy.
A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the cell or population of cells of any one of claims 35 to 48 to the subject.
The method of any one of claims 59 to 62, further comprising administering an additional therapy to the subject.
The method of any one of claims 59 to 63, wherein the subject is a human.
The use or method of any one of claims 52 to 64, wherein the fusion protein, the cell, population of cells, or pharmaceutical composition reduces cancer-induced immunosuppression.
The use or method of any one of claims 52 to 65, wherein the cancer is a hematological cancer.
The use or method of any one of claims 52 to 66, wherein the cancer is a solid tumor.
A method of genetically engineering an immune effector cell comprising transferring the polynucleotide of claim 32 into the cell.
The method of claim 68, wherein the polynucleotide is transferred via electroporation.
The method of claim 68, wherein the polynucleotide is transferred via viral transduction.
The method of claim 68, wherein the polynucleotide is transferred using a transposon system.
The method of claim 68, wherein the polynucleotide is transferred using gene-editing.
The method of claim 72, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.
The method of any one of claims 68 to 73, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.