CD20-binding single domain antibody

Abstract

This application relates, in part, to agents that bind to CD20 and their use as therapeutic agents. This application further relates to pharmaceutical compositions comprising the CD20-binding agents and their use in treating various diseases. [Selection diagram] No diagram

Description

[Technical field]

[0001] The present invention relates, in part, to binding agents (for example, but not limited to, antibodies such as VHHs) that bind to CD20 and their use as therapeutic agents. [Background technology]

[0002] CD20 is a transmembrane protein expressed on the surface of B lymphocytes. It is expressed during B lymphocyte development from early pre-B stage to terminal differentiation into plasma cells, a stage at which CD20 expression disappears. CD20 is also expressed on malignant B cells. In particular, CD20 is expressed on more than 90% of B cell non-Hodgkin's lymphoma (NHL) cells and more than 95% of B type chronic lymphocytic leukemia (B-CLL) cells.

[0003] Antibodies directed against CD20 are used to treat B-cell derived leukemias and lymphomas. Specifically, rituximab (Rituxan) is a genetically engineered chimeric mouse / human monoclonal antibody directed against CD20. Rituximab is currently approved for the treatment of relapsed or refractory follicular lymphoma. Weekly infusions of rituximab have been reported to result in an overall response rate of 48%. However, many patients do not respond to rituximab treatment. Furthermore, patients who respond often develop resistance to rituximab and eventually relapse. In addition, rituximab is believed to nonspecifically kill normal CD20-positive cells, which results in significant toxicity and side effects.

[0004] Thus, there is a need for therapeutically effective CD20 binding agents that can bind to CD20-expressing malignant cells with high specificity while minimizing side effects. Summary of the Invention

[0005] In various aspects, the present invention relates to CD20 binding agents having at least one targeting moiety that specifically binds to CD20. In one embodiment, the targeting moiety is a single domain antibody (VHH or nanobody). In some embodiments, these CD20 binding agents bind to CD20 but do not functionally modulate (e.g., partially or completely neutralize) CD20. In some embodiments, the CD20 binding agents bind to CD20-positive cells, resulting in the death of the CD20-positive cells.

[0006] In various embodiments, the CD20 binding agent further comprises a signaling agent that can be modified, such as, but not limited to, interferon, interleukin, and tumor necrosis factor. In various embodiments, the CD20 binding agent comprises an additional targeting moiety that binds to another target of interest (e.g., antigen, receptor). In one embodiment, the other target of interest (e.g., antigen, receptor) is present on a tumor cell. In another embodiment, the other target of interest (e.g., antigen, receptor) is present on an immune cell.

[0007] In various embodiments, the CD20 binding agents of the present invention find use in treating a variety of diseases or disorders involving cells expressing CD20. In some embodiments, such diseases or disorders include cancer, infectious diseases, immune disorders, and other diseases and disorders. In various embodiments, the present invention encompasses a variety of methods of treatment. [Brief description of the drawings]

[0008] [Figure 1]Figure 1 shows the results of a cell ELISA assay using periplasmic extracts of positive colonies. For each set of histograms, the first bar is CHO-K1-hCD20 and the second bar is CHO-K1. A clone is considered specific if it gives a signal in transfected cells at least 2-fold higher than the signal obtained in the negative control parental cells. Furthermore, the relative intensity of the signal may not reflect the relative quality of the nanobodies, since in these experiments crude periplasmic extracts were used and differences in the ELISA signal may be related to factors such as the amount of VHH used, rather than to quality such as affinity, actual yield, etc. [Diagram 2] Figure 1 shows the nucleotide sequences of 17 different VHHs specific for human CD20. The VHHs are: 2HCD16, 2HCD17, 2HCD22, 2HCD25, 2HCD35, 2HCD40, 2HCD42, 2HCD43, 2HCD59, 2HCD68, 2HCD73, 2HCD78, 2HCD81, 2HCD88, R3CD7, R3CD18, R3CD105. Gaps were introduced to align the sequences. [Diagram 3] Figure 1 shows the amino acid sequences of 17 different VHHs specific for human CD20. The VHHs are 2HCD16, 2HCD17, 2HCD22, 2HCD25, 2HCD35, 2HCD40, 2HCD42, 2HCD43, 2HCD59, 2HCD68, 2HCD73, 2HCD78, 2HCD81, 2HCD88, R3CD7, R3CD18, R3CD105. Complementarity determining regions (CDR1, CDR2 and CDR3) are underlined and defined according to Kabat. The peptide AAA sequence is a linker connecting the VHH sequence to the HA tag (shown in bold) and the His6 tag (carboxy terminus). Gaps were introduced to align the sequences. [Figure 4]Figure 1 shows the results of a cell ELISA assay using periplasmic extracts of positive colonies. For each set of histograms, the first bar is CHO-K1-mCD20 and the second bar is CHO-K1. A clone is considered specific if it gives a signal in the transfected cells that is at least 2-fold higher than the signal obtained in the negative control parental cells. Furthermore, the relative intensity of the signal may not reflect the relative quality of the VHHs, since in these experiments crude periplasmic extracts were used and differences in the ELISA signal may be related to factors such as the amount of VHH used, rather than to quality such as affinity, actual yield, etc. The nanobodies used in this figure correspond to Camelidae VHHs. [Diagram 5] Figure 1 shows the results of a cell ELISA assay using periplasmic extracts of positive colonies screened with biotinylated mouse CD20 peptide. For each set of histograms, the first bar is CHO-K1-mCD20 and the second bar is CHO-K1. A clone is considered specific if it gives a signal in transfected cells at least 2-fold higher than the signal obtained in the negative control parental cells. Furthermore, the relative intensity of the signal may not reflect the relative quality of the VHHs, since in these experiments crude periplasmic extracts were used and differences in the ELISA signal may be related to factors such as the amount of VHH used, rather than to quality such as affinity, actual yield, etc. The nanobodies used in this figure correspond to Camelidae VHHs. [Figure 6] Figure 1 shows the nucleotide sequences of 21 different VHHs specific for mouse CD20. The VHHs are: R2MUC21, R2MUC36, R2MUC70, R2MUC85, R3MUC17, R3MUC22, R3MUC56, R3MUC57, R3MUC58, R3MUC66, R3MUC75, 2MC20, 2MC38, 2MC39, 2MC42, 2MC51, 2MC52, 2MC57, 2MC82, R3MCD22, R3MCD137. Gaps were introduced to align the sequences. [Figure 7]Figure 1 shows the amino acid sequences of 21 different VHHs specific for mouse CD20. The VHHs are R2MUC21, R2MUC36, R2MUC70, R2MUC85, R3MUC17, R3MUC22, R3MUC56, R3MUC57, R3MUC58, R3MUC66, R3MUC75, 2MC20, 2MC38, 2MC39, 2MC42, 2MC51, 2MC52, 2MC57, 2MC82, R3MCD22, R3MCD137. The complementarity determining regions (CDR1, CDR2 and CDR3) are underlined and defined according to Kabat. The peptide AAA sequence is a linker connecting the VHH sequences to the HA tag (shown in bold) and the His6 tag (carboxy terminus). Gaps were introduced to align the sequences. [Figure 8] Figure 2 shows binding data for selected anti-human CD20 VHHs. The left peak represents the untransfected parental CHO-K1. The middle peak represents mouse CD20-expressing CHO-K1 and the right peak represents human CD20-expressing CHO-K1. [Figure 9] Figure 2 shows the binding data of selected anti-mouse CD20 VHHs. The left peak represents the parental untransfected CHO-K1. The middle peak represents human CD20 expressing CHO-K1 and the right peak represents mouse CD20 expressing CHO-K1. [Figure 10] Figure 1 shows the in vivo antitumor potential of chimeric CD20 VHHs in the A20 lymphoma model. hCD20Nb refers to the 2HCD25 VHH against human CD20, and mCD20Nb refers to the 2MC57 VHH against mouse CD20. mIFN refers to wild type mouse IFN. AcTAferon refers to mutant Q124R interferon. Nb is the abbreviation for Nanobody, which is used herein as the equivalent of VHH. [Figure 11]Figure 1 shows the in vivo antitumor potential of chimeric CD20 VHHs in a B16 melanoma model. hCD20Nb refers to the 2HCD25 VHH against human CD20, and mCD20Nb refers to the 2MC57 VHH against mouse CD20. mIFN refers to wild type mouse IFN. AcTAferon refers to mutant Q124R interferon. Nb is the abbreviation for nanobody, which is used herein as the equivalent of VHH. [Figure 12] FIG. 12 shows that the antitumor effect of chimeric CD20 VHH in the B16 melanoma model shown in FIG. 11 does not simultaneously cause life-threatening side effects. Nb is the abbreviation for nanobody and is used herein as the equivalent of VHH. [Figure 13] FIG. 1 shows that chimeric CD20 VHH induce partial B cell depletion. [Figure 13-2] FIG. 1 shows that chimeric CD20 VHH induce partial B cell depletion. [Figure 14] Figure 1 shows the antitumor potential of chimeric CD20 VHH in combination with doxorubicin (dox). Nb is the abbreviation for nanobody, used herein as equivalent to VHH. [Figure 15] FIG. 15 shows that the antitumor potential of chimeric CD20 VHH in combination with doxorubicin as shown in FIG. 14 does not simultaneously cause life-threatening side effects. [Figure 15-2] FIG. 15 shows that the antitumor potential of chimeric CD20 VHH in combination with doxorubicin as shown in FIG. 14 does not simultaneously cause life-threatening side effects. [Figure 15-3] FIG. 15 shows that the antitumor potential of chimeric CD20 VHH in combination with doxorubicin as shown in FIG. 14 does not simultaneously cause life-threatening side effects. [Figure 16] Figure 1 shows the antitumor potential of chimeric CD20 VHH in combination with tumor necrosis factor.Nb is the abbreviation for nanobody, used herein as equivalent to VHH. [Figure 17]Figure 17 shows that the antitumor potential of chimeric CD20 VHH in combination with tumor necrosis factor as shown in Figure 16 does not simultaneously cause life-threatening side effects. Nb is the abbreviation for nanobody and is used herein as the equivalent of VHH. [Figure 17-2] Figure 17 shows that the antitumor potential of chimeric CD20 VHH in combination with tumor necrosis factor as shown in Figure 16 does not simultaneously cause life-threatening side effects. Nb is the abbreviation for nanobody and is used herein as the equivalent of VHH. [Figure 17-3] Figure 17 shows that the antitumor potential of chimeric CD20 VHH in combination with tumor necrosis factor as shown in Figure 16 does not simultaneously cause life-threatening side effects. Nb is the abbreviation for nanobody and is used herein as the equivalent of VHH. [Figure 18] Figure 1 shows the anti-tumour potential of chimeric CD20 VHH in combination with anti-PD-L1 camelid VHH. Nb is the abbreviation for nanobody and is used herein as equivalent to VHH. [Figure 19] FIG. 14. Antitumour potential of chimeric CD20 VHH in combination with anti-PD-L1 camelid VHH and Treg cell depletion. [Figure 20] Figure 1 shows the antitumor potential of systemically administered chimeric CD20 VHH in a B16 melanoma model. Nb is the abbreviation for nanobody, used herein as equivalent to VHH. [Figure 21] FIG. 1 shows that the anti-tumor effect of chimeric CD20 VHH fused to mutant interferon (i.e., hCD20-AcTa) does not require B cell depletion. [Figure 22] FIG. 1 shows that the anti-tumor effect of chimeric CD20 VHH fused to mutant interferon (i.e., hCD20-AcTa) does not require B cell depletion. [Diagram 23] FIG. 1 shows the efficacy of chimeric CD20 VHHs using an experimental autoimmune encephalomyelitis (EAE) model. [Figure 23-2]FIG. 1 shows the efficacy of chimeric CD20 VHHs using an experimental autoimmune encephalomyelitis (EAE) model. [Figure 24] Figure 16 shows the anti-tumor effect of chimeric CD20 VHH fused to IFNα2 in combination with anti-PD-L1 camelid VHH. Experiments were performed as depicted in Example 4. Panel A complements the data shown in Figure 18 by demonstrating that combination therapy with chimeric CD20 VHH (i.e., 2MC57 VHH fused to AcTaferon: mutant IFNα2-Q124R) and anti-PD-L1 camelid VHH exhibited potent anti-tumor activity with no tumor recurrence by day 38 (or day 23 post-dosing). Panel B shows that the combination therapy was well tolerated with no significant weight loss compared to treatment with wild-type IFN. Nb is an abbreviation for nanobody and is used herein as equivalent to VHH. [Figure 24-2] Figure 16 shows the anti-tumor effect of chimeric CD20 VHH fused to IFNα2 in combination with anti-PD-L1 camelid VHH. Experiments were performed as depicted in Example 4. Panel A complements the data shown in Figure 18 by demonstrating that combination therapy with chimeric CD20 VHH (i.e., 2MC57 VHH fused to AcTaferon: mutant IFNα2-Q124R) and anti-PD-L1 camelid VHH exhibited potent anti-tumor activity with no tumor recurrence by day 38 (or day 23 post-dosing). Panel B shows that the combination therapy was well tolerated with no significant weight loss compared to treatment with wild-type IFN. Nb is an abbreviation for nanobody and is used herein as equivalent to VHH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present application is based, in part, on the discovery of agents that recognize and bind to CD20 (e.g., antibodies, such as VHHs or nanobodies, as non-limiting examples). In various embodiments, these CD20 binding agents bind to CD20 but do not functionally regulate CD20. In various embodiments, these CD20 binding agents can bind and directly or indirectly recruit immune cells to sites requiring therapeutic action (e.g., tumors or tumor microenvironment). In various embodiments, the CD20 binding agents bind to CD20 positive cells, resulting in the death of such cells. The present application provides pharmaceutical compositions comprising the CD20 binding agents and their use in treating various diseases. In various embodiments, the CD20 binding agents are part of a chimeric protein with a modified signaling agent as described herein.

[0010] CD20-binding agents In various embodiments, the CD20 binding agent of the present invention is a protein-based agent that can specifically bind to CD20. In various embodiments, the CD20 binding agent of the present invention is a protein-based agent that can specifically bind to CD20 without neutralizing CD20. CD20 is a non-glycosylated member of the transmembrane 4-A (MS4A) family. It functions as a B cell-specific differentiation antigen in both mice and humans (Stashenko et al., 1980; Oettgen et al., 1983; Ishibashi et al., 2001). In particular, human CD20 cDNA encodes a transmembrane protein that consists of four hydrophobic transmembrane domains, two extracellular loops and intracellular N- and C-terminal regions (Einfield et al., 1988).

[0011] In various embodiments, the CD20 binding agent of the present application comprises a targeting moiety having an antigen recognition domain that recognizes an epitope present on CD20. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on CD20. As used herein, a linear epitope refers to any continuous amino acid sequence present on CD20. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on CD20. As used herein, a conformational epitope refers to one or more sections of amino acids (which may be discontinuous) that form a three-dimensional surface having characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0012] In various embodiments, the CD20 binding agents of the present application may bind to full length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other naturally occurring or synthetic analogs, variants, or mutants of CD20 (e.g., human CD20). In various embodiments, the CD20 binding agents of the present application may bind to any form of CD20 (e.g., human CD20), such as monomers, dimers, trimers, tetramers, heterodimers, multimers, and associated forms. In one embodiment, the CD20 binding agent binds to the monomeric form of CD20. In another embodiment, the CD20 binding agent binds to the dimeric form of CD20. In another embodiment, the CD20 binding agent binds to the tetrameric form of CD20. In a further embodiment, the CD20 binding agent binds to the phosphorylated form of CD20, which may be either monomeric, dimeric, or trimeric.

[0013] In one embodiment, a CD20 binding agent of the invention comprises a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human CD20. In one embodiment, human CD20 comprises the following amino acid sequence: MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKM ESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP.

[0014] In various embodiments, the CD20 binding agent of the present invention comprises a targeting moiety capable of specific binding. In various embodiments, the CD20 binding agent comprises a targeting moiety having an antigen recognition domain, such as an antibody or a derivative thereof. In one embodiment, the CD20 binding agent comprises a targeting moiety that is an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises one variable region (V L ) and one constant region (C L ). The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs, designated CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0015] In some embodiments, the CD20 binding agents comprise a targeting moiety that is an antibody derivative or format. In some embodiments, the CD20 binding agents of the present invention comprise a targeting moiety, the targeting moiety being a single domain antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a shark heavy chain only antibody (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin; a tetranectin; an affibody; an affimer, a transbody; anticalin; an adnectin; an affilin; a microbody; a peptide aptamer; an alterase, a plastic antibody; a phylomer (p hylomer); stradobody; maxibody; evibody; fynomer, armadillo repeat protein, Kunitz-type domain, avimer, atrimer, probody, immune antibody, triomab, troybody; pepbody; vaccibody, UniBody; DuoBody, Fv, Fab, Fab', F(ab')2, peptidomimetic molecule, or synthetic molecule. These include U.S. patents or U.S. patent publications nos. 7,417,130, 2004 / 132094, 5,831,012, 2004 / 023334, 7,250,297, 6,818,418, 2004 / 209243, 7,838,629, 7,186,524, 6,004,746, 5,475, 096, 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446, and / or 7,166,697, the contents of which are incorporated herein by reference in their entireties. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0016] In some embodiments, the CD20 binding agent comprises a targeting moiety that is a single domain antibody, such as a VHH. The VHH can be derived from organisms that produce VHH antibodies, such as camelids, sharks, or the VHH can be a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy chain antibodies are composed of a single variable domain (VHH). H VHHs comprise a constant domain (CH) and two constant domains (CH2 and CH3). VHHs are commercially available under the trademark NANOBODIES. In one embodiment, the CD20 binding agent comprises a nanobody. In some embodiments, the single domain antibodies described herein are immunoglobulin single variable domains (ISVDs).

[0017] In some embodiments, the CD20 binding agent comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework (FR)" regions and three "complementarity determining regions (CDRs)". As used herein, "framework region" or "FR" refers to the region in the variable domain located between the CDRs. As used herein, "complementarity determining region" or "CDR" refers to the variable region in the VHH that comprises an amino acid sequence capable of specifically binding to an antigenic target.

[0018] In various embodiments, the CD20 binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence.

[0019] In some embodiments, the CDR1 sequence is selected from the following: GRTFSRQSMG (SEQ ID NO:35); GRTFSGQSMG (SEQ ID NO:36); GRTFSSYAMG (SEQ ID NO:37); GRTFSSYNMG (SEQ ID NO:38); GRTFSNYNMG (SEQ ID NO:39); GRTFSNSNMG (SEQ ID NO:40); GRSFSSVNMG (SEQ ID NO:41); GRTFSMG (SEQ ID NO:42); GRTVGSYSMG (SEQ ID NO:43); RFTLDYYAIG (SEQ ID NO:44); GFTLDYYAIG (SEQ ID NO: 45); GRDFATYSMA (SEQ ID NO: 106); GRDFATYSMT(SEQ ID NO:107); GRDFSTYSMG (SEQ ID NO: 108); GRTFNTYSMG (SEQ ID NO: 109); GRTFSTYSMG (SEQ ID NO: 110); GRDFSTYSMG (SEQ ID NO: 111); GNTFDTRAMG (SEQ ID NO: 112); GRTRDANAMG (SEQ ID NO: 113); or GSTFSIKAMG (sequence number 114).

[0020] In some embodiments, the CDR2 sequence is selected from the following: VITWSGGSPYYADSVRG (SEQ ID NO:46); VITWSGGSPYYADSVKG (SEQ ID NO:47); VISWSGGSPYYADSVKG (SEQ ID NO:48); AIDWSGGSPYYAASVRG(SEQ ID NO:49); VIDWSGGSPYYTDSVRG(SEQ ID NO:50); AITYSGGSPYYASSVRG (SEQ ID NO:51); AVIWSGASPYYADSVKG (SEQ ID NO:52); AVTWSGASPYYADSVKG (SEQ ID NO:53); AVTRSGASPYYADSVKG (SEQ ID NO:54); CISSSGGSTNYADSVKG (SEQ ID NO:55); TISWSGQRTRYADSVKG (SEQ ID NO: 115); SISWSGQRSRYADSVKG(SEQ ID NO:116); IISWSGQRTRYADSVKG (SEQ ID NO:117); AISRSSFNTYYSDSVTG (SEQ ID NO: 118); AISWSGSRTYYADSVKG (SEQ ID NO: 119); AFISGRGSTKYADSVKG (SEQ ID NO: 120); or GFISGRGSAKYADSVKG (sequence number 121).

[0021] In some embodiments, the CDR3 sequence is selected from the following: PVSYGSQWLADY (SEQ ID NO:56); PVSYGSSWLADY (SEQ ID NO:57); PLSYGSTWLADY (SEQ ID NO:58); GVSFGSRWLSDY (SEQ ID NO:59); GVSYGSRWLGDY (SEQ ID NO:60); NPTYGSDWNAEN (SEQ ID NO:61); NPTYSGGWHAEY (SEQ ID NO:62); ERTWVSNYYCSGDGDGYDYD (SEQ ID NO:63); PRTWGEFPPTQYDS (SEQ ID NO: 122); GKYGMKWRDGADY (SEQ ID NO: 123); DRSIEVQIADYDY (SEQ ID NO: 124); VLPTGGGSAMDY (SEQ ID NO: 125); or VLTTGGGSAMDY (sequence number 126).

[0022] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:35, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0023] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:35, a CDR2 comprising the amino acid sequence of SEQ ID NO:47, and a CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0024] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0025] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:37, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:56.

[0026] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:38, a CDR2 comprising the amino acid sequence of SEQ ID NO:48, and a CDR3 comprising the amino acid sequence of SEQ ID NO:57.

[0027] In various embodiments, the CD20-binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:39, a CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:58.

[0028] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:40, a CDR2 comprising the amino acid sequence of SEQ ID NO:50, and a CDR3 comprising the amino acid sequence of SEQ ID NO:59.

[0029] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:41, a CDR2 comprising the amino acid sequence of SEQ ID NO:50, and a CDR3 comprising the amino acid sequence of SEQ ID NO:60.

[0030] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:42, a CDR2 comprising the amino acid sequence of SEQ ID NO:51, and a CDR3 comprising the amino acid sequence of SEQ ID NO:61.

[0031] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:43, a CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR3 comprising the amino acid sequence of SEQ ID NO:62.

[0032] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:43, a CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a CDR3 comprising the amino acid sequence of SEQ ID NO:62.

[0033] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:43, a CDR2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR3 comprising the amino acid sequence of SEQ ID NO:62.

[0034] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:44, a CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a CDR3 comprising the amino acid sequence of SEQ ID NO:62.

[0035] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:45, a CDR2 comprising the amino acid sequence of SEQ ID NO:55, and a CDR3 comprising the amino acid sequence of SEQ ID NO:63.

[0036] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:106, a CDR2 comprising the amino acid sequence of SEQ ID NO:115, and a CDR3 comprising the amino acid sequence of SEQ ID NO:122.

[0037] In various embodiments, the CD20-binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:107, a CDR2 comprising the amino acid sequence of SEQ ID NO:115, and a CDR3 comprising the amino acid sequence of SEQ ID NO:122.

[0038] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:108, a CDR2 comprising the amino acid sequence of SEQ ID NO:115, and a CDR3 comprising the amino acid sequence of SEQ ID NO:122.

[0039] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:109, a CDR2 comprising the amino acid sequence of SEQ ID NO:116, and a CDR3 comprising the amino acid sequence of SEQ ID NO:122.

[0040] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:110, a CDR2 comprising the amino acid sequence of SEQ ID NO:117, and a CDR3 comprising the amino acid sequence of SEQ ID NO:122.

[0041] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:111, a CDR2 comprising the amino acid sequence of SEQ ID NO:115, and a CDR3 comprising the amino acid sequence of SEQ ID NO:122.

[0042] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:112, a CDR2 comprising the amino acid sequence of SEQ ID NO:118, and a CDR3 comprising the amino acid sequence of SEQ ID NO:123.

[0043] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:113, a CDR2 comprising the amino acid sequence of SEQ ID NO:119, and a CDR3 comprising the amino acid sequence of SEQ ID NO:124.

[0044] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:114, a CDR2 comprising the amino acid sequence of SEQ ID NO:120, and a CDR3 comprising the amino acid sequence of SEQ ID NO:125.

[0045] In various embodiments, the CD20 binding agent comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:114, a CDR2 comprising the amino acid sequence of SEQ ID NO:121, and a CDR3 comprising the amino acid sequence of SEQ ID NO:126.

[0046] In various embodiments, the CD20-binding agent comprises an amino acid sequence selected from the following sequences: 2HCD16: QVQLQESGGGLVQAGGSLRLSCAASGRTFSRQSMGWFRQAPGKEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 18) 2HCD22: QVQLQESGGGLVQAGDSLRLSCAASGRTFSRQSMGWFRQAPGKEREFVAVITWSGGSPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 19) 2HCD35: QVQLQESGGGLVQAGGSLRLSCAASGRTFSGQSMGWFRQAPGKEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 20) 2HCD42: QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 21) 2HCD73: QVQLQESGGGLVQAGGSLRLSCAASGRTFSRQSMGWFRQAPGEEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 22) 2HCD81: QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYNMGWFRQAPGKEREFVAVISWSGGSPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSSWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 23) R3CD105: QVQLQESGGGLVQAGGSLRLSCAASGRTFSNYNMGWFRQAPGKEREFVAAIDWSGGSPYYAASVRGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAAPLSYGSTWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 24) R3CD18: QVQLQESGGGLVQAGGSLRLSCAASGRTFSNSNMGWFRQAPGKEREFVAVIDWSGGSPYYTDSVRGRFTISRDNAKNTVYLQMNRLKPEDTAVYYCAGGVSFGSRWLSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 25) R3CD7: QVQLQESGGGLVQAGGSLRLSCAASGRSFSSVNMGWFRQAPGKEREFVAVIDWSGGSPYYTDSVRGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGVSYGSRWLGDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 26) 2HCD25: QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 27) 2HCD78: QVQLQESGGGLVQPGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 28) 2HCD17: QVQLQESGGGLVQAGGSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVIWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 29) 2HCD40: QVQLQESGGGLVQAGGSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVTWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 30) 2HCD88: QVQLQESGGGLVQAGDSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVTWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 31) 2HCD59: QVQLQESGGGSEQPGGSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVTRSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 32) 2HCD68: QVQLQESGGGLVQPGGSLRLSCAASRFTLDYYAIGWFRQAPGKEREFVAAVTWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 33) 2HCD43: QVQLQESGGGLVQPGGSLRLSCTASGFTLDYYAIGWLRQAPGKEREGVSCISSSGGSTNYADSVKGRFTISRDNAKNTVYLLMNSLKPEDTAVYYCAAERTWVSNYYCSGDGDGYDYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 34) 2MC57: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAMPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 85) R2MUC70: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 86) R3MUC17: QVQLQESGGGLVPAGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 87) R3MUC56: QVQLQESGGGLVQAGDSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 88) R3MUC57: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMAWFRQAPGEERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 89) R3MUC58: QVQLQESGGGLVQPGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 90) R2MUC85: QVQLQESGGGLVQPGGSLRLSCATSGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 91) R3MUC66: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMTWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 92) R2MUC21: QVQLQESGGGLVQAGDSLRLSCAASGRDFSTYSMGWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 93) 2MC52: QVQLQESGGGLVQAGGSLRLSCVASGRTFNTYSMGWFRQAPGKEREFVASISWSGQRSRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (SEQ ID NO: 94) R3MUC22: QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYSMGWFRQAPGKEREFVAIISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 95). R3MUC75: QVQLQESGGGSVQTGGSLRLSCAASGRDFSTYSMGWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 96). 2MC39: QVQLQESGGGLAQAGNSLRISCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 97). 2MC51: QVQLQESGGGLAQAGNSLRISCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 98). 2MC38: QVQLQESGGGLVQAGESLRISCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 99). 2MC82: QVQLQESGGGLAQEGGSLRLSCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 100). 2MC20: QVQLQESGGGLVQTGGSLRLSCAASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 101). 2MC42: QVQLQESGGGSVQTGGTLTLSCVASGNTFDTRAMGWFRQAPGEEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 102). R2MUC36: QVQLQESGGGLVQAEGSLRLSCAASGRTRDANAMGWFRQAPGKERELVAAISWSGSRTYYADSVKGRFTISRDNVMHTVYLSMNSLKPEDTAVYYCAADRSIEVQIADYDYWGRGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 103). R3MCD137: QVQLQESGGGSVQAGGSLRLSCAASGSTFSIKAMGWYRQAPGKQRELVAAFISGRGSTKYADSVKGRFAISRDNAKNTMYLQMDSLEPEDTAVYYCYIVLPTGGGSAMDYWGEGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 104). R3MCD22: QVQLQESGGGVVQAGGSLRLSCAASGSTFSIKAMGWYRQAPGKQRDLVAGFISGRGSAKYADSVKGRFAISRDNAKNTMYLQMDSLKPEDTAVYYCYIVLTTGGGSAMDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHH (sequence number 105).

[0047] In various embodiments, the present application contemplates the use of any naturally occurring or synthetic analogs, mutants, variants, alleles, homologs and orthologues (collectively referred to herein as "analogs") of the CD20 binding agents for the uses described herein. In various embodiments, the amino acid sequences of the CD20 binding agents further comprise amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0048] In various embodiments, the CD20 binding agent comprises a targeting moiety comprising a sequence at least 60% identical to any one of SEQ ID NOs: 18-34 or 85-105. In various embodiments, the CD20 binding agent comprises a targeting moiety comprising a sequence at least 60% identical to any one of SEQ ID NOs: 18-34 or 85-105 minus the linker sequence, the HA tag, and / or the HIS6 tag. For example, the CD20 binding agent may be selected from the group consisting of SEQ ID NOs: 2-87 and at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 80%, or about 82%, or about 84%, or about 86%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 10 ... or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% identical to the sequence of the targeting moiety.

[0049] In various embodiments, the CD20 binding agent comprises a targeting moiety that comprises an amino acid sequence having one or more amino acid mutations relative to SEQ ID NO: 18-34 or 85-105. In various embodiments, the CD20 binding agent comprises a targeting moiety that comprises 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 15, or 20 amino acid mutations relative to SEQ ID NO: 18-34 or 85-105. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0050] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0051] "Conservative substitutions" can be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the amino acid residues involved. The 20 naturally occurring amino acids can be classified into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0052] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups above. For example, the replacement of Asp with Glu retains one negative charge in the polypeptide modified in this way. Furthermore, glycine and proline can be substituted for each other based on their ability to break α-helix.

[0053] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) above.

[0054] In various embodiments, substitutions may include non-classical amino acids (e.g., selenocysteine, pyrrolidine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcocyme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general).

[0055] In various embodiments, the amino acid mutations may be present in the CDRs (e.g., CDR1, CDR2 or CDR3 regions) of the targeting moiety. In another embodiment, the amino acid changes may be present in the framework regions (FRs) of the targeting moiety, e.g., the FR1, FR2, FR3 or FR4 regions.

[0056] The modification of amino acid sequence can be achieved by using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis.Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0057] In various embodiments, the mutations do not substantially reduce the ability of the CD20 binding agents of the invention to specifically bind to CD20. In various embodiments, the mutations do not substantially reduce the ability of the CD20 binding agents of the invention to specifically bind to CD20 without neutralizing CD20.

[0058] In various embodiments, the binding affinity of the CD20 binding agents of the present application to the full-length, and / or mature forms, and / or isoforms, and / or splice variants, and / or fragments, and / or monomeric, and / or dimeric, and / or tetrameric forms of human CD20, and / or any other naturally occurring or synthetic analogs, variants, or mutants (such as monomeric and / or dimeric and / or tetrameric forms) is determined by the equilibrium dissociation constant (K D In various embodiments, the CD20-binding agent has a K of less than about 1 uM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or about 4.5 nM, or about 1 nM. D and / or any other naturally occurring or synthetic analog, variant or mutant (such as monomeric, dimeric and / or tetrameric forms) of human CD20, having the formula:

[0059] In various embodiments, the CD20 binding agent comprises a targeting moiety that binds to, but does not functionally modulate, the antigen of interest, i.e., CD20. For example, in various embodiments, the targeting moiety of the CD20 binding agent simply targets the antigen but does not substantially functionally modulate (e.g., substantially inhibit, reduce, or neutralize) the biological effect that the antigen has. In various embodiments, the targeting moiety of the CD20 binding agent binds to an epitope (e.g., an active site of the antigen) that is physically separated from the antigen site important for its biological activity.

[0060] Such binding without significant functional modulation finds use in various embodiments of the present application. In various embodiments, the CD20 binding agents of the present invention bind to CD20 positive cells and induce the death of such cells. In some embodiments, the CD20 binding agents induce cell death mediated by one or more of apoptosis or direct cell death, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the CD20 binding agents of the present invention induce the translocation of CD20 to large lipid microdomains or "lipid rafts" in the plasma membrane upon CD20 binding. This clustering process enhances complement activation and exerts potent complement-dependent cytotoxicity (CDC). In other embodiments, the CD20 binding agents of the present invention induce direct cell death. In alternative embodiments, the therapeutic effect of the CD20 binding agents is not dependent on B cell depletion.

[0061] In various embodiments, the CD20 binding agents of the present invention can be used to directly or indirectly recruit active immune cells to required sites via effector antigens. For example, in various embodiments, the CD20 binding agents of the present invention can be used to directly or indirectly recruit immune cells to cancer or tumor cells in a method of reducing or eliminating cancer or tumor (e.g., the CD20 binding agent may include a targeting moiety with an anti-CD20 antigen recognition domain and a targeting moiety with a recognition domain (e.g., an antigen recognition domain) directed against Clec9A, an antigen expressed on dendritic cells). In these embodiments, CD20 signaling is an important part of the effect of reducing or eliminating cancer. In various embodiments, the CD20 binding agents of the present invention can recruit T cells, B cells, dendritic cells, macrophages, and natural killer (NK) cells.

[0062] Therapeutic Agents Comprising the CD20-Binding Agents of the Present Invention Chimeras and Fusions with Signaling Agents In various embodiments, the CD20 binding agents of the present application are part of a chimera or fusion with one or more signaling agents. Thus, the present application provides, for example, a chimera or fusion protein that includes a targeting moiety for CD20 and one or more signaling agents.

[0063] In various embodiments, the signaling agent is modified to reduce affinity or activity for one or more of its receptors, allowing for attenuated activity (such as agonism or antagonism) and / or preventing non-specific signaling or undesired sequestration of the chimeric or fusion protein. In various embodiments, the signaling agent is antagonistic in its wild-type form and carries one or more mutations that attenuate its antagonistic action. In various embodiments, the signaling agent is antagonistic due to one or more mutations, e.g., an agonist signaling agent is converted to an antagonistic signaling agent, such converted signaling agent carrying one or more mutations that attenuate its antagonistic action (e.g., as described in WO 2015 / 007520, which is incorporated by reference in its entirety).

[0064] Thus, in various embodiments, the signaling agent is a modified (e.g., mutated) form of the signaling agent having one or more mutations. In various embodiments, the modification (e.g., mutation) allows the modified signaling agent to have one or more weakened activities, such as one or more of a reduced binding affinity, a reduced intrinsic activity, and a reduced specific biological activity, compared to the unmodified or unmutated, i.e., wild-type form of the signaling agent (e.g., compared to the same signaling agent in wild-type form vs. modified or mutant form). In some embodiments, mutations that weaken or reduce binding or affinity include those mutations that substantially reduce or eliminate binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity are different from mutations that substantially reduce or eliminate binding or activity. As a result, in various embodiments, mutations allow for improved safety of the signaling agent, such as reduced systemic toxicity, reduced side effects, and reduced off-target effects, relative to the unmutated, i.e., wild-type, signaling agent (e.g., compared to the same signaling agent in wild-type form vs. modified (e.g., mutant) form).

[0065] As described herein, an agent may have improved safety due to one or more modifications, e.g., mutations. In various embodiments, improved safety means that the chimeric protein of the present invention provides lower toxicity (e.g., systemic toxicity, and / or tissue / organ-related toxicity), and / or reduced or substantially eliminated side effects, and / or increased tolerability, reduced or substantially eliminated adverse events, and / or reduced or substantially eliminated off-target effects, and / or an increased therapeutic window.

[0066] In various embodiments, the signaling agent is modified to have one or more mutations that reduce its binding affinity or activity to one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate its binding affinity or activity to the receptor. In some embodiments, the activity provided by the wild-type signaling agent is agonism at the receptor (e.g., activation of a cellular effect at the treatment site). For example, the wild-type signaling agent can activate its receptor. In such embodiments, the mutation results in the modified signaling agent having reduced or eliminated activation activity at the receptor. For example, the mutation results in the modified signaling agent delivering a reduced activation signal to the target cell or eliminating the activation signal. In some embodiments, the activity provided by the wild-type signaling agent is antagonism at the receptor (e.g., blocking or attenuating a cellular effect at the treatment site). For example, the wild-type signaling agent can antagonize or inhibit the receptor. In these embodiments, the mutation results in the modified signaling agent having reduced or eliminated antagonism activity at the receptor. For example, the mutations may result in the modified signaling agent delivering a reduced inhibitory signal to the target cell or may eliminate the inhibitory signal. In various embodiments, the signaling agent is antagonistic by one or more mutations, e.g., an agonist signaling agent is converted to an antagonist signaling agent (e.g., as described in WO 2015 / 007520, the entire contents of which are incorporated herein by reference), and the signaling agent thus converted also optionally carries one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or that substantially reduce or eliminate its binding affinity or activity to one or more of its receptors.

[0067] In some embodiments, the reduced affinity or activity at the receptor is salvageable by attachment to one or more of the targeting moieties described herein (e.g., a targeting moiety for CD20, or any other targeting moiety described herein). In other embodiments, the reduced affinity or activity at the receptor is not substantially salvageable by the activity of one or more of the targeting moieties.

[0068] In various embodiments, the chimeric proteins of the present application reduce off-target effects because their signaling agents have mutations at the receptor that weaken or eliminate binding affinity or activity. In various embodiments, such reduction in side effects is observed, for example, compared to wild-type signaling agents. In various embodiments, the signaling agents are active on the target cells because the targeting moiety(s) required for substantial activation compensate for missing / insufficient binding (e.g., limited and / or no avidity). In various embodiments, the modified signaling agents are substantially inactive until the site of therapeutic activity and have substantially their effect on the specifically targeted cell type, greatly reducing undesirable side effects.

[0069] In some embodiments, the signaling agent may contain one or more mutations that weaken or reduce binding or affinity at one receptor (i.e., therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity at a second receptor. In such embodiments, these mutations may be at the same position or different positions (i.e., the same mutation or multiple mutations). In some embodiments, the mutation(s) that reduce binding and / or activity at one receptor are different from the mutation(s) that substantially reduce or eliminate binding at another receptor. In some embodiments, the mutation(s) that reduce binding and / or activity at one receptor are the same as the mutation(s) that substantially reduce or eliminate binding at another receptor. In some embodiments, the chimeric proteins of the invention have modified signaling agents that have both a mutation that weakens binding and / or activity at a therapeutic receptor, thus allowing for more controlled on-target therapeutic effects (e.g., relative to the wild-type signaling agent), and a mutation that substantially reduces or eliminates binding and / or activity at another receptor, thus reducing side effects (e.g., compared to the wild-type signaling agent).

[0070] In some embodiments, the substantial reduction or elimination of binding or activity is not substantially reversible by a targeting moiety (e.g., a targeting moiety for CD20 or any other targeting moiety described herein). In some embodiments, the substantial reduction or elimination of binding or activity is reversible by a targeting moiety. In various embodiments, substantially reducing or eliminating binding or activity at a second receptor may also prevent adverse effects mediated by other receptors. Alternatively or additionally, substantially reducing or eliminating binding or activity at other receptors may improve therapeutic efficacy by reducing or eliminating sequestration of the therapeutic chimeric protein away from the therapeutic site of action. For example, in some embodiments, this eliminates the need for high doses of the chimeric protein of the invention to compensate for losses at other receptors. This ability to reduce doses results in less potential side effects.

[0071] In various embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced, substantially reduced, or eliminated affinity, e.g., binding (e.g., K D ), and / or (e.g., the modified signaling agent is, e.g., K A and / or E.C. 50 activation (e.g., when a signaling modifier is a K receptor agonist), and / or I and / or I.C. 50The modified agent will have measurable inhibition (if the agent is an antagonist of that receptor) at one or more of its receptors. In various embodiments, the reduced affinity of the agent at the receptor allows for attenuated activity (including agonism or antagonism). In such embodiments, the modified signaling agent has about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% affinity for the receptor compared to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower, relative to the wild-type signaling agent.

[0072] In embodiments where the chimeric protein has mutations that reduce binding at one receptor and substantially reduce or eliminate binding at a second receptor, the weakened or reduced binding affinity of the modified signaling agent at one receptor is less than the substantial reduction or elimination of affinity at the other receptor. In some embodiments, the attenuation or reduction of the binding affinity of the modified signaling agent at one receptor is less than the substantial reduction or elimination of affinity at the other receptor by about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In various embodiments, substantial reduction or elimination refers to a greater reduction in binding affinity and / or activity than attenuation or reduction.

[0073] In various embodiments, the modified signaling agent comprises one or more mutations that reduce the intrinsic activity of the signaling agent, e.g., to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, compared to the wild-type signaling agent.

[0074] In some embodiments, the modified signaling agent comprises one or more mutations that result in the signaling agent having a lower binding affinity of the targeting moiety(s) to its (their) receptor(s) for which the affinity of the receptor(s) is reduced. In some embodiments, the difference in binding affinity is between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, the difference in binding affinity results in the signaling agent, e.g., the mutated signaling agent, having a localized on-target effect and minimizing the off-target effects that are the basis of side effects observed with wild-type signaling agents. In some embodiments, the binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold lower, or at least about 25-fold, or at least about 50-fold lower, or at least about 100-fold, or at least about 150-fold.

[0075] Receptor binding activity can be measured using methods known in the art.For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis and computer fitting of binding data (e.g., Scatchard, 1949), or by reflectance interferometry under flow-through conditions as described in Brecht et al. (1993), the entire contents of which are incorporated herein by reference.

[0076] In various embodiments, the signaling agent is an immunomodulatory agent, such as one or more of an interleukin, an interferon, and a tumor necrosis factor.

[0077] In some embodiments, the signaling agent is an interleukin or modified interleukin, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, or fragments, variants, analogs, or family members thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulating the proliferation of immune cells (e.g., T helper cells, B cells, eosinophils and lymphocytes), chemotaxis of neutrophils and T lymphocytes and / or inhibition of interferon.Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds., IRL Press, Washington, DC 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20.

[0078] In some embodiments, the signaling agent is an interferon or a modified form of an interferon, such as type I, type II, and type III interferon. Exemplary interferons include, for example, interferon-α1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon-β, and interferon-γ, interferon-κ, interferon-ξ, interferon-τ, and interferon-ω.

[0079] In some embodiments, the signaling agent is tumor necrosis factor (TNF), or a modified form of tumor necrosis factor (TNF), or a protein of the TNF family, including, but not limited to, TNF-α, TNF-β, LT-β, ​​CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

[0080] The amino acid sequences of the wild-type signaling agents described herein are known in the art. Thus, in various embodiments, the modified signaling agent has a similar affinity to the known wild-type amino acid sequence of a signaling agent described herein, at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or At least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 90%, 3%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to the amino acid sequence.

[0081] In various embodiments, the modified signaling agent is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to the amino acid sequence.

[0082] In various embodiments, the modified signal transduction agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions, as described elsewhere herein.

[0083] In various embodiments, substitutions can also include non-classical amino acids as described elsewhere herein.

[0084] As described herein, modified signaling agents carry mutations that affect affinity and / or activity at one or more receptors. As described herein, receptors for any signaling agent are known in the art.

[0085] Exemplary mutations that result in reduced affinity and / or activity (e.g., agonistic activity) at the receptor can be found in WO 2013 / 107791 (e.g., for interferons), WO 2015 / 007542 (e.g., for interleukins), and WO 2015 / 007903 (e.g., for TNF), the entire contents of each of which are incorporated herein by reference. Exemplary mutations that result in reduced affinity and / or activity (e.g., antagonistic activity) at the receptor can be found in WO 2015 / 007520, the entire contents of which are incorporated herein by reference.

[0086] In one embodiment, the modified signal transduction agent is interferon alpha. In such embodiments, the modified IFN-alpha agent has reduced affinity and / or activity for the IFN-alpha / beta-1 receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN-alpha agent has substantially reduced or eliminated affinity and / or activity for the IFN-alpha / beta-1 receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0087] Mutant forms of interferon alpha are known to those of skill in the art. In an exemplary embodiment, the modified signaling agent is an allelic form of IFN-α2a having the following amino acid sequence: IFN-α2a: CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 127)

[0088] In an exemplary embodiment, the modified signaling agent is an allelic IFN-α2b having the following amino acid sequence (which differs from IFN-α2a at amino acid position 23): IFN-α2b: CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 128)

[0089] In some embodiments, the IFN-α2 mutant (IFN-α2a or IFN-α2b) is mutated at one or more amino acids at positions 144-154, such as amino acid positions 148, 149, and / or 153. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO 2013 / 107791 and Piehler et al., (2000) J. Biol. Chem, 275:40425-33, the entire contents of which are incorporated herein by reference.

[0090] In some embodiments, the IFN-α2 mutant has reduced affinity and / or activity for IFNAR1. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO 2010 / 030671, the entire contents of which are incorporated herein by reference.

[0091] In some embodiments, the IFN-α2 mutant includes one or more mutations selected from K133A, R144A, R149A, and L153A, as described in International Publication No. WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0092] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from R120E and R120E / K121E, as described in WO 2015 / 007520 and WO 2010 / 030671, the entire contents of which are incorporated herein by reference. In such embodiments, the IFN-α2 mutant antagonizes the activity of wild-type IFN-α2. In such embodiments, the mutant IFN-α2 has reduced affinity and / or activity for IFNAR1 and retains affinity and / or activity for IFNR2.

[0093] In some embodiments, the IFN-α2 mutant includes (1) one or more mutations selected from R120E and R120E / K121E, which, without wishing to be bound by theory, result in an antagonistic effect, and (2) one or more mutations selected from K133A, R144A, R149A, and L153A, which, without wishing to be bound by theory, enable a weakening effect, for example, in IFNAR2.

[0094] In one embodiment, the modified signal transduction agent is interferon beta. In such embodiments, the modified interferon beta agent has reduced affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified interferon beta agent has substantially reduced or eliminated affinity and / or activity for the IFN-α / β-1 receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0095] In one embodiment, the modified signal transduction agent is interferon gamma. In such an embodiment, the modified interferon gamma agent has reduced affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon gamma agent has substantially reduced or eliminated affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains.

[0096] In one embodiment, the modified signal transduction agent is TNF-α. TNF is a pleiotropic cytokine with many diverse functions, including regulation of cell proliferation, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation and septic shock. It binds to two different membrane receptors on target cells, TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, whereas TNFR2 is preferentially expressed on specific populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes and mesenchymal stem cells. Very different biological pathways are activated in response to receptor activation, although there is some overlap. As a general rule, and without wishing to be bound by theory, TNFR1 signal transduction is associated with the induction of apoptosis (cell death) and TNFR2 signal transduction is associated with the activation of cell survival signals (e.g., activation of the NFkB pathway). Administration of TNF is systemic toxic, which is mainly due to the involvement of TNFR1. However, it should be noted that activation of TNFR2 is also associated with a broad range of activities and, similar to TNFR1, targeting and control over TNF activity is important in the context of developing TNF-based therapeutics.

[0097] In some embodiments, the modified signaling agent has a reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling, while TNFR2 is involved in cell survival signaling. Thus, in embodiments relating to methods for treating cancer, the modified signaling agent has a reduced affinity and / or activity for TNFR1 and / or has a substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, the chimeric protein may be targeted to cells where apoptosis is desired, such as tumor cells or tumor vascular endothelial cells. For example, in embodiments relating to methods for promoting cell survival in neurogenesis for the treatment of neurodegenerative diseases, the modified signaling agent has a reduced affinity and / or activity for TNFR2 and / or has a substantially reduced or eliminated affinity and / or activity for TNFR1. In other words, the chimeric protein of the present invention, in some embodiments, comprises a modified TNF-α agent that favors either death or survival signals.

[0098] In some embodiments, the chimeric protein has a modified TNF with reduced affinity and / or activity for TNFR1 and / or a modified TNF with substantially reduced or eliminated affinity and / or activity for TNFR2. Such chimeras are, in some embodiments, more potent inducers of apoptosis compared to chimeras carrying only wild-type TNF and / or a mutation(s) that cause reduced affinity and / or activity for TNFR1. Such chimeras, in some embodiments, find use in inducing tumor cell death or tumor vasculature endothelial cell death (e.g., in the treatment of cancer). Also, in some embodiments, these chimeras inhibit TNF via, for example, TNFR2. regThis avoids or reduces cellular activation, thus further supporting TNFR1-mediated anti-tumor activity in vivo.

[0099] In some embodiments, the chimeric protein has a modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. In some embodiments, such chimeras are more potent activators of cell survival in some cell types, which may be of specific therapeutic interest in various disease settings, including, but not limited to, stimulating neurogenesis. In some embodiments, the chimeras target autoreactive T cells. In some embodiments, the chimeras target T reg Promotes cell activation and indirect suppression of cytotoxic T cells.

[0100] In some embodiments, the chimeras (e.g., modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1) cause the death of autoreactive T cells, for example, by activating TNFR2 and / or evading TNFR1. Without wishing to be bound by theory, these autoreactive T cells have altered apoptosis / survival signals, for example, by altering NFkB pathway activity / signaling.

[0101] In some embodiments, TNFR-2 based chimeras have additional therapeutic applications in diseases such as various cardiac diseases, demyelinating and neurodegenerative disorders, and infectious diseases, among others.

[0102] In one embodiment, the wild-type TNF-α has the following amino acid sequence: TNF-α VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL.

[0103] In such embodiments, the modified TNF-α agent has mutations at one or more of amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147 to produce modified TNF-α with reduced receptor binding affinity. See, e.g., U.S. Patent No. 7,993,636, the entire contents of which are incorporated herein by reference.

[0104] In some embodiments, the modified TNF-α agent has mutations at one or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, and A145 (numbering according to human TNF sequence, Genbank Accession No. BAG70306, version BAG70306.1 GI:197692685), e.g., as described in WO 2015 / 007903, the entire contents of which are incorporated herein by reference. In some embodiments, the modified TNF-α agent has a substitution mutation selected from R32G, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, Y87Q, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, and A145T.

[0105] In some embodiments, the modified TNF-α agent has one or more mutations selected from N39Y, S147Y, and Y87H, as described in International Publication No. WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0106] In one embodiment, the modified signaling agent is TNF-β. TNF-β can form homotrimers or heterotrimers with LT-β (LT-α1β2). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for TNFR1, and / or TNFR2, and / or herpes virus entry mediator (HEVM), and / or LT-βR.

[0107] In one embodiment, the wild-type TNF-β has the following amino acid sequence: TNF-β LPGVGLTPSAAQTARQHPKMHLAHSNLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL.

[0108] In such embodiments, the modified TNF-β agent may include a mutation at one or more amino acids at positions 106-113, which produces a modified TNF-β agent with reduced receptor binding affinity for TNFR2. In one embodiment, the modified signaling agent has one or more substitution mutations at amino acid positions 106-113. In an exemplary embodiment, the substitution mutations are selected from Q107E, Q107D, S106E, S106D, Q107R, Q107N, Q107E / S106E, Q107E / S106D, Q107D / S106E, and Q107D / S106D. In another embodiment, the modified signaling agent has an insertion of about 1 to about 3 amino acids at positions 106-113.

[0109] In some embodiments, the modifying agent is a TNF family member (e.g., TNF-alpha, TNF-beta) that may be in a single-chain trimer form, as described in International Publication No. WO 2015 / 007903, the entire contents of which are incorporated herein by reference.

[0110] In some embodiments, the modified agent is a TNF family member (e.g., TNF-alpha, TNF-beta) that has reduced affinity and / or activity, i.e., antagonist activity, at TNFR1 (e.g., natural antagonist activity or antagonist activity that is the result of one or more mutations (see, e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF-alpha, TNF-beta) that optionally also has substantially reduced or eliminated affinity and / or activity for TNFR2. In some embodiments, the modified agent is a TNF family member (e.g., TNF-alpha, TNF-beta) with reduced affinity and / or activity, i.e., antagonist activity, at TNFR2 (e.g., natural antagonist activity or antagonist activity that is the result of one or more mutations (see, e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF-alpha, TNF-beta) that optionally also has substantially reduced or eliminated affinity and / or activity for TNFR1. Constructs of such embodiments are used, for example, in methods of attenuating TNF responses in a cell-specific manner. In some embodiments, the antagonist TNF family member (e.g., TNF-alpha, TNF-beta) is a single-chain trimer as described in WO 2015 / 007903.

[0111] In one embodiment, the modified signal transduction agent is TRAIL. In some embodiments, the modified TRAIL agent has reduced affinity and / or activity for DR4 (TRAIL-RI) and / or DR5 (TRAIL-RII) and / or DcR1 and / or DcR2. In some embodiments, the modified TRAIL agent has substantially reduced or eliminated affinity and / or activity for DR4 (TRAIL-RI), and / or DR5 (TRAIL-RII), and / or DcR1 and / or DcR2.

[0112] In one embodiment, wild-type TRAIL has the following amino acid sequence: TRAIL MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNS KNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG.

[0113] In such embodiments, the modified TRAIL agent may contain mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-1220, K224-A226, W231, E236-L239, E249-K251, T261-H264, and H270-E271 (numbering based on the human sequence, Genbank accession number NP_003801, version 10NP_003801.1, GI:4507593; see above).

[0114] In one embodiment, the modified signaling agent is an interleukin. In one embodiment, the modified signaling agent is IL-1. In one embodiment, the modified signaling agent is IL-1α or IL-1β. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-1R2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-1R2. For example, in some embodiments, the modified IL-1 agent of the invention avoids interaction at IL-1R2, thus substantially reducing its function as a decoy and / or sink for therapeutic agents.

[0115] In one embodiment, the wild-type IL-1β has the following amino acid sequence: IL-1β (mature form, wild type) APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS.

[0116] IL-1 is a proinflammatory cytokine and an important regulator of the immune system. It is a potent activator of CD4 T cell responses, increasing the proportion of Th17 cells and increasing the expansion of IFNγ- and IL-4-producing cells. IL-1 also activates CD8 + A potent regulator of T cells, antigen-specific CD8 +It promotes T cell expansion, differentiation, and migration to the periphery and memory. IL-1 receptors include IL-1R1 and IL-1R2. Binding to and signaling through IL-1R1 constitutes the mechanism by which IL-1 mediates many of its biological (and pathological) activities. IL-1R2 can function as a decoy receptor, thereby reducing the availability of IL-1 for interaction and signaling through IL-1R1.

[0117] In some embodiments, the modified IL-1 has reduced affinity and / or activity (e.g., agonist activity) for IL-1R1. In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, there is restorable IL-1 / IL-1R1 signaling at IL-R2 and prevention of loss of the therapeutic chimera, thus reducing the dose of IL-1 required (e.g., for IL-R1, compared to a chimera with only wild type or attenuating mutations). Such constructs find use in methods of treating cancer, including, for example, stimulating the immune system to mount an anti-cancer response.

[0118] In some embodiments, the modified IL-1 has reduced affinity and / or activity for IL-1R1 (e.g., antagonist activity, e.g., naturally occurring antagonist activity or antagonist activity that is the result of one or more mutations, see, e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference). In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, there is no recovery of IL-1 / IL-1R1 signaling at IL-R2 and thus prevention of loss of the therapeutic chimera, thus reducing the dose of IL-1 required (e.g., for IL-R1, compared to wild type or chimeras with only attenuating mutations).

[0119] In such embodiments, the modified signaling agent has a deletion of amino acids 52-54 to produce a modified IL-1β with reduced binding affinity to type I IL-1R and reduced biological activity. See, e.g., WO 1994 / 000491, the entire contents of which are incorporated herein by reference. In some embodiments, the modified IL-1β is selected from the group consisting of A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, E221 and have one or more substitution mutations selected from S / N224A, N224S / K225S, E244K, N245Q (wherein X may be any change (e.g., a non-conservative change) of an amino acid) and exhibit reduced binding to IL-1R, e.g., as described in WO 2015 / 007542 and WO 2015 / 007536, the entire contents of which are incorporated by reference (numbering based on human IL-1β sequence, Genbank Accession No. NP_000567, version NP-000567.1, GI:10835145). In some embodiments, the modified IL-1β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K.

[0120] In one embodiment, the modified signaling agent is IL-2. In such embodiments, the modified signaling agent has reduced affinity and / or activity for IL-2Rα, and / or IL-2Rβ, and / or IL-2Rγ. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-2Rα. Such embodiments may be relevant for the treatment of cancer, for example, where the modified IL-2 is an agonist at IL-2Rβ and / or IL-2Rγ. For example, the constructs of the invention may be used to inhibit activated CD8 with IL-2 receptors β and γ. + T cells (which may provide antitumor effects) that have IL2 receptors α, β, and γ reg (which may provide immunosuppressive, pro-tumor effects). Moreover, in some embodiments, the selection of IL-2Rβ and / or IL-2Rγ over IL-2Rα avoids IL-2 side effects such as pulmonary edema. Also, IL-2-based chimeras are useful for treating diseases, for example, when the modified IL-2 is antagonistic (e.g., naturally occurring antagonistic or antagonistic as a result of one or more mutations, see e.g., WO 2015 / 007520, the entire contents of which are incorporated herein by reference) at IL-2Rβ and / or IL-2Rγ. For example, the constructs of the present invention may be used to express CD8 with IL2 receptors β and γ, which have attenuated suppression (and thus attenuated immune responses). + T cells have a preference for IL2 receptors α, β, and γ. reg Alternatively, in some embodiments, the IL-2 bearing chimera may be reg activation and thus immunosuppression, favoring CD8 + They do not favor T cell activation. For example, these constructs find use in the treatment of disease(s) that would benefit from immunosuppression.

[0121] In some embodiments, the chimeric protein is CD8 + T cells, and modified IL-2 agents that have reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ, and / or substantially reduced or eliminated affinity and / or activity for IL-2Rα. + These provide T cell activity. reg Generally inactive (or have substantially reduced activity) against cells. In some embodiments, such constructs have improved immune stimulatory effects compared to wild-type IL-2 (e.g., by not stimulating Tregs, without wishing to be bound by theory) and eliminate or reduce systemic toxicity associated with IL-2.

[0122] In one embodiment, the wild-type IL-2 has the following amino acid sequence: IL-2 (mature form, wild type) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

[0123] In such embodiments, the modified IL-2 agents have one or more mutations at amino acids L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K), and Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K). Without wishing to be bound by theory, it is believed that these modified IL-2 agents have reduced affinity for the high affinity IL-2 receptor and preserved affinity for the intermediate affinity IL-2 receptor compared to wild-type IL-2. See, for example, U.S. Patent Application Publication No. 2012 / 0244112, the entire contents of which are incorporated herein by reference.

[0124] In one embodiment, the modified signaling agent is IL-3. In some embodiments, the modified signaling agent has reduced affinity and / or activity for the IL-3 receptor, which is a heterodimer with a unique alpha chain paired with a common beta (beta c or CD131) subunit. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the IL-3 receptor, which is a heterodimer with a unique alpha chain paired with a common beta (beta c or CD131) subunit.

[0125] In one embodiment, the modified signaling agent is IL-4. In such embodiments, the modified signaling agent has reduced affinity and / or activity for type 1 and / or type 2 IL-4 receptors. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for type 1 and / or type 2 IL-4 receptors. Type 1 IL-4 receptors are composed of IL-4Rα subunits with a common gamma chain and specifically bind IL-4. Type 2 IL-4 receptors comprise an IL-4Rα subunit bound to a different subunit known as IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for type 2 IL-4 receptors.

[0126] In one embodiment, the wild-type IL-4 has the following amino acid sequence: IL-4 (mature form, wild type) HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS.

[0127] In such an embodiment, the modified IL-4 agent has one or more mutations at amino acids R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), E122 (E122F), Y124 (Y124A, Y124Q, Y124R, Y124S, Y124T), and S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL-4 agents maintain the activity mediated by the type I receptor, but significantly reduce the biological activity mediated by other receptors. See, for example, U.S. Patent No. 6,433,157, the entire contents of which are incorporated herein by reference.

[0128] In one embodiment, the modified signaling agent is IL-6. IL-6 signals through a cell surface type I cytokine receptor complex, including the ligand-bound IL-6R chain (CD126) and the signaling component gp130. IL-6 can also bind to the soluble form of IL-6R (sIL-6R), which is the extracellular portion of IL-6R. The sIL-6R / IL-6 complex is believed to be involved in neurite outgrowth and neuronal survival, and thus may be important in nerve regeneration by remyelination. Thus, in some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-6R / gp130 and / or sIL-6R. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-6R / gp130 and / or sIL-6R.

[0129] In one embodiment, the wild-type IL-6 has the following amino acid sequence: IL-6 (mature form, wild type) APVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLTTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM.

[0130] In such an embodiment, the modified signal transduction agent has one or more mutations at amino acid 58, 160, 163, 171 or 177. Without wishing to be bound by theory, it is believed that these modified IL-6 agents exhibit reduced binding affinity to IL-6Rα and reduced biological activity. See, for example, International Publication No. WO 97 / 10338, the entire contents of which are incorporated herein by reference.

[0131] In one embodiment, the modified signaling agent is IL-10. In such embodiments, the modified signaling agent has reduced affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2. In some embodiments, the modified signaling agent has substantially reduced affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2.

[0132] In one embodiment, the modified signaling agent is IL-11. In such embodiments, the modified signaling agent has reduced affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130. In such embodiments, the modified signaling agent has eliminated or substantially reduced affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0133] In one embodiment, the modified signaling agent is IL-12. In such embodiments, the modified signaling agent has reduced affinity and / or activity for IL-12R-β1 and / or IL-12R-β2. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-12R-β1 and / or IL-12R-β2.

[0134] In one embodiment, the modified signaling agent is IL-13. In such embodiments, the modified signaling agent has reduced affinity and / or activity for the IL-4 receptor (IL-4Rα) and IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the IL-4 receptor (IL-4Rα) or IL-13Rα1.

[0135] In one embodiment, the wild-type IL-13 has the following amino acid sequence: IL-13 (mature form, wild type) SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN.

[0136] In such embodiments, the modified IL-13 agents have one or more mutations at amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114. Without wishing to be bound by theory, it is believed that these modified IL-13 agents exhibit reduced biological activity. See, for example, International Publication No. WO 2002 / 018422, the entire contents of which are incorporated herein by reference.

[0137] In one embodiment, the modified signaling agent is IL-18. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for IL-18Rα II, an isoform of IL-18Rα lacking the TIR domain required for signal transduction.

[0138] In one embodiment, wild-type IL-18 has the following amino acid sequence: IL-18 (wild type) MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDL.

[0139] In such embodiments, the modified IL-18 agent may include one or more mutations at amino acids or amino acid regions selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183, and Q190-N191 (numbering based on the human IL-18 sequence, Genbank Accession No. AAV38697, version AAV38697.1, GI:54696650), as described in WO 2015 / 007542, the entire contents of which are incorporated herein by reference.

[0140] In one embodiment, the modified signaling agent is IL-33. In such embodiments, the modified signaling agent has reduced affinity and / or activity for the ST-2 receptor and IL-1RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the ST-2 receptor and IL-1RAcP.

[0141] In one embodiment, wild-type IL-33 has the following amino acid sequence: MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSIT FALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET.

[0142] In such embodiments, the modified IL-33 agent may include one or more mutations at amino acids or amino acid regions selected from I113-Y122, S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227, and T260-E269, as described in WO 2015 / 007542, the entire contents of which are incorporated herein by reference (numbering based on human sequence, Genbank Accession No. NP_254274, version NP_254274.1, GI:15559209).

[0143] In one embodiment, a chimeric protein of the invention has (i) a targeting moiety to CD20 and (ii) a targeting moiety directed to a tumor cell, along with any of the modified or mutated signal agents described herein. In one embodiment, a chimeric protein of the invention has a targeting moiety to CD20 on a tumor cell and a second targeting moiety to PD-L1 or PD-L2.

[0144] Multispecific Chimeras and Fusions with Signal Transduction Agents - Patent application In various embodiments, the CD20-binding agents of the present application are part of a chimera or fusion with one or more signaling agents described herein and / or one or more additional targeting moieties. Thus, the present application provides chimera or fusion proteins that include one or more signaling agents and a targeting moiety for CD20 and / or one or more additional targeting moieties.

[0145] In various embodiments, the chimeric proteins of the present application have targeting moieties that target two different cells (e.g., to create a synapse) or the same cell (e.g., to obtain a more concentrated signaling agent effect).

[0146] In various embodiments, the CD20 binding agents of the present application are multispecific, i.e., the CD20 binding agents comprise two or more targeting moieties having recognition domains (e.g., antigen recognition domains) that recognize and bind to two or more targets (e.g., antigens, or receptors, or epitopes). In such embodiments, the CD20 binding agents of the present application may comprise two or more targeting moieties having recognition domains that recognize and bind to two or more epitopes on the same antigen, or on different antigens, or on different receptors. In various embodiments, such multispecific CD20 binding agents exhibit advantageous properties such as increased avidity and / or improved selectivity. In one embodiment, the CD20 binding agents of the present application comprise two targeting moieties and are bispecific, i.e., they bind to and recognize two epitopes on the same antigen, or on different antigens, or on different receptors.

[0147] In various embodiments, the multispecific CD20 binding agent of the present application comprises two or more targeting moieties, each targeting moiety being an antibody or antibody derivative as described herein. In one embodiment, the multispecific CD20 binding agent of the present application comprises at least one VHH comprising an antigen recognition domain for CD20, and one antibody or antibody derivative comprising a recognition domain for a tumor antigen or an immune cell.

[0148] In various embodiments, a multispecific CD20 binding agent of the invention has two or more targeting moieties that target different antigens or receptors, where one targeting moiety may be weakened for that antigen or receptor, e.g., the targeting moiety binds to that antigen or receptor with low affinity or avidity (e.g., a lower affinity or avidity for that antigen or receptor than the other targeting moiety has, e.g., the difference in binding affinity may be about 10-fold, or 25-fold, or 50-fold, or 100-fold, or 300-fold, or 500-fold, or 1000-fold, or 5000-fold, e.g., at the lower affinity or avidity, the targeting moiety has a K in the medium to high nM, or low to medium μM range. DAt higher affinity or avidity, the targeting moiety may bind to its antigen or receptor with a K in the moderate to high pM or low to moderate nM range. D For example, in some embodiments, the multispecific CD20 binding agents of the invention include a weakened targeting moiety that is directed to a promiscuous antigen or receptor. This can improve targeting to cells of interest (e.g., via other targeting moieties) and prevent effects across multiple cell types, including those not targeted for therapy (e.g., by binding the promiscuous antigen or receptor with a higher affinity than provided in these embodiments).

[0149] The multispecific CD20 binding agents of the present application may be constructed using methods known in the art. See, for example, U.S. Pat. No. 9,067,991, U.S. Pat. Publication No. 20110262348, and WO 2004 / 041862, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the multispecific CD20 binding agents of the present application comprising two or more targeting moieties may be constructed by chemical cross-linking, for example, by reacting amino acid residues with organic derivatizing agents, as described in Blattler et al., Biochemistry 24,1517-1524 and European Patent Specification No. 294703, the entire contents of which are incorporated herein by reference. In another exemplary embodiment, the multispecific CD20 binding agents comprising two or more targeting moieties are constructed by gene fusion, i.e., constructing a single polypeptide comprising the polypeptides of the individual targeting moieties. For example, a single polypeptide construct may be formed that encodes a first VHH having an antigen recognition domain for CD20 and a second antibody or antibody derivative having a recognition domain for a tumor antigen. Methods for producing bivalent or multivalent VHH polypeptide constructs are disclosed in International Patent Application No. 96 / 34103, the entire contents of which are incorporated herein by reference. In further exemplary embodiments, the multispecific CD20 binding agent of the present application may be constructed using a linker. For example, the carboxy terminus of a first VHH having an antigen recognition domain for CD20 may be linked to the amino terminus of a second antibody or antibody derivative having a recognition domain for a tumor antigen (or vice versa). Exemplary linkers that may be used are described herein. In some embodiments, the components of the multispecific CD20 binding agent of the present application are directly linked to each other without the use of a linker.

[0150] In various embodiments, the multispecific CD20 binding agents of the present application recognize and bind to CD20 and one or more antigens found on one or more immune cells. These binding agents include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes, T helper cells, natural killer T cells), B lymphocytes, plasma cells, dendritic cells, or subsets thereof. In some embodiments, the CD20 binding agents specifically bind to an antigen of interest, effectively directly or indirectly recruiting one or more immune cells. In exemplary embodiments, the CD20 binding agents of the present invention can directly or indirectly recruit immune cells (e.g., dendritic cells) to a site of action (such as, but not limited to, the tumor microenvironment).

[0151] In various embodiments, the multispecific CD20 binding agents of the present application recognize and bind to CD20 and one or more antigens found on cancer or tumor cells. In these embodiments, the CD20 binding agents of the present invention may have enhanced selectivity for CD20 positive cancer or tumor cells by binding to two or more antigens on the cancer or tumor cells (i.e., CD20 and another cancer or tumor antigen).

[0152] In some embodiments, the CD20 binding agents of the invention can be used or find use in methods involving shifting the balance of immune cells for immune attack of tumors. For example, the CD20 binding agents of the invention can shift the proportion of immune cells at clinically significant sites in favor of cells that can kill and / or suppress tumors (e.g., T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, antitumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, and tumor-protecting cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor-associated neutrophils (TANs), M2 macrophages, tumor-associated macrophages (TAMs), or subsets thereof). In some embodiments, the CD20 binding agents of the invention can increase the ratio of effector T cells to regulatory T cells.

[0153] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a cancer or tumor cell. In some embodiments, the targeting moiety directly or indirectly recruits tumor cells. For example, in some embodiments, recruitment of tumor cells is to one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress the tumor cells.

[0154] Tumor cell or cancer cell refers to the uncontrolled proliferation of cells or tissues and / or the abnormally increased cell viability and / or inhibition of apoptosis, which interferes with the normal function of the body's organs and systems.For example, tumor cell includes benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Exemplary tumor cells include, but are not limited to, the following cells: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; renal cancer; laryngeal cancer; leukemia; hepatoma; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; lymphomas such as Hodgkin lymphoma and non-Hodgkin lymphoma, as well as B-cell lymphomas (such as low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; Bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD) as well as abnormal blood vessel proliferation, edema (e.g., associated with brain tumors), and Meigs syndrome associated with phakomatosis.

[0155] Tumor cells, or cancer cells, include, but are not limited to, carcinomas, such as various subtypes, e.g., adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma, sarcomas (e.g., bone and soft tissue), leukemias (e.g., acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, hairy cell, etc.), lymphomas and myelomas (e.g., Hodgkin's and non-Hodgkin's lymphoma, light chain, non-secretory, MGUS, and plasmacytoma, etc.), and cancers of the central nervous system, such as brain (e.g., gliomas (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningiomas, pituitary adenomas, neuromas, etc., and spinal tumors (e.g., meningiomas and neurofibromas).

[0156] In certain embodiments, cancer or tumor cells refer to leukemia or lymphoma cells, such as the following cells: B-cell lymphoma, non-Hodgkin's lymphoma (NHL), lymphocytic predominant subtype Hodgkin's lymphoma, precursor B-cell lymphoblastic leukemia / lymphoma, mature B-cell neoplasms, B-cell chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), low grade leukemia, ... Follicular lymphoma (FL) such as grade, intermediate and high grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma, MALT-type marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic-type marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, multiple myeloma, and anaplastic large cell lymphoma (ALCL).

[0157] Exemplary tumor antigens include, but are not limited to, MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE ​​family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE -4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, viral products such as p15, gp75, GM2 and GD2 gangliosides, human papillomavirus proteins, Smad family tumor antigens, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1These include CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, and PMSA. In various embodiments, the CD20-binding agent comprises a targeting moiety that binds to one or more of these tumor cell antigens.

[0158] In some embodiments, the multispecific CD20 binding agents of the invention recognize and bind to CD20 and antigens on tumor cells, hi some embodiments, the multispecific CD20 binding agents directly or indirectly recruit immune cells to the tumor cells or tumor microenvironment.

[0159] In some embodiments, the multispecific CD20 binding agents of the present application include a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a T cell. In some embodiments, the targeting moiety directly or indirectly recruits a T cell. In one embodiment, the antigen recognition domain specifically binds to an effector T cell. In some embodiments, the antigen recognition domain directly or indirectly recruits an effector T cell, for example, in some embodiments, to a therapeutic site (e.g., a locus having one or more diseased cells or cells to be modulated for a therapeutic effect). Exemplary effector T cells include cytotoxic T cells (e.g., αβTCR, CD3 + , CD8 + , CD45RO + );CD4 + Effector T cells (e.g., αβTCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL-7R / CD127 + );CD8 + Effector T cells (e.g., αβTCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL-7R / CD127 + ); effector memory T cells (e.g., CD62Llow, CD44+ , TCR, CD3 + , IL-7R / CD127 + , IL-15R + , CCR7low); central memory T cells (e.g., CCR7 + , CD62L + , CD27 + ; or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL-7R / CD127 + , IL-15R + );CD62L + Effector T cells; early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - ) (TemE and TemL, respectively) + Effector memory T cells (TEM); CD127 ( + )CD25(low / -) effector T cells;CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + ; or αβTCR, CD3 + , CD4 + , IL-12R + , IL-IFNγR + , CXCR3 + ), TH2 effector T cells (e.g., CCR3 + , CCR4 + and CCR8 + ; or αβTCR, CD3 + , CD4 + , IL-4R + , IL-33R + , CCR4 + , IL-17RB +, CRTH2 + ); TH9 effector T cells (e.g., αβTCR, CD3 + , CD4 + ); TH17 effector T cells (e.g., αβTCR, CD3 + , CD4 + , IL-23R + , CCR6 + , IL-1R + );CD4 + CD45RO + CCR7 + Effector T cells, ICOS + Effector T cells; CD4 + CD45RO + CCR7( - ) effector T cells; including effector T cells that secrete IL-2, IL-4 and / or IFN-γ.

[0160] The main ingredient in the T-speed range is a (small-scale spell). Protein:CD8, CD3, SLAMF4, IL-2Rα,4 -1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM 1, IL-6R, CCR3, IL-7Rα, CCR4, CXCRl / IL-SRA, CCR5, CCR6, IL-10Rα, CCR7, I L-l0Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4. ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, Linkα4 / CD49d, CDS CatalystE / CD103 CD6, CatalystM / CD 11b, CDS, scaffoldαX / CD11c, scaffoldβ2 / CDlS、KIR / CD15S、CD27 / TNFRSF7、KIR2DL1、CD2S、KIR2DL3、CD30 / TNFRSFS 、KIR2DL4 / CD15Sd 、CD31 / PECAM-1 、KIR2DS4 、CD40リ / TNFSF5 、LAG-3 、CD43 LAIR1 CD45 LAIR2 CDS3 B4-R1 CDS4 / SLAMF5 NCAM-L1 CD94N KG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NT B-A / SLAMF6, IL-2Rγ, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1 CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-セセチン, CXCR3, SIRPβ1, CXCR4 SLAM, CXCR6, TCCR / WSX-1, DNAM-1, EMPRIN / CD147, TIM-1, Eph B6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, T.S IM-6 TNFR1 / TNFRSF1A TNFRIII / TNFRSF1B TRAILRI / TNFRSFlO A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C.These include IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLPR. In various embodiments, the CD20 binding agent comprises a targeting moiety that binds to one or more of these exemplary T cell antigens.

[0161] As non-limiting examples, in various embodiments, the chimeric proteins of the invention have a targeting moiety directed against checkpoint markers expressed on T cells, such as one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0162] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with B cells. In some embodiments, the targeting moiety directly or indirectly recruits B cells, for example, in some embodiments, to a therapeutic site (e.g., a locus having one or more diseased cells or cells to be modulated for a therapeutic effect). Exemplary B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, and CDw150. In various embodiments, the CD20 binding agents comprise a targeting moiety that binds to one or more of these exemplary B cell antigens.

[0163] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a natural killer cell. In some embodiments, the targeting moiety directly or indirectly recruits natural killer cells, for example, in some embodiments, to a therapeutic site (e.g., a locus having one or more diseased cells or cells to be modulated for a therapeutic effect). Exemplary natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, Kir1α, DNAM-1, LMIR5 / CD300LB, Fc-epsilon RII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NK G2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1 epsilon, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In various embodiments, the CD20-binding agents comprise a targeting moiety that binds to one or more of these exemplary NK cell antigens.

[0164] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with macrophages / monocytes. In some embodiments, the targeting moiety directly or indirectly recruits macrophages / monocytes, for example, in some embodiments, to a therapeutic site (e.g., a locus having one or more diseased cells or cells to be modulated for a therapeutic effect). Exemplary macrophage / monocyte antigens of interest include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin alpha 4 / CD49d, BLAME / SLAMF8, integrin alpha X / CDllc, CCL6 / C10, integrin beta 2 / CD18, CD155 / PVR, integrin beta 3 / CD61, CD31 / PECAM-1, latexin, CD36 / SR-B3, leukotriene B4 R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, Endoglin / CD105, Osteoactivin / GPNMB, Fc-γ RI / CD64, osteopontin, Fc-γ RIIB / CD32b, PD-L2, Fc-γ RIIC / CD32c, Siglec-3 / CD33, Fc-γ RIIA / CD32a, SIGNR1 / CD209, Fc-γ RIII / CD16, SLAM, GM-CSF R α, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γ Rl, TLR4, IFN-γR2, TREM-l, IL-l RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF 4, IL-10 R α, ALCAM, IL-10 R β, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α 4 / CD49d, CCR5, integrin α M / CDll b, CCR8, integrin α X / CDllc, CD155 / PVR, integrin β 2 / CD18, CD14, integrin β 3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotrienes B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD3 00LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γ RI / CD64, PSGL-1, Fc-γ RIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF R alpha, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL-6 R, TREM-2, CXCR1 / IL-8 RA, TREM-3, and TREML1 / TLT-1. In various embodiments, the CD20-binding agent comprises a targeting moiety that binds to one or more of these exemplary macrophage / monocyte antigens.

[0165] In some embodiments, the multispecific CD20 binding agents of the present application include a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a dendritic cell. In some embodiments, the targeting moiety directly or indirectly recruits dendritic cells, for example, in some embodiments, to a therapeutic site (e.g., a diseased cell or a locus having one or more cells to be modulated for a therapeutic effect). Exemplary dendritic cell antigens of interest include, for example, Clec9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-Pl / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23 p40, 4-amino-1,8-Naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, integrin α 4 / CD49d, Aag, integrin β 2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, Clq R1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LB CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, osteoactivin, GPNMB, Chern 23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, DCE205, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC, SLAM, EMPRINS / CD147, TCCR / WSX-1, Fc-γ R1 / CD64, TLR3, Fc-γ RIIB / CD32b, TREM-1, Fc-γ These include RIIC / CD32c, TREM-2, Fc-gamma RIIA / CD32a, TREM-3, Fc-gamma RIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, and vanilloid R1. In various embodiments, the CD20 binding agent comprises a targeting moiety that binds to one or more of these exemplary DC antigens. In some embodiments, the CD20 binding agent comprises a targeting moiety that binds to Clec9A, and such CD20 binding agents find use, for example, in the treatment of multiple sclerosis.

[0166] In some embodiments, a multispecific CD20-binding agent of the present application comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof. In some embodiments, the antigen recognition domain directly or indirectly recruits, for example, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof, in some embodiments, to a treatment site (e.g., a locus having one or more diseased cells or cells that modulate the therapeutic effect).

[0167] In some embodiments, the multispecific CD20-binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with megakaryocytes and / or platelets. Exemplary megakaryocyte and / or platelet antigens of interest include, for example, GP IIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the CD20-binding agents comprise a targeting moiety that binds to one or more of these exemplary megakaryocyte and / or platelet antigens.

[0168] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with an erythrocyte. Exemplary erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and rhesus monkey antigens. In various embodiments, the CD20 binding agents comprise a targeting moiety that binds to one or more of these exemplary erythrocyte antigens.

[0169] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a mast cell. Exemplary mast cell antigens of interest include, for example, SCFR / CD117, Fc ε These include RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCERL1, FCER2, TPSABl. In various embodiments, the CD20 binding agent comprises a targeting moiety that binds to one or more of these mast cell antigens.

[0170] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with basophils. Exemplary basophil antigens of interest include, for example, Fc ε These include RI, CD203c, CD123, CD13, CD107a, CD107b, and CD 164. In various embodiments, the CD20-binding agent comprises a targeting moiety that binds to one or more of these basophil antigens.

[0171] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with neutrophils. Exemplary neutrophil antigens of interest include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 protein (LFA-1, CR3, and p150,95), CD45, CD67, and CD177. In various embodiments, the CD20 binding agents comprise a targeting moiety that binds to one or more of these neutrophil antigens.

[0172] In some embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with eosinophils. Exemplary eosinophil antigens of interest include, for example, CD35, CD44, and CD69. In various embodiments, the CD20 binding agents comprise a targeting moiety that binds to one or more of these eosinophil antigens.

[0173] In various embodiments, the multispecific CD20 binding agents of the present application comprise a targeting moiety having a recognition domain that specifically binds to a suitable antigen or cell surface marker known to those skilled in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Exemplary tissue-specific markers include, but are not limited to, endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMl, PROCR, SELE, SELP, TEK, THBD, VCAMl, VWF; smooth muscle cell surface markers such as ACTA2, MYHlO, MYHl1, MYH9, MYOCD; ALCAM, CD34, COLlAl, COL1A2, COL3A fibroblast (stromal) cell surface markers such as 1, FAP, PH-4; epithelial cell surface markers such as CD1D, K6IRS2, KRT1O, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCl, TACSTD1; neovasculature markers such as CD13, TFNA, alpha-v beta-3 (αVβ3), E-selectin; adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the CD20 binding agent comprises a targeting moiety that binds to one or more of these antigens. In various embodiments, the targeting moiety of the chimeric protein binds to one or more cells that bear these antigens.

[0174] In various embodiments, the multispecific CD20-binding agents of the present application have one or more targeting moieties directed against one or more of checkpoint markers, e.g., PD-1 / PD-L1 or PD-L2, CD28 / CD80 or CD86, CTLA4 / CD80 or CD86, ICOS / ICOSL or B7RP1, BTLA / HVEM, KIR, LAG3, CD137 / CD137L, OX40 / OX40L, CD27, CD40L, TIM3 / Gal9, and A2aR.

[0175] As non-limiting examples, in various embodiments, the chimeric proteins of the invention (i) comprise a targeting moiety directed against a checkpoint marker expressed on T cells, e.g., one or more of PD-1, PD-L1, PD-L2, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aR, and (ii) the targeting moiety is directed against a cancer cell or tumor cell (e.g., CD20 on a cancer or tumor cell) in conjunction with any of the modified (e.g., mutant) signaling agents described herein.

[0176] As a non-limiting example, in various embodiments, the chimeric protein of the invention comprises the polypeptide of PD-1: EVQLVESGGGLVQAGKSLRLSCAASGSIFSIHAMGWFRQAPGKEREFVAAITWSGGITYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADRAESSWYDYWGQGTQVTVSS, or at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, or sequences that are about 98%, at least about 99%, or 100% identical to the above sequences (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% identical to the sequence of the targeting moiety.

[0177] As a further non-limiting example, in various embodiments, the chimeric protein of the invention comprises the polypeptide of PD-L1: EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAKCWFRQAPGKEREWVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAARHGGPLTVEYFFDYWGQGTQVTVSS, or at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, or sequences that are about 98%, at least about 99%, or 100% identical to the above sequences (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% identical to the sequence of the targeting moiety.

[0178] Linkers and Functional Groups In various embodiments, the CD20 binding agent may include one or more functional groups, residues, or moieties. In various embodiments, one or more functional groups, residues, or moieties are attached or genetically fused to any of the signaling agents or targeting moieties described herein. In some embodiments, such functional groups, residues, or moieties impart one or more desired properties or functionalities to the CD20 binding agent of the present application. Examples of such functional groups and examples of techniques for introducing them into CD20 binding agents are known in the art, see, for example, Remington's Pharmaceutical Sciences, 16th Edition, Mack Publishing Co., Easton, Pa (1980).

[0179] In some embodiments, the functional group, residue, or moiety comprises a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG). In some embodiments, the attachment of a PEG moiety increases the half-life and / or reduces the immunogenicity of the CD20 binding protein. Generally, any suitable form of PEGylation can be used, such as PEGylation used in the art, for example, for antibodies and antibody fragments (including, but not limited to, single domain antibodies such as VHHs). See, for example, Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003), and WO 04060965, the entire contents of which are incorporated herein by reference. A variety of reagents for protein PEGylation are also commercially available, for example from Nektar Therapeutics (USA). In some embodiments, site-specific PEGylation is used, particularly via cysteine ​​residues (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003), the entire contents of which are incorporated herein by reference). For example, for this purpose, PEG may be attached to a cysteine ​​residue naturally occurring in the CD20 binding agent of the present application. In some embodiments, the CD20 binding agent of the present application is modified to suitably introduce one or more cysteine ​​residues for attaching PEG, or an amino acid sequence containing one or more cysteine ​​residues for attaching PEG may be fused to the amino- and / or carboxy-terminus of the CD20 binding agent using techniques known in the art.

[0180] In some embodiments, the functional group, residue, or moiety comprises N-linked or O-linked glycosylation. In some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and / or post-translational modification.

[0181] In some embodiments, the functional group, residue, or moiety comprises one or more detectable labels or other signal-generating groups or moieties. Suitable labels and techniques for attaching, using, and detecting them are known in the art and include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and fluorescent metals, such as Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels, or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate esters, dioxetanes, or GFP and its analogs), metals, metal chelates, or metal cations, or in vivo, in vitro, or in vivo detection. Other metals or metal cations that are particularly suitable for use in in situ diagnosis and imaging include chromophores and enzymes such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Other suitable labels include moieties that can be detected using NMR or ESR spectroscopy. Such labelled VHHs and polypeptides of the present application may be used, for example, for in vitro, in vivo or in situ assays (such as immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays"), as well as for in vivo diagnostic and imaging purposes, depending on the choice of the particular label.

[0182] In some embodiments, the functional group, residue, or moiety comprises a tag attached or genetically fused to the CD20-binding agent. In some embodiments, the CD20-binding agent may comprise a single tag or multiple tags. The tag is, for example, a peptide, sugar, or DNA molecule that does not inhibit or prevent binding of the CD20-binding agent to CD20 or other antigens of interest, such as tumor antigens. In various embodiments, the tag is at least about 3-5 amino acids in length, at least about 5-8 amino acids in length, at least about 8-12 amino acids in length, at least about 12-15 amino acids in length, or at least about 15-20 amino acids in length. Exemplary tags are described, for example, in U.S. Patent Publication No. 2013 / 0058962. In some embodiments, the tag is an affinity tag, such as glutathione-S-transferase (GST) and histidine (His) tags. In one embodiment, the CD20-binding agent comprises a His tag. In one embodiment, the CD20-binding agent comprises an HA tag.

[0183] In some embodiments, the functional group, residue, or moiety comprises a chelating group, for example, to chelate one of a metal or metal cation. Suitable chelating groups include, for example, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0184] In some embodiments, the functional group, residue, or moiety comprises a functional group that is part of a specific binding pair, such as a biotin-(strept)avidin binding pair. Such functional groups can be used to link the CD20 binding agent of the present application to another protein, polypeptide, or compound by binding to the other half of the binding pair, i.e., through the formation of a binding pair. For example, the CD20 binding agent of the present application can be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such a conjugated CD20 binding agent can be used as a reporter, for example, in a diagnostic system in which a detectable signal generating agent is conjugated to avidin or streptavidin. Such binding pairs can be used to bind the CD20 binding agent to a carrier, such as a carrier that is suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described by Cao and Suresh, Journal of Drug Targeting, 8,4,257 (2000). Such binding pairs can also be used to link therapeutically active agents to the CD20-binding agents of the present application.

[0185] In some embodiments, the CD20 binding agents of the present invention optionally include one or more linkers. In some embodiments, the CD20 binding agents include a linker connecting each binding region and / or targeting moiety. In some embodiments, the CD20 binding agents include a linker connecting each signaling agent and targeting moiety (or, if there is more than one targeting moiety, connecting the signaling agent to one of the targeting moieties). In some embodiments, linkers can be utilized to link various functional groups, residues, or moieties described herein to the CD20 binding agents. In some embodiments, the linker is a single amino acid or multiple amino acids that do not affect or reduce the stability, orientation, binding, neutralization, and / or clearance properties of the binding region and binding protein. In various embodiments, the linker is selected from a peptide, a protein, a sugar, or a nucleic acid.

[0186] In some embodiments, the CD20 binding agents of the invention comprise a linker connecting the targeting moiety and the signaling agent, hi some embodiments, the chimeric proteins of the invention comprise a linker within the signaling agent (e.g., in the case of single chain TNF, two linkers can be included to generate a trimer).

[0187] The present application contemplates the use of various linker sequences. In various embodiments, the linker is derived from a naturally occurring multidomain protein or is an empirical linker, for example, as described in Chichili et al. (2013), Protein Sci. 22(2): 153-167, Chen et al. (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker may be designed using a linker design database and computer programs such as those described in Chen et al. (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, and Crasto et al. (2000), Protein Eng. 13(5): 309-312, the entire contents of which are incorporated herein by reference. In various embodiments, the linker may be functional. For example, but not limited to, a linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve biological activity of a CD20 binding agent of the invention.

[0188] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids in length. For example, the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is a polypeptide. In some embodiments, the linker is more than about 100 amino acids in length. For example, the linker may be greater than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is flexible. In other embodiments, the linker is rigid.

[0189] In some embodiments, the length of the linker allows the targeting moiety and the signal transduction agent to effectively bind to their receptor.For example, in some embodiments, the length of the linker allows one of the targeting moieties and the signal transduction agent to effectively bind to the receptor on the same cell, and the other targeting moiety to effectively bind to another cell.Exemplary paired cells are provided elsewhere herein.

[0190] In some embodiments, the length of the linker is at least equal to the minimum distance between one of the targeting moieties and the binding site of the signaling agent for the receptor on the same cell.In some embodiments, the length of the linker is at least 2 times, 3 times, or 4 times, or 5 times, or 10 times, or 20 times, or 25 times, or 50 times, or 100 times or more between the minimum distance between one of the targeting moieties and the binding site of the signaling agent for the receptor on the same cell.

[0191] In some embodiments, a linker connects two targeting moieties to one another, the linker having a short length, and a linker connects a targeting moiety and a signaling agent, the linker being longer than the linker connecting the two targeting moieties. For example, the difference in amino acid length between the linker connecting two targeting moieties and the linker connecting the targeting moiety and the signaling agent may be about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids. In some embodiments, the linker is flexible. In another embodiment, the linker is rigid.

[0192] In one embodiment, the linker is AAA.

[0193] In various embodiments, the linker substantially comprises glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly4Ser)n, where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8. In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS. Additional exemplary linkers include, but are not limited to, the sequences LE, GGGGS, (GGGGS) n (n=1~4), (Gly)8, (Gly)6, (EAAAK) n (n=1-3), A(EAAAK) n A(n=2~5), AEAAAKEAAAKA, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, and (XP) nIn various embodiments, the linker has the formula:

[0194] In some embodiments, the linker is a hinge region of an antibody (eg, IgG, IgA, IgD, and IgE (including subclasses, eg, IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker is an antibody hinge region (e.g., IgG, IgA, IgD, and IgE (including subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, as well as IgA1 and IgA2). The hinge region found in antibodies of the IgG, IgA, IgD, and IgE classes serves as a flexible spacer that allows the Fab portion to move freely in space. In contrast to constant regions, hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region differs among IgG subclasses. The hinge region of IgG1 contains 216-231 amino acids, and this region is freely flexible, allowing the Fab fragments to rotate about their axis of symmetry and move within a sphere central to the first of the two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and 4 1 disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid poly-proline double helix stabilized by an extra inter-heavy chain disulfide bridge. These properties limit the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by a unique extended hinge region (approximately four times longer than the IgG1 hinge). This extended hinge region contains 62 amino acids (including 21 prolines and 11 cysteines) that form an inflexible poly-proline double helix. In IgG3, the Fab fragment is relatively far from the Fc fragment, conferring greater flexibility to the molecule. The extended hinge of IgG3 also results in a higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge region reportedly decreases in the order IgG3>IgG1>IgG4>IgG2.

[0195] According to crystallographic studies, immunoglobulin hinge regions can be functionally further subdivided into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al. (1992), Immunological Reviews 130:87. The upper hinge region is H1 The upper hinge region includes the amino acids from the carboxyl terminus of the heavy chain to the first residue in the hinge that restricts movement, with the first cysteine ​​residue generally forming an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of that segment of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region includes the C H2 Binds to the amino terminus of the domain, C H2 (ibid.). The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys, which when dimerized by disulfide bond formation results in a cyclic octapeptide that is believed to act as a pivot and thus confer flexibility. In various embodiments, the linkers of the invention comprise one or two or three upper hinge, core, and lower hinge regions of any antibody (e.g., IgG, IgA, IgD, and IgE (including subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, as well as IgA1 and IgA2)). The hinge region can also contain one or more glycosylation sites, including several structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, which is believed to be an advantageous property of secreted immunoglobulins. In various embodiments, the linker of the present application comprises one or more glycosylation sites, hi various embodiments, the linker is the hinge-CH2-CH3 domain of a human IgG4 antibody.

[0196] If desired, the CD20 binding agents of the invention can be H 2 domain and C HIt can be linked to an antibody Fc region comprising one or both of the three domains, and optionally a hinge region. For example, a vector encoding the CD20 binding agent of the present invention linked to an Fc region as a single nucleotide sequence can be used to prepare such a polypeptide.

[0197] In some embodiments, the linker is a synthetic linker, such as PEG.

[0198] In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve the biological activity of the CD20 binding agent of the present invention. In another example, the linker may function to target the CD20 binding agent to a particular cell type or location.

[0199] Modification and production of CD20-binding agents In various embodiments, the CD20 binding agent comprises a targeting moiety that is a VHH. In various embodiments, the VHH is not limited to a specific biological source or a specific preparation method. For example, the VHH can generally be obtained by: (1) the VHH of a naturally occurring heavy chain antibody; H (2) isolating the naturally occurring V H (3) expressing a nucleotide sequence encoding a naturally occurring V domain; H "Humanizing" the H domain, or such a humanized V H(4) "camelizing" a naturally occurring VH domain from any animal species, e.g. a mammalian species such as from human, or expressing a nucleic acid encoding such a camelized VH domain; (5) "camelizing" a "domain antibody" or "Dab" as described in the art, or expressing a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known in the art; (7) preparing a nucleic acid encoding a VHH using nucleic acid synthesis techniques known in the art and then expressing the nucleic acid so obtained; and / or (8) any combination of one or more of the above.

[0200] In one embodiment, the CD20 binding agent is a V-chain of a naturally occurring heavy chain antibody directed against human CD20. H In some embodiments, such a VHH comprises a VHH corresponding to the H domain. H The H sequence is generally prepared by suitably immunizing a camelid species with a CD20 molecule (i.e. so as to elicit an immune response and / or heavy chain antibodies directed against CD20), obtaining a suitable camelid-derived biological sample (such as a blood sample, or any sample of B cells), and isolating VH sequences directed against CD20 starting from the sample using any suitable known technique. H In some embodiments, the VH sequence can be generated or obtained by generating a naturally occurring VH sequence for CD20. H The H domain is the V domain of camelids. HThe naive VH sequences can be obtained, for example, by screening such libraries with CD20 or at least one part, fragment, antigenic determinant or epitope thereof, using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 9937681, WO 0190190, WO 03025020, and WO 03035694, the entire contents of which are incorporated herein by reference. In some embodiments, the naive VH sequences can be obtained by screening such libraries using CD20 or at least one part, fragment, antigenic determinant or epitope thereof, using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 9937681, WO 0190190, WO 03025020, and WO 03035694, the entire contents of which are incorporated herein by reference. H Improved synthetic or semi-synthetic libraries derived from H libraries, such as naive VH libraries, by techniques such as random mutagenesis and / or CDR shuffling, as described in International Publication No. WO 0043507, the entire contents of which are incorporated herein by reference. H V obtained from the H library H In some embodiments, a VH library directed against CD20 may be used. H Another technique for obtaining H sequences is to suitably immunize a transgenic mammal capable of expressing heavy chain antibodies (i.e., to elicit an immune response and / or heavy chain antibodies directed against CD20), obtain a suitable biological sample from the transgenic mammal (e.g., a blood sample, or any sample of B cells), and then, starting from the sample, to obtain V sequences directed against CD20, using any suitable known technique. H For example, for this purpose, heavy chain antibody expressing mice and further methods and techniques described in WO02085945 and WO04049794, the entire contents of which are incorporated herein by reference, can be used.

[0201] In one embodiment, the CD20 binding agent is a "humanized" VHH, i.e., a naturally occurring V HThe VHHs include those that are "humanized" by replacing one or more amino acid residues in the amino acid sequence of the H sequence (and in particular the framework sequences) with one or more of the amino acid residues present at the corresponding position(s) in the VH domain from a conventional four-chain antibody of human origin. This can be done using humanization techniques known in the art. In some embodiments, possible humanizing substitutions, or combinations of humanizing substitutions, can be determined by methods known in the art, for example, by comparing the sequence of the VHH with the sequence of a naturally occurring human VH domain. In some embodiments, the humanizing substitutions are selected such that the resulting humanized VHH still retains advantageous functional properties. Generally, as a result of humanization, the VHHs of the present application have a similar structure to the corresponding naturally occurring VH domains. H In comparison with the H domain, it can be more "human-like" while still retaining favorable properties such as reduced immunogenicity. In various embodiments, the humanized VHH of the present application can be obtained by any suitable method known in the art and thus resembles a naturally occurring VHH. H It is not strictly limited to polypeptides obtained using a polypeptide containing an H domain as a starting material.

[0202] In one embodiment, the CD20 binding agent is a "camelized" VHH, i.e., one or more amino acid residues within the amino acid sequence of a naturally occurring VH domain from a traditional four chain antibody are replaced with the VH domain of a camelid heavy chain antibody. HThese include VHHs that are "camelized" by substitution at one or more of the amino acid residues present at the corresponding position(s) in the H domain. In some embodiments, such "camelization" substitutions are made at and / or inserted at amino acid positions present at the VH-VL interface and / or at so-called camelid-characteristic residues (see, for example, WO 9404678, the entire contents of which are incorporated herein by reference). In some embodiments, the VH sequence used as starting material or starting point for generating or designing a camelized VHH is a VH sequence of mammalian origin, such as a human VH sequence, for example a VH3 sequence. In various embodiments, the camelized VHH can be obtained in any suitable manner known in the art (i.e. as indicated in points (1) to (8) above) and is therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VH domain as starting material.

[0203] In various embodiments, both "humanized" and "camelized" refer to naturally occurring V H This can be done by providing a nucleotide sequence encoding the H or VH domain, respectively, and then altering one or more codons in the nucleotide sequence in a manner known in the art in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. This nucleic acid can then be expressed in a manner known in the art, thereby providing the desired VHH of the present application. Alternatively, the naturally occurring VHH, respectively, can be altered in a manner known in the art. H Based on the amino acid sequence of the H domain or VH domain, the amino acid sequence of the desired humanized or camelized VHH of the present application can be designed and then synthesized de novo using peptide synthesis techniques known in the art. HBased on the amino acid or nucleotide sequence of the H or VH domain, a nucleotide sequence encoding the desired humanized or camelized VHH, respectively, can be designed and then synthesized de novo using nucleic acid synthesis techniques known in the art, and the nucleic acid thus obtained can then be expressed in a manner known in the art, resulting in the desired VHH of the present application. H Other suitable methods and techniques for obtaining the VHHs of the present application and / or nucleic acids encoding same, starting from a VH sequence, are known in the art and can include, for example, one or more parts of one or more naturally occurring VH sequences (e.g. one or more FR sequences and / or CDR sequences), one or more naturally occurring VH sequences, H It may comprise a combination of one or more parts of the H sequence (e.g. one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner, resulting in a VHH of the present application or a nucleotide sequence or nucleic acid encoding same.

[0204] The method of producing the CD20 binding agent of the present application is described herein. For example, the DNA sequence encoding the CD20 binding agent of the present application can be chemically synthesized using methods known in the art. The synthetic DNA sequence can be ligated to other suitable nucleotide sequences, such as expression control sequences, to produce a gene expression construct encoding the desired CD20 binding agent. Thus, in various embodiments, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding the CD20 binding agent of the present application.

[0205] The nucleic acid encoding the CD20 binding agent of the present application may be incorporated (ligated) into an expression vector and introduced into a host cell by transfection, transformation or transduction techniques. For example, the nucleic acid encoding the CD20 binding agent of the present application may be introduced into a host cell by retroviral transduction. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cell may be grown under conditions that allow the host cell to express the gene encoding the CD20 binding agent of the present application. Thus, in various embodiments, the present application provides an expression vector comprising a nucleic acid encoding the CD20 binding agent of the present application. In various embodiments, the present application further provides a host cell comprising such an expression vector.

[0206] The specific expression and purification conditions vary depending on the expression system used.For example, when expressing a gene in E. coli, the gene is first cloned into an expression vector by placing a suitable bacterial promoter, such as Trp or Tac, and the engineered gene downstream from a prokaryotic signal sequence.In another example, when expressing an engineered gene in a eukaryotic host cell, such as a CHO cell, for example, the gene is first inserted into an expression vector that includes a suitable eukaryotic promoter, secretion signal, enhancer, and various introns.The gene construct can be introduced into the host cell using transfection, transformation, or transduction techniques.

[0207] The CD20 binding agent of the present application can be produced by growing a host cell transfected with an expression vector encoding the CD20 binding agent under conditions that allow expression of the protein. After expression, the protein can be harvested and purified using techniques well known in the art, for example affinity tags such as glutathione-S-transferase (GST) and histidine (His) tags, or by chromatography. In one embodiment, the CD20 binding agent comprises a His tag. In one embodiment, the CD20 binding agent comprises a His tag and a proteolytic site that allows cleavage of the His tag.

[0208] Thus, in various embodiments, the application provides a nucleic acid encoding a CD20-binding agent of the application. In various embodiments, the application provides a host cell comprising a nucleic acid encoding a CD20-binding agent of the application.

[0209] Pharmaceutically acceptable salts and excipients The CD20 binding agent described herein has a sufficiently basic functional group that can react with inorganic or organic acids, or a carboxyl group that can react with inorganic or organic bases, and can form pharmaceutically acceptable salts.Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids, as is well known in the art.Such salts include, for example, the pharmaceutically acceptable salts described in Journal of Pharmaceutical Science, 66,2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002.These are incorporated herein in their entirety by reference.

[0210] Non-limiting examples of pharma- ceutically acceptable salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, salts of naphthalene-2-benzoate, isobutyrate, phenylbutyrate, alpha-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0211] The term "pharmaceutically acceptable salt" also refers to a salt of a composition of the present application having an acidic functional group (such as a carboxylic acid functional group) and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium and lithium; hydroxides of alkaline earth metals such as calcium, magnesium; hydroxides of other metals such as aluminum and zinc; unsubstituted or hydroxy-substituted mono-, di- or trialkylamines, ammonia such as dicyclohexylamine, organic amines; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis- or tris-(2-OH-lower alkylamines), such as mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

[0212] In some embodiments, the compositions described herein are in the form of a pharma- ceutically acceptable salt.

[0213] Pharmaceutical Compositions and Formulations In various embodiments, the present application relates to a pharmaceutical composition comprising a CD20 binding agent as described herein and a pharma- ceutically acceptable carrier or excipient. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition that comprises a pharma- ceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharma- ceutically acceptable excipient to provide a form for proper administration.

[0214] In various embodiments, the pharmaceutical excipients may be liquids such as water and oils, such as those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients may be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickening agents, lubricants and coloring agents may be used. In one embodiment, the pharmaceutically acceptable excipients are sterilized when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline and aqueous dextrose and glycerol solutions may also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Any agent described herein can also contain, if desired, a small amount of a wetting agent, or an emulsifying agent, or a pH buffering agent. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (edited by Alfonso R. Gennaro, 19th Edition 1995), which is incorporated herein by reference.

[0215] The present application includes the described pharmaceutical compositions (and / or additional therapeutic agents) in various formulations. Any of the inventive pharmaceutical compositions (and / or additional therapeutic agents) described herein may take the form of a solution, suspension, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, gelatin capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, freeze suspensions, dry powders, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In a further embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

[0216] If necessary, the pharmaceutical composition of the present invention (and / or additional agent) can also contain a solubilizing agent.The agent can also be delivered using a suitable vehicle or delivery device known in the art.The combination therapy outlined herein can be delivered simultaneously in a single delivery vehicle or delivery device.

[0217] The formulations containing the pharmaceutical composition (and / or additional agent) of the present application may be conveniently provided in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agent into association with the carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately contacting the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation (e.g., as wet or dry granules, powder blends, etc., followed by tableting using conventional methods known in the art).

[0218] In various embodiments, any pharmaceutical composition described herein (and / or additional agents) are formulated in accordance with routine procedures into compositions compatible with the modes of administration described herein.

[0219] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. Administration may be local or systemic. In some embodiments, administration is orally. In other embodiments, administration is by parenteral injection. The mode of administration may be at the discretion of the practitioner and will depend in part on the site of the medical condition. In most cases, administration will result in the release of any agent described herein into the bloodstream.

[0220] In one embodiment, the CD20 binding agent described herein is formulated according to routine procedures as a composition suitable for oral administration. Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents, such as sweeteners, such as fructose, aspartame, or saccharin; flavorings, such as peppermint, wintergreen oil, or cherry; coloring agents; and preservatives, to provide a pharma-ceutical palatable preparation. Additionally, in tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. A selectively permeable membrane surrounding any of the CD20 binding agents described herein that are driven by osmotic activity is also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compounds, which expand and displace the agent or agent composition through an opening. These delivery platforms can provide an essentially zero-order delivery profile, as opposed to the spiked profiles of immediate release formulations. Time-delay materials such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. Suspensions can contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and mixtures thereof.

[0221] Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be prepared in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injectable medium immediately before use. They can include, for example, suspending or dispersing agents known in the art. Suitable formulation components for parenteral administration include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as sodium chloride or dextrose.

[0222] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and preserved against microorganisms. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof.

[0223] The compositions provided herein, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation (i.e., "nebulized"). The aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0224] Any pharmaceutical composition of the present invention (and / or additional agent) described herein can be administered by controlled release or sustained release means or by delivery devices known to those skilled in the art.Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556 and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms may be useful, for example, using hydropropyl cellulose, hydropropyl methyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide controlled or sustained release of one or more active ingredients, and to provide desired release profiles at various rates.Suitable controlled or sustained release formulations known to those skilled in the art, such as those described herein, can be easily selected for use with the active ingredients of the agents described herein.Thus, the present application provides single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel caps, and caplets, adapted for controlled or sustained release.

[0225] Controlled or sustained release of an active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, changes in temperature, stimulation with light of appropriate wavelengths, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0226] In another embodiment, a controlled release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose. (See, e.g., Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems (discussed in the review by Langer, 1990, Science 249:1527-1533) can be used.

[0227] The pharmaceutical formulation is preferably sterile. Sterilization can be achieved, for example, by filtration through a sterile filtration membrane. If the composition is lyophilized, filter sterilization can be performed prior to or after lyophilization and reconstitution.

[0228] Administration and Dosage It will be understood that the actual dose of the CD20 binding agent administered according to the present application will vary according to the particular formulation and mode of administration. Many factors that may modify the action of the CD20 binding agent (e.g., body weight, sex, diet, administration time, administration route, excretion rate, subject condition, drug combination, genetic configuration and reaction sensitivity) can be taken into consideration by those skilled in the art. Administration can be performed continuously or in one or more separate doses within the maximum tolerated dose. The optimal administration rate for a given set of conditions can be ascertained by those skilled in the art using conventional dosage tests.

[0229] In some embodiments, a suitable dosage of a CD20-binding agent is within the range of about 0.01 mg / kg to about 10 g / kg of the subject's body weight, about 0.01 mg / kg to about 1 g / kg of the subject's body weight, about 0.01 mg / kg to about 100 mg / kg of the subject's body weight, about 0.01 mg / kg to about 10 mg / kg of the subject's body weight, e.g., about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0. ...8 mg / kg, about 0.9 mg / kg, about 0.9 mg / kg, about 0.1 mg / kg 0.5 mg / kg body weight, about 0.6 mg / kg body weight, about 0.7 mg / kg body weight, about 0.8 mg / kg body weight, about 0.9 mg / kg body weight, about 1 mg / kg body weight, about 1.1 mg / kg body weight, about 1.2 mg / kg body weight, about 1.3 mg / kg body weight, about 1.4 mg / kg body weight, about 1.5 mg / kg body weight, about 1.6 mg / kg body weight, about 1.7 mg / kg body weight, about 1.8 mg / kg body weight, 1.9 mg / kg body weight, about 2 mg / kg body weight, about 3 mg / kg body weight, about 4 mg / kg body weight, about 5 mg / kg body weight, about 6 mg / kg body weight, about 7 mg / kg body weight, about 8 mg / kg body weight, about 9 mg / kg body weight, about 10 mg / kg body weight, about 100 mg / kg body weight, about 1 g / kg body weight, about 10 g / kg body weight, including all values ​​and ranges therebetween.

[0230] The individual doses of the CD20 binding agents are, for example, about 0.01 mg to about 100 g, about 0.01 mg to about 75 g, about 0.01 mg to about 50 g, about 0.01 mg to about 25 g, about 0.01 mg to about 10 g, about 0.01 mg to about 7.5 g, about 0.01 mg to about 5 g, about 0.01 mg to about 2.5 g, about 0.01 mg to about 1 g, about 0.01 mg to about 100 mg, about 0.1 mg to about 100 mg, about 0.1 mg The present invention can be administered in a unit dosage form containing about 0.1 mg to about 90 mg, about 0.1 mg to about 80 mg, about 0.1 mg to about 70 mg, about 0.1 mg to about 60 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 40 mg, about 0.1 mg to about 30 mg, about 0.1 mg to about 20 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 1 mg, or about 5 mg to about 80 mg of active ingredient. For example, unit dosage forms may include about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, The amount may be about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g, about 5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g, including all values ​​and ranges therebetween.

[0231] In one embodiment, the CD20 binding agent is administered at a dose of about 0.01 mg / day to about 100 g / day, about 0.01 mg / day to about 75 g / day, about 0.01 mg / day to about 50 g / day, about 0.01 mg / day to about 25 g / day, about 0.01 mg / day to about 10 g / day, about 0.01 mg / day to about 7.5 g / day, about 0.01 mg / day to about 5 g / day, about 0.01 mg / day to about 0.01 mg / day ~ about 2.5g / day, about 0.01mg / day to about 1g / day, about 0.01mg / day to about 100mg / day, about 0.1mg / day to about 100mg / day, about 0.1mg / day to about 95mg / day, about 0.1mg / day to about 90mg / day, about 0.1mg / day to about 85mg / day, about 0.1mg / day to about 80mg / day, about 0.1mg / day to about 75mg / day , about 0.1 mg / day to about 70 mg / day, about 0.1 mg / day to about 65 mg / day, about 0.1 mg / day to about 60 mg / day, about 0.1 mg / day to about 55 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 45 mg / day, about 0.1 mg / day to about 40 mg / day, about 0.1 mg / day to about 35 ...50 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 50 mg / day It is administered in an amount of about 30 mg / day, about 0.1 mg / day to about 25 mg / day, about 0.1 mg / day to about 20 mg, about 0.1 mg / day to about 15 mg / day, about 0.1 mg / day to about 10 mg, about 0.1 mg / day to about 5 mg / day, about 0.1 mg / day to about 3 mg / day, about 0.1 mg / day to about 1 mg / day, or about 5 mg / day to about 80 mg / day. In various embodiments, the CD20-binding agent is administered in a daily dose of about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, The amount may be administered in an amount including about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g, about 5 g, about 7.5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100, and all values ​​and ranges therebetween.

[0232] According to certain embodiments of the application, a pharmaceutical composition comprising a CD20-binding agent may be administered, for example, once or more times per day (e.g., about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, about nine times, or about ten times per day), about once per day, about once every other day, about once every third day, about once per week, about once every two weeks, about once per month, about once every two months, about once every three months, about once every six months, or about once per year.

[0233] Combination Therapies and Additional Therapeutic Agents In various embodiments, the pharmaceutical compositions of the present application are co-administered in combination with an additional therapeutic agent(s). Co-administration can be simultaneous or sequential.

[0234] In one embodiment, the additional therapeutic agent and the CD20 binding agent of the present application are administered to the subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the CD20 binding agent are administered at a time interval of about 60 minutes or less, for example, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 1 minute or less. The administration of the additional therapeutic agent and the CD20 binding agent may be by simultaneously administering a single formulation (e.g., a formulation including the additional therapeutic agent and the CD20 binding agent) or separate formulations (e.g., a first formulation including the additional therapeutic agent and a second formulation including the CD20 binding agent).

[0235] In co-administration, the therapeutic agents do not need to be administered simultaneously if the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the CD20 binding agent overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the CD20 binding agent can be administered sequentially. The term "sequentially" as used herein means that the additional therapeutic agent and the CD20 binding agent are administered more than about 60 minutes apart. For example, the time between the sequential administration of the additional therapeutic agent and the CD20 binding agent can be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, more than about 1 week, or more than about 2 weeks, or more than about 1 month apart. The optimal administration time depends on the metabolism, excretion rate and / or pharmacodynamic activity of the additional therapeutic agent and the CD20 binding agent administered. Either the additional therapeutic agent or the CD20 binding agent cells can be administered first.

[0236] Co-administration also does not require that the therapeutic agents be administered to a subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, e.g., parenterally or non-parenterally.

[0237] In some embodiments, the CD20 binding agents described herein act synergistically when co-administered with another therapeutic agent. In such embodiments, the CD20 binding agent and the additional therapeutic agent may be administered at lower doses than would be used if the agents were used in a monotherapy context.

[0238] In some embodiments, the present application relates to chemotherapeutic agents as additional therapeutic agents. For example, and not by way of limitation, such combinations of CD20-binding agents of the present invention and chemotherapeutic agents find use in the treatment of cancer, as described elsewhere herein. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cytoxan cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelanin; acetogenins (e.g., bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; cally statin; CC-1065 (and its adzenecin, carzelesin, and bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; chlorambucil, chlornaphazine, clophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride hydrochloride), nitrogen mustards such as melphalan, novembitine, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin gamma II, and calicheamicin ωll (e.g., Agnew, Chem. Intl., ed. Engl., 33:183-186 (1994)); dynemicins such as dynemicin A;bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, automycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalacid, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; and carasterone and dromostanolone propionate. propionate, epithiostanol, mepitostanol, testolactone, and other androgens; adrenal anti-insulins such as minoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine;Maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oregon); lesoxan; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., taxol paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Abraxane Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and Taxotere docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitors RFS 2000;difluoromethylornithine (DMFO);retinoids such as retinoic acid;capecitabine;combretastatin;leucovorin (LV);Oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation, as well as pharmacologic acceptable salts, acids or derivatives of any of the above. Additionally, the method of treatment can further include the use of photodynamic therapy.

[0239] In some embodiments, the present application relates to combination therapy using a CD20 binding agent and a chemotherapeutic agent. In some embodiments, the present application relates to administration of a CD20 binding agent to a patient undergoing treatment with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a DNA intercalating agent, such as, but not limited to, doxorubicin, cisplatin, daunorubicin, and epirubicin. In one embodiment, the DNA intercalating agent is doxorubicin.

[0240] In an exemplary embodiment, the CD20 binding agent acts synergistically when co-administered with doxorubicin. In an exemplary embodiment, the CD20 binding agent acts synergistically when co-administered with doxorubicin for use in treating tumors or cancer. For example, co-administration of the CD20 binding agent and doxorubicin may act synergistically to reduce or eliminate tumors or cancers, or to slow the growth, and / or progression, and / or metastasis of tumors or cancers. In an exemplary embodiment, the combination of the CD20 binding agent and doxorubicin may exhibit an improved safety profile compared to the agent used alone in a monotherapy setting. In an exemplary embodiment, the CD20 binding agent and doxorubicin may be administered at a lower dose than the dose used when the agent is used in a monotherapy setting. In some embodiments, the CD20 binding agent comprises a mutant interferon, such as a mutant IFNα. In an exemplary embodiment, the mutant IFNα comprises one or more mutations at positions 148, 149 and 153, such as substitutions M148A, R149A, and L153A, relative to SEQ ID NO:127 or SEQ ID NO:128.

[0241] In some embodiments, the present application relates to combination therapy with one or more immunomodulatory agents, including but not limited to agents that modulate immune checkpoints. In various embodiments, the immunomodulatory agent targets one or more of PD-1, PD-L1, and PD-L2. In various embodiments, the immunomodulatory agent is a PD-1 inhibitor. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of PD-1, PD-L1, and PD-L2. For example, in some embodiments, the immunomodulatory agent is an antibody, such as, but not limited to, nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL328OA (ROCHE). In some embodiments, the immunomodulatory agent targets one or more of CD137 or CD137L. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of CD137 or CD137L. For example, in some embodiments, the immunomodulatory agent is an antibody, such as, but not limited to, urelumab (also known as BMS-663513 and anti-4-1BB antibody). In some embodiments, the chimeric protein of the present invention is combined with urelumab (optionally one or more of nivolumab, lirilumab, and urelumab) for the treatment of solid tumors, and / or B-cell non-Hodgkin's lymphoma, and / or head and neck cancer, and / or multiple myeloma. In some embodiments, the immunomodulatory agent is an agent that targets one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2, and PPP2R5A. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2, and PPP2R5A. For example, in some embodiments, the immunomodulatory agent is an antibody, such as, but not limited to, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and / or tremelimumab (Pfizer).In some embodiments, the chimeric proteins of the present invention are combined with ipilimumab (optionally bavituximab) for the treatment of one or more of melanoma, prostate cancer, and lung cancer. In various embodiments, the immunomodulator targets CD20. In various embodiments, the immunomodulator is an antibody specific for CD20. For example, in some embodiments, the immunomodulator is an antibody such as, but not limited to, ofatumumab ((GENMAB), obinutuzumab (GAZYVA), AME-133v (APPLIED MOLECULAR EVOLUTION), ocrelizumab (GENENTECH), TRU-015 (TRUBION / EMERGENT), veltuzumab (IMMU-106). In one embodiment, the immunomodulator is an antibody that targets OX40.

[0242] In some embodiments, the present application relates to combination therapy using a CD20 binding agent and a checkpoint inhibitor. In some embodiments, the present application relates to administration of a CD20 binding agent to a patient undergoing treatment with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an agent that targets one or more of PD-1, PD-L1, PD-L2, and CTLA-4. In some embodiments, the checkpoint inhibitor is one or more of nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL328OA (ROCHE), ipilimumab (MDX-010, MDX-101, Yervoy, BMS), and tremelimumab (Pfizer). In one embodiment, the checkpoint inhibitor is an antibody against PD-L1.

[0243] In exemplary embodiments, the CD20 binding agent acts synergistically when co-administered with an anti-PD-L1 camelid VHH. In exemplary embodiments, the CD20 binding agent acts synergistically when co-administered with an anti-PD-L1 camelid VHH for use in treating a tumor or cancer. For example, co-administration of a CD20 binding agent and an anti-PD-L1 camelid VHH may act synergistically to reduce or eliminate a tumor or cancer, or to slow the growth, and / or progression, and / or metastasis of a tumor or cancer. In some embodiments, the combination of a CD20 binding agent and an anti-PD-L1 camelid VHH may exhibit an improved safety profile compared to the agent used alone in a monotherapy setting. In some embodiments, the CD20 binding agent and the anti-PD-L1 camelid VHH may be administered at a lower dose than would be used if the agent were used in a monotherapy setting. In some embodiments, the CD20 binding agent comprises a mutant interferon, such as a mutant IFNα. In an exemplary embodiment, the mutant IFNα comprises one or more mutations at positions 148, 149 and 153, such as substitutions M148A, R149A, and L153A, relative to SEQ ID NO:127 or SEQ ID NO:128.

[0244] In some embodiments, the application relates to the administration of a CD20-binding agent in combination with depletion of Treg cells.

[0245] In some embodiments, the present application relates to combination therapy with one or more chimeric agents described in WO 2013 / 10779, WO 2015 / 007536, WO 2015 / 007520, WO 2015 / 007542, and WO 2015 / 007903, which are incorporated by reference in their entireties.

[0246] In some embodiments, including but not limited to infectious disease applications, the present application relates to an anti-infective as an additional therapeutic agent, in some embodiments, the anti-infective is an antiviral agent, such as but not limited to abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, foscarnet, etc. In some embodiments, the anti-infective is an antibacterial agent such as, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); carbapenem antibiotics (ertapenem, doripenem, imipenem / cilastatin, and meropenem). In some embodiments, anti-infectives include antimalarials (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether / lumefantrine, atovaquone / proguanil, and sulfadoxine / pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.

[0247] In some embodiments, including but not limited to autoimmune applications, the additional therapeutic agent is an immunosuppressant. In some embodiments, the immunosuppressant is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a nonsteroidal anti-inflammatory drug (NSAID). Steroids, particularly corticosteroids and their synthetic analogs, are well known in the art. Examples of corticosteroids useful in the present application include, but are not limited to, hydroxyltriamcinolone, α-methyldexamethasone, β-methylbetamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoximetasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorone acetonide, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl ester (flucortine butyl ester). butylester), fluocortolone, fluprednidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide acetonide, medrysone, amcinafide, amcinafide, beta-methasone and its ester residues, chloroprednisone, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate.(NSAIDS) that may be used in this disclosure include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoate, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin. In some embodiments, the immunosuppressant may be a cytostatic agent such as an alkylating agent, antimetabolite (e.g., azathioprine, methotrexate), a cytotoxic antibiotic, an antibody (e.g., basiliximab, daclizumab, and muromonab), an anti-immunophilin (e.g., cyclosporine, tacrolimus, sirolimus), an interferon, an opioid, tumor necrosis factor (TNF), a TNF binding protein, mycophenolate, a small biological agent (e.g., fingolimod, myriocin), etc. Additional anti-inflammatory agents are described, for example, in U.S. Patent No. 4,537,776, the entire contents of which are incorporated herein by reference.

[0248] In some embodiments, the present application relates to combination therapy using a CD20 binding agent and an immunosuppressant. In some embodiments, the present application relates to administration of a CD20 binding agent to a patient undergoing treatment with an immunosuppressant. In one embodiment, the immunosuppressant is TNF.

[0249] In an exemplary embodiment, the CD20 binding agent acts synergistically when co-administered with TNF. In an exemplary embodiment, the CD20 binding agent acts synergistically when co-administered with TNF for use in treating tumors or cancer. For example, co-administration of a CD20 binding agent and TNF may act synergistically to reduce or eliminate tumors or cancers, or to slow the growth, and / or progression, and / or metastasis of tumors or cancers. In some embodiments, the combination of a CD20 binding agent and TNF may exhibit an improved safety profile compared to the agent used alone in a monotherapy setting. In some embodiments, the CD20 binding agent and TNF may be administered at a lower dose than the dose used when the agent is used in a monotherapy setting. In some embodiments, the CD20 binding agent comprises a mutant interferon, such as a mutant IFNα. In an exemplary embodiment, the mutant IFNα comprises one or more mutations at positions 148, 149 and 153, such as substitutions M148A, R149A, and L153A, relative to SEQ ID NO:127 or SEQ ID NO:128.

[0250] In some embodiments, the CD20 binding agents described herein include derivatives that are modified, i.e., by covalent attachment of any type of molecule to the composition, such that the covalent attachment does not interfere with the activity of the composition. For example, and without limitation, derivatives include compositions modified by glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands, or other proteins, among others. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0251] In yet other embodiments, the CD20-binding agents described herein further comprise a cytotoxic agent, which in exemplary embodiments includes toxins, chemotherapeutic agents, and agents that cause apoptosis or cell death. Such agents can be conjugated to the compositions described herein.

[0252] Thus, the CD20 binding agents described herein may be post-translationally modified to add effector moieties such as chemical linkers, detectable moieties such as fluorochromes, enzymes, substrates, bioluminescent agents, and chemiluminescent moieties, or functional moieties such as streptavidin, avidin, biotin, cytotoxins and cytotoxic agents.

[0253] Exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine; mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin alkylating agents such as cisplatin C, cis-dichlorodiamineplatinum(II) (DDP) cisplatin, and carboplatin (paraplatin); anthracyclines such as daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisantrene; antibiotics such as dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, anthramycin (AMC); and antimitotic agents such as the vinca alkaloids, vincristine, and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicine, dihydroxyanthracindione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.

[0254] Additional cytotoxic agents include, but are not limited to, carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins, taxol, irinotecan, 5-fluorouracil, gemcitabine, leucovorine, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine). ), mustine, tyrosine kinase inhibitors, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g., IL-12 or IL-2), IL-12R antagonists, toxin-conjugated monoclonal antibodies, tumor antigen-specific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies, Rituxan, Ocrelizumab, Ofatumumab, DXL625, HERCEPTIN®, or any combination thereof. Toxic enzymes from plants and bacteria, such as ricin, diphtheria toxin, and Pseudomonas toxin, may be conjugated to therapeutic agents (e.g., antibodies) to generate cell-type specific killing reagents (Youle et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)). Other cytotoxic agents include cytotoxic ribonucleases, as described by Goldenberg, U.S. Patent No. 6,653,104.

[0255] Exemplary detectable moieties further include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, β-galactosidase and luciferase.Exemplary fluorescent substances further include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride.Exemplary chemiluminescent moieties further include, but are not limited to, luminol.Exemplary bioluminescent substances further include, but are not limited to, luciferin and aequorin.

[0256] Treatment method The methods and compositions described herein have application to the treatment of various diseases and disorders involving CD20 positive cells.In various embodiments, the diseases and disorders include, but are not limited to, cancer, infectious diseases, immune disorders, inflammatory diseases or conditions, autoimmune diseases and neurological disorders.

[0257] Additionally, any of the agents of the present invention may be for use in the treatment of various diseases and disorders in which CD20 positive cells are involved (including but not limited to cancer, infectious diseases, immune disorders, inflammatory diseases or conditions, autoimmune diseases and neurological disorders) or for use in the manufacture of a therapeutic medicament.

[0258] In some embodiments, the CD20 binding agents of the present application are used for the treatment of diseases in which depletion of CD20+ cells is therapeutically beneficial, such as Waldenstrom's macroglobulinemia, multiple myeloma, plasma cell dyscrasias, chronic lymphocytic leukemia, transplantation therapy, hairy cell leukemia, ITP, Epstein-Barr virus lymphoma following stem cell transplantation, and kidney transplantation. In other embodiments, the CD20 binding agents of the present application are used for the treatment of diseases selected from B cell lymphoma, leukemia, myeloma, autoimmune diseases, transplantation, graft-versus-host disease, infectious diseases involving B cells, lymphoproliferative diseases, and the treatment of any disease or condition in which suppression of B cell activity and / or humoral immunity is desirably suppressed. In certain embodiments, the CD20 binding agents of the present application are used to treat a disease selected from the group consisting of B cell lymphoma, leukemia, myeloma, transplantation, graft-versus-host disease, autoimmune diseases, lymphoproliferative conditions, and other therapeutic diseases and conditions in which inhibition of humoral immunity, B cell function, and / or proliferation is therapeutically beneficial. In further embodiments, the CD20 binding molecules of the present application are used to treat a disease selected from the group consisting of B-ALL, hairy cell leukemia, multiple myeloma, Richter's syndrome, acquired factor VIII inhibitors, antiphospholipid syndrome, autoimmune hemolytic anemia, autoimmune thrombocytopenia, bullous pemphigoid, cold hemagglutinin disease, Evans syndrome, Goodpasture's syndrome, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgM-associated polyneuropathy, Kaposi's sarcoma-associated Used for the treatment of herpes simplex virus (KSHV)-associated multicentric Castleman disease (MCD), myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, pure red cell aplasia, rheumatoid arthritis, Sjogren's syndrome, systemic immune complex vasculitis, systemic lupus erythematosus, mixed cryoglobulinemia type II, Wegener's granulomatosis, allograft rejection, post-transplant lymphoproliferative disorder, or purging of stem cells for bone marrow transplantation.

[0259] In some embodiments, the present application relates to the treatment of cancer or the treatment of patients with cancer. As used herein, cancer refers to any uncontrolled growth of cells that may interfere with the normal function of the body's organs and systems, including both primary and metastatic tumors. When a primary tumor or cancer migrates from its original location and seeds vital organs, it may eventually lead to the death of the subject due to functional deterioration of the affected organ. A metastasis is a cancer cell or group of cancer cells that is different from the location of the primary tumor as a result of the spread of cancer cells from the primary tumor to other parts of the body. It may eventually lead to the death of the subject. For example, cancer includes benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases.

[0260] Exemplary cancers that may be treated include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; renal cancer; laryngeal cancer; leukemia; liver cancer; lung Cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; Hodgkin's lymphoma and non-Hodgkin's lymphoma, as well as B-cell lymphomas (such as low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; as well as Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; other carcinomas and sarcomas; as well as post-transplant lymphoproliferative disorder (PTLD) and abnormal blood vessel proliferation associated with phakomatosis, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0261] In some embodiments, the cancer is a leukemia or lymphoma. Exemplary leukemias or lymphomas include, but are not limited to, B-cell lymphoma, non-Hodgkin's lymphoma (NHL), such as low-grade and intermediate-grade non-Hodgkin's lymphoma (NHL), relapsed Hodgkin's disease, high-grade resistant Hodgkin's disease, lymphocyte-predominant subtypes of Hodgkin's lymphoma, precursor B-cell lymphoblastic leukemia / lymphoma, mature B-cell neoplasms, B-cell chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCLL), and / or other lymphomas. follicular lymphoma (FL), such as low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma, MALT-type marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic-type marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, multiple myeloma, and anaplastic large cell lymphoma (ALCL).

[0262] In various embodiments, the compositions of the present invention are used to treat or prevent one or more immune disorders, such as, for example, an inflammatory disease or condition, e.g., inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh's syndrome, glycerol kinase deficiency, familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis.

[0263] In some embodiments, exemplary immune disorders include, but are not limited to, rheumatoid arthritis (RA), juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), vasculitis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, diabetes mellitus, Raynaud's syndrome, Crohn's disease. , ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis C-related cryoglobulinemic vasculitis, chronic focal encephalitis, hemophilia A, membranoproliferative glomerulonephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, neuromyelitis optica, Graves' thyroid disease, bullous skin diseases, bullous pemphigoid, pemphigus, Churg-Strauss syndrome, asthma, psoriatic arthritis, dermatitis, respiratory distress syndrome, meningitis, encephalitits, anti-NMDA receptor Encephalitis, uveitis, eczema, atherosclerosis, leukocyte adhesion deficiency, juvenile onset diabetes, Reiter's disease, Behçet's disease, hemolytic anemia, atopic dermatitis, Wegener's granulomatosis, Omenn's syndrome, chronic renal failure, acute infectious mononucleosis, HIV and herpes-related diseases, systemic sclerosis, Sjögren's syndrome and glomerulonephritis, dermatomyositis, ANCA vasculitis, aplastic anemia, autoimmune anemia, autoimmune hemolytic anemia (AIHA), pure red cell aplasia, Evans syndrome, factor VIII deficiency, These include hemophilia A, autoimmune neutropenia, Castleman syndrome, Goodpasture's syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant), Guillain-Barré syndrome, large-vessel vasculitis, giant cell (Takayasu) arteritis, medium-sized vasculitis, Kawasaki disease, polyarteritis nodosa, Devic's disease, autoimmune pancreatitis, opsoclonus-myoclonus syndrome (OMS), IgG4-related disease, scleroderma, and chronic fatigue syndrome.

[0264] In some embodiments, the CD20 binding agents of the present application may be utilized to treat vasculitis and other vascular diseases, such as microscopic polyangiitis, Churg-Strauss syndrome, and other ANCA-associated vasculitis, polyarteritis nodosa, essential cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, Kawasaki disease, Takayasu's arteritis, giant cell arthritis, Henoch-Schönlein purpura, primary or isolated cerebral vasculitis, erythema nodosum, thromboangiitis obliterans, thrombotic thrombocytopenic purpura (such as hemolytic uremic syndrome), and secondary vasculitis including cutaneous leukocytoinfarct vasculitis (e.g., secondary to hepatitis B, hepatitis C, Waldenstrom's macroglobulinemia, B-cell neoplasms, rheumatoid arthritis, Sjogren's syndrome, or systemic lupus erythematosus). Further examples are erythema nodosum, allergic vasculitis, pancreatitis, Weber-Christian disease, purpuric hyperglobulinemia, and Buerger's disease.

[0265] In some embodiments, the CD20-binding agents of the present application may be utilized to treat skin diseases such as contact dermatitis, linear IgA dermatosis, vitiligo, pyoderma gangrenosum, epidermolysis bullosa acquisita, pemphigus vulgaris (such as cicatricial pemphigoid and bullous pemphigoid), alopecia areata (such as alopecia universalis and alopecia totalis), dermatitis herpetiformis, erythema multiforme, and chronic autoimmune urticaria (such as vasomotor edema and urticarial vasculitis).

[0266] In some embodiments, the CD20-binding agents of the present application may be utilized to treat immune-mediated cytopenias, such as autoimmune neutropenia and pure red cell aplasia.

[0267] In some embodiments, the CD20-binding agents of the present application may be used to treat connective tissue disorders such as CNS lupus, discoid lupus erythematosus, CREST syndrome, mixed connective tissue disease, polymyositis / dermatomyositis, inclusion body myositis, secondary amyloidosis, cryoglobulinemia types I and II, fibromyalgia, phospholipid syndrome, secondary hemophilia, relapsing polychondromatosis, sarcoidosis, stiff man syndrome, rheumatic fever, etc. A further example is eosinophilic fasciitis.

[0268] In some embodiments, the CD20-binding agents of the present application may be utilized to treat arthritis, such as ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease, and SAPHO syndrome. Further examples are sacroileitis, reactive arthritis, Still's disease, and gout.

[0269] In some embodiments, the CD20 binding agents of the present application may be utilized to treat hematological disorders such as aplastic anemia, primary hemolytic anemia (such as cold agglutinin syndrome), hemolytic anemia secondary to CLL or systemic lupus erythematosus; POEMS syndrome, pernicious anemia, and Waldemstrom's hyperglobulinemic purpura. Further examples are granulocytopenia, autoimmune neutropenia, Franklin's disease, Seligman's disease, μ-chain disease, paraneoplastic syndromes secondary to thymoma and lymphoma, formation of factor VIII inhibitors.

[0270] In some embodiments, the CD20 binding agents of the present application may be utilized to treat endocrine disorders, such as polyendocrinopathy and Addison's disease. Further examples are autoimmune hypoglycemia, autoimmune hypothyroidism, autoimmune insulin syndrome, de Quervain's thyroiditis, and insulin receptor antibody-mediated insulin resistance.

[0271] In some embodiments, the CD20 binding agents of the present application may be utilized to treat hepato-gastrointestinal disorders such as celiac disease, Whipple's disease, primary biliary cirrhosis, chronic active hepatitis, and primary sclerosing cholangitis. A further example is autoimmune gastritis.

[0272] In some embodiments, the CD20 binding agents of the present application can be utilized to treat nephropathy such as rapidly progressive glomerulonephritis, poststreptococcal glomerulonephritis, Goodpasture's syndrome, membranous glomerulonephritis, and cryoglobulinemic nephritis. A further example is minimal change disease.

[0273] In some embodiments, the present application relates to the treatment of neurological disorders or the treatment of patients with neurological disorders. Exemplary neurological disorders include, but are not limited to, multiple sclerosis (MS; including but not limited to, benign multiple sclerosis, relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), progressive relapsing multiple sclerosis (PRMS), and primary progressive multiple sclerosis (PPMS)), Alzheimer's disease (including but not limited to, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease (FAD), Parkinson's disease and parkinsonism (including but not limited to, idiopathic Parkinson's disease, vascular parkinsonism, drug-induced parkinsonism, Lewy body disease, inherited Parkinson's disease, and pulmonary hypertension, including but not limited to, pulmonary hypertension, pulmonary hypertension, and pulmonary hypertension, including but not limited to ... Parkinson's), juvenile Parkinson's disease), Huntington's disease, amyotrophic lateral sclerosis (ALS, including but not limited to sporadic ALS, familial ALS, Western Pacific ALS, juvenile ALS, Hirayama disease), autoimmune neuropathy, mononeuritis multiplex, Lambert-Eaton amyotrophic lateral sclerosis syndrome, Sydenham chorea, tabes dorsalis, Guillain-Barre syndrome, further examples include myelopathy / tropical spastic paresis, myasthenia gravis, acute inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy.

[0274] In some embodiments, the CD20-binding agents of the present application may be utilized to treat cardiac and pulmonary disorders such as fibrosing alveolitis, obliterative bronchiolitis, allergic aspergillosis, cystic fibrosis, Loeffler's syndrome, myocarditis and pericarditis. Further examples are hypersensitivity pneumonitis, and paraneoplastic syndromes secondary to lung cancer.

[0275] In some embodiments, the CD20 binding agents of the present application may be utilized to treat allergic diseases such as bronchial asthma and hyper-IgE syndrome. A further example is amaurosis fugax.

[0276] In some embodiments, the CD20-binding agents of the present application may be utilized to treat ocular diseases such as idiopathic chorioretinitis.

[0277] In some embodiments, the CD20 binding agents of the present application may be utilized to treat transplant-derived disorders such as allograft and xenograft rejection and graft-versus-host disease, hi some embodiments, the CD20 binding agents of the present application may be utilized post-transplant.

[0278] In some embodiments, the present application relates to the treatment of or patients with microbial infections and / or chronic infections (e.g., infections of B cells). Exemplary infections include, but are not limited to, HIV / AIDS, tuberculosis, osteomyelitis, Hepatitis B, Hepatitis C, Epstein-Barr virus or parvovirus, T-cell leukemia virus, bacterial overgrowth syndrome, fungal infections, or parasitic infections.

[0279] kit The present application also provides kits (e.g., with or without additional therapeutic agents) for administration of any of the CD20 binding agents described herein. The kit is a collection of materials or components, including at least one of the pharmaceutical compositions of the present invention described herein. Thus, in some embodiments, the kit includes at least one of the pharmaceutical compositions described herein.

[0280] The exact nature of the components configured in the kit will vary depending on its intended purpose, hi one embodiment, the kit is configured for treating a human subject.

[0281] Instructions for use may be included in the kit. Instructions for use typically include specific wording that describes the techniques to be used in using the components of the kit to achieve a desired therapeutic outcome, such as for treating cancer. Optionally, the kit also includes other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipettes or measuring instruments, dressings or other useful instruments as would be readily recognized by those skilled in the art.

[0282] The materials and components assembled in the kit can be stored and provided to the practitioner in any convenient and appropriate manner that preserves their operability and usefulness. For example, the components can be provided at room temperature, refrigerated or frozen temperatures. These components are typically included in suitable packaging materials. In various embodiments, the packaging materials are constructed by well-known methods to provide a preferably sterile, contaminant-free environment. The packaging materials may have an exterior label indicating the contents and / or purpose of the kit and / or its components.

[0283] definition As used herein, "a," "an," or "the" can mean one or more.

[0284] Furthermore, the term "about" when used in connection with a reference numeral means plus or minus up to 10% of the reference numeral. For example, the language "about 50" covers the range of 45 to 55.

[0285] An "effective amount" as used in relation to medical use is an amount effective to effect a measurable treatment, prevention or reduction in the etiological incidence of the disease of interest.

[0286] As used herein, an activity and / or effect readout is "decreased" in the presence of an agent or stimulus relative to the absence of such modulation if the readout is decreased by a significant amount, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least 100%. As will be appreciated by those of skill in the art, in some embodiments, activity is decreased and some downstream readouts are decreased while others may be increased.

[0287] Conversely, activity is "increased" if the activity and / or effect readout is increased by a significant amount, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of the agent or stimulus relative to the absence of that agent or stimulus.

[0288] As referred to herein, all composition percentages are by weight of the total composition unless otherwise specified. As used herein, the term "include" and variations thereof are non-limiting, and thus the recitation of items in a list is not intended to exclude other similar items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and variations thereof are non-limiting, and thus a reference that an embodiment may or may include certain elements or features is not intended to exclude other embodiments of this technology that do not include these elements or features.

[0289] Although the open-ended term "comprising," which is synonymous with terms such as "including," "containing," or "having," is used herein to describe and claim the invention, the invention or embodiments thereof may alternatively be described using alternative terms, such as "consisting of" or "consisting essentially of."

[0290] As used herein, the terms "preferred" and "preferably" refer to embodiments of the technology that provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[0291] The amount of the compositions described herein required to achieve a therapeutic effect can be empirically determined according to conventional procedures for a particular purpose. Generally, to administer a therapeutic agent for therapeutic purposes, the therapeutic agent is administered in a pharmacologically effective amount. "Pharmacologically effective amount", "pharmacologically effective dose", "therapeutically effective amount", "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired result, particularly for treating a disorder or disease. As used herein, an effective amount includes, for example, an amount sufficient to delay the onset of a symptom of a disorder or disease, alter the course of a symptom of a disorder or disease (e.g., slow the progression of a symptom of a disease), reduce or eliminate one or more symptoms or signs of a disorder or disease, or reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[0292] Effective doses, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. Therapeutically effective doses can be estimated initially from in vitro assays, such as, for example, cell culture assays. Also, when determined in cell cultures or suitable animal models, doses can be formulated in animal models to achieve a circulating plasma concentration range, such as IC50. Plasma levels of the described compositions can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay. Dosages can be determined by a physician and adjusted, if necessary, to suit the observed therapeutic effect.

[0293] In certain embodiments, the effect results in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect results in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[0294] As used herein, "method of treatment" is equally applicable to the use of a composition to treat a disease or disorder described herein and / or the use of a composition for use in the manufacture of a medicament for treating a disease or disorder described herein.

[0295] Working Example Example 1. Construction and evaluation of VHH specific for human CD20 Cloning of the human CD20 gene Human CD20 was amplified from the Orfeome v5.1 collection (ID11051) using the forward primer 5'-GATAAGATCTCAGGCGGATCCACAACACCCAGAAATTCAG (O-7954) and reverse primer 5'-GGTTTTTTCTCTAGATCAAGGAGAGCTGTCATTTTCTATTGG (O-7956). The amplified product was cut with BglII and XbaI and ligated into the mammalian expression vector pMet7. This plasmid was used for transient transfection of Hek293T cells and for generation of CHO-K1 clones stably expressing human CD20.

[0296] Isolation of antigen-specific VHHs A VHH library was constructed and subjected to three consecutive rounds of panning (in solution) on stably transfected CHO-K1 cells expressing human CD20. Parallel panning was performed on parental (non-transfected CHO-K1) cells to serve as a negative control for calculating the enrichment of CD20-specific phages. Enrichment of antigen-specific phages was assessed by comparing the number of phagemid particles eluted from transfected cells with the number of phagemid particles eluted from parental cells after each round of panning. These experiments suggested that the phage population was enriched approximately 2-, 8- and 4-fold after the first, second and third rounds of panning (for antigen-specific phages), respectively. In total, approximately 95 colonies were randomly selected from the second round of panning and their crude periplasmic extracts (such as soluble VHHs) were analyzed by cell ELISA for specific binding to transfected CHO-K1, compared to parental cells. 62 of the 95 colonies were positive in this assay. Based on the sequence data, the 62 positive colonies represented 14 different VHHs (Figure 1, top panel).

[0297] A VHH library was constructed and subjected to three consecutive rounds of panning (in solution) on stably transfected CHO-K1 cells expressing human CD20 as described above. Here, the phage population was enriched approximately 7-fold (for antigen-specific phages) after the third panning. No enrichment was observed after the first and second pannings. Approximately 142 randomly picked colonies from the third panning were tested by cell ELISA as described above, with 8 colonies showing positive. Based on the sequence data, the 8 positive colonies represented 3 different VHHs (Figure 1, lower panel). The 3 different VHHs belong to the same group. The group identified here is the same as one of the groups identified previously.

[0298] The following table provides a description of the 17 clones representing 17 different anti-human CD20 VHH genes. E. coli TG1 harboring the recombinant phagemid pMECS containing the anti-human CD20 VHH sequences was stored at -80°C. The vector pMECS encodes ampicillin resistance. [Table 1]

[0299] In summary, 17 different VHHs were identified that belong to four different groups, as shown in the table below: The nucleotide and amino acid sequences of the 17 anti-human CD20 VHHs are shown in Figures 2 and 3, respectively. [Table 2]

[0300] Transformation of non-suppressor strains (e.g., WK6) with recombinant pMECS The VHH genes cloned into the pMECS vector contained a PelB signal sequence at the N-terminus and an HA tag and a His6 tag at the C-terminus (PelB leader-VHH-HA-His6). The PelB leader sequence targets the VHH to the periplasmic space of E. coli, and the HA and His6 tags can be used for purification and detection of the VHH (e.g., by ELISA, Western blot, etc.).

[0301] In the pMECS vector, the His6 tag was followed by an amber stop codon (TAG), which was followed by gene III of the M13 phage. In suppressor E. coli strains (e.g., TG1), the amber stop codon was read as a glutamine and therefore the VHH was expressed as a fusion protein with protein III of the phage allowing display of the VHH on the phage coat for panning. In non-suppressor E. coli strains (e.g., WK6), the amber stop codon was read as a stop codon and therefore the resulting VHH was not fused to protein III.

[0302] To express and purify VHH cloned into pMECS vector, pMECS containing the gene of the VHH of interest was prepared and transformed into a non-suppressor strain (e.g., WK6). The VHH of the resulting clone was sequenced using MP057 primer (5'-TTATGCTTCCGGCTCGTATG-3') to confirm the identity of the clone. The antigen binding ability was retested by ELISA or any other suitable assay. The non-suppressor strain (e.g., WK6) containing the recombinant pMECS vector with the VHH gene was then used to express and purify the VHH described herein.

[0303] Recloning of VHH genes from pMECS into pHEN6c vector In the pMECS vector, the His6 tag is cleaved upon storage of the VHH (even for short periods and at -20°C). Thus, the VHH genes were subcloned from pMECS into the pHEN6c vector, e.g., when the His6 tag was used for detection.

[0304] The VHH genes were amplified by PCR using E. coli containing recombinant pMECS carrying the VHH genes as a template and primers A6E and PMCF, which are primers for framework 1 and framework 4, respectively. The primer sequences were as follows: [ka]

[0305] The amplification protocol included approximately 30 cycles of PCR, each consisting of 30 s at 94°C, 30 s at 55°C, and 45 s at 72°C, followed by a 10 min extension at 72°C at the end of the PCR. A fragment of approximately 400 bp was amplified.

[0306] The PCR products were purified (e.g., by Qiaquick PCR Purification Kit (Qiagen)) and digested overnight with PstI. The purified PCR products were digested overnight with BstEII (or Eco91I (Fermentas)). The temperature used for digestion was varied. For example, digestion with BstEII was performed at 50° C. or 60° C. depending on the enzyme supplier.

[0307] For ligation, the pHEN6c vector was digested with PstI for 3 h, purified as above, and then digested with BstEII for 2-3 h. Alternatively, digestion was performed with Eco91I (Fermentas). The digested vector was run on a 1% agarose gel and the vector band was excised from the gel and purified (e.g., by Qiaquick Gel Extraction Kit (Qiagen)). The PCR fragment was then ligated into the vector.

[0308] Electrocompetent WK6 cells were transformed with the ligation reaction and transformants were selected using LB / agar / ampicillin (100 μg / ml) / glucose (1-2%) plates. Positive clones were screened by PCR using universal reverse and universal forward primers. If an insert was present, a fragment of approximately 550 bp was amplified. At least two clones per each VHH were sequenced using the universal reverse primer to confirm the identity of the clones. Antigen binding ability was retested by ELISA or any other appropriate assay.

[0309] Following the protocol described above, we generated VHH genes cloned into the pHEN6c vector, which contains a PelB signal sequence at the N-terminus and a His6-tail at the C-terminus. The PelB leader sequence targets the VHH to the periplasmic space of E. coli, and the His tag was used for purification and detection of the VHH (e.g., by ELISA, Western blot, etc.).

[0310] Expression and purification of VHHs was performed. Specifically, on day 1, 10–20 ml of LB + ampicillin (100 μg / ml) + glucose (1%) was inoculated with a freshly transformed WK6 colony. This preculture was incubated overnight at 37 °C with shaking at 200–250 rpm. On day 2, TB medium was used for expression of VHHs. TB medium contained per liter 2.3 g KH2PO4, 16.4 g K2HPO4 3H2O, 12 g tryptone (Duchefa Biochemie), 24 g yeast (Duchefa Biochemie) and 4 ml 100% glycerol (Duchefa Biochemie).

[0311] A 1-liter baffled shake flask was filled with 330 ml of TB and autoclaved. KH2PO4 and K2HPO4·3H2O were not autoclaved. Instead, KH2PO4 and K2HPO4·3H2O were prepared, filter sterilized, and then added to the remaining medium that had already been autoclaved. Approximately 1 ml of the preculture was added to 330 ml of TB supplemented with 100 μg / ml ampicillin, 2 mM MgCl2, and 0.1% glucose, followed by incubation at OD with shaking (200–250 rpm). 600 The cells were grown at 37°C until the OD reached 0.6-0.9. VHH expression was induced by adding IPTG (final concentration 1 mM). The cultures were incubated overnight (approximately 16-18 hours) with shaking at 28°C. The OD after overnight induction was 600 The yield was usually 25-30. At least 1 liter of culture (3 bottles) was prepared per clone with an average yield of 1-15 mg / l.

[0312] Extraction of VHHs from the periplasm of E. coli was performed on day 3. The solution used contained TES: 0.2 M Tris pH 8.0, 0.5 mM EDTA, 0.5 M sucrose, and TES / 4:TES diluted 4-fold in water.

[0313] Overnight induced cultures were centrifuged at 8000 rpm for 8 min. Cell pellets from 1 liter cultures were resuspended in 12 ml TES by pipetting up and down and shaking on ice for 1 h. For each 12 ml TES used, approximately 18 ml TES / 4 was added and incubated on ice with shaking for an additional hour, followed by centrifugation at 8000 rpm for 30 min at 4°C. The supernatant containing proteins extracted from the periplasmic space was transferred to a fresh Falcon tube.

[0314] VHHs were subsequently purified by IMAC utilizing the following solutions: HIS-select (SIGMA), PBS, and 50 mM NaAcetate pH 4.6.

[0315] His-select was equilibrated with PBS. Specifically, 1 ml of resin (approximately 2 ml of His-select solution) was added to a 50 ml Falcon tube for each periplasmic extract derived from 1 liter of culture. PBS was added to a final volume of 50 ml and mixed. Centrifugation was performed at 2000 rpm for 2 min and the supernatant was discarded. The resin was washed twice with PBS as described above. The periplasmic extract was added to the resin and incubated at room temperature for 30 min to 1 h with gentle shaking. The sample was loaded onto a PD-10 column with a filter at the bottom (GE healthcare, cat.No.17-0435-01) and washed with 50-100 ml of PBS (50-100 ml of PBS was used per ml of resin). Elution was performed three times, each time with 1 ml of PBS / 0.5 M imidazole per ml of resin used (for efficient elution, the beads were resuspended and the column was left overnight at 4 °C with the bottom closed). Dialysis was performed overnight at 4 °C against PBS (cutoff 3500 Daltons) to remove imidazole. For efficient dialysis, the dialysis buffer (PBS) was changed 2-3 times. Alternatively, instead of elution with imidazole, bound VHHs could be eluted with 10 ml of 50 mM sodium acetate pH 4.6. When 50 mM sodium acetate pH 4.6 was used to elute the VHHs, the eluted VHHs were immediately neutralized with 1 M Tris pH 8.0 and no dialysis was required.

[0316] The amount of protein was determined by the OD of the eluted sample. 280 The extinction coefficient of each clone was estimated by OD measurements. The extinction coefficient of each clone was determined by the protParam tool under primary structure analysis of the Expasy proteomics server. Further purification of the VHHs could be achieved by different methods. For example, samples could be concentrated by centrifugation at 2000 rpm at 4°C until a suitable volume (up to 4 ml) was obtained for loading onto a Superdex 75 16 / 60 (Vivaspin 5000 MW cutoff, Vivascience). The concentrated sample was loaded onto a Superdex 75 16 / 60 column equilibrated with PBS. Peak fractions were pooled and analyzed by OD for quantification. 280Measurements were performed. Typically, VHHs eluted after 85-95 min when run at 1 ml / min. Aliquots of concentrated VHH samples were stored at -20°C at a concentration of approximately 1 mg / ml.

[0317] Example 2. Construction and evaluation of VHH specific for mouse CD20 Cloning of the mouse CD20 gene Mouse CD20 was purchased from Imagenes cat#IRAVP968C1280D and amplified with forward primer 5'-gataagatctcaGGCGGATCCAGTGGACCTTTCCCAGCAGAGC (O-7962) and reverse primer 5'-GGTTTTTTCTCTAGATCAAGGAGCGATCTCATTTTCCACTG (O-7964). The amplified product was cut with BglII and XbaI and ligated into the mammalian expression vector pMet7. This plasmid was used for transient transfection of Hek293T cells and for generation of CHO-K1 clones stably expressing mouse CD20.

[0318] Isolation of antigen-specific VHHs The VHH library was constructed from peripheral blood lymphocytes (PBLs). Specifically, total RNA from PBLs was used as a template for first strand cDNA synthesis using oligo(dT) primers. Using this cDNA, sequences encoding VHHs were amplified by PCR, digested with PstI and NotI, and cloned into the PstI and NotI sites of the phagemid vector pMECS. Approximately 10 7 A VHH library of 100 independent transformants was obtained. Approximately 90% of the transformants contained vectors with the correct insert size.

[0319] Similarly, about 10 8 A VHH library with independent transformants sizes of 1000 was obtained from peripheral blood lymphocytes (PBLs). Approximately 65% ​​of the transformants carried vectors with the correct insert size.

[0320] The library was panned (in solution) three consecutive times on stably transfected CHO-K1 cells expressing mouse CD20. Parallel panning was performed on parental (non-transfected CHO-K1) cells to serve as a negative control for calculating the enrichment of CD20-specific phages. Enrichment of antigen-specific phages was assessed by comparing the number of phagemid particles eluted from transfected cells with the number of phagemid particles eluted from parental cells after each round of panning. These experiments showed approximately 2-fold, 2-fold and 10-fold enrichment after the first, second and third rounds of panning, respectively (for antigen-specific phages). 3 fold, suggesting that the phage population was enriched. In total, 285 colonies (95 from each panning) were randomly selected and their crude periplasmic extracts (e.g., soluble VHHs) were analyzed by cell ELISA for specific binding to transfected CHO-K1 compared to parental cells. Of the 285 colonies, 124 colonies (0 in the first round, 40 in the second round, and 84 in the third round, respectively) were positive in this assay. Based on the sequence data, the 124 positive colonies represented 11 different VHHs (Figure 4, top panel). The 11 different VHHs belong to two different groups.

[0321] The library was panned (in solution) two consecutive times on stably transfected CHO-K1 cells expressing mouse CD20 as described above. Here, the phage population was enriched approximately 2-fold and 45-fold (for antigen-specific phages) after the first and second rounds of panning, respectively. Here, 190 randomly picked colonies from the second round of panning were tested by cell ELISA as described above, with 10 colonies showing positive. Based on the sequence data, the 10 positive colonies represented 8 different VHHs (Figure 4, bottom panel). The 8 different VHHs belong to two different groups. One of the groups identified here is the same group as one of the groups identified above.

[0322] This library was subjected to four successive rounds of panning, which were performed on solid-phase coated biotinylated mouse CD20 peptide (3 μg / well). Coating of the peptide to the wells was mediated by streptavidin. Enrichment of antigen-specific phages was assessed by comparing the number of phagemid particles eluted from (streptavidin-mediated) antigen-coated blocked wells to the number of phagemid particles eluted from wells coated with streptavidin and then blocked with blocking buffer. These experiments suggested that the phage population was only slightly enriched for antigen-specific phages after the third panning. 190 randomly picked colonies from the third panning on peptide were analyzed by ELISA (ELISA with crude periplasmic extracts containing soluble VHHs) for specific binding to biotinylated mouse CD20 peptide. Five colonies out of 190 were positive in this assay. Based on the sequence data, the five positive colonies represented two different VHHs (Figure 5). The two different VHHs belong to the same group, albeit in different groups, compared to all groups identified above by cell panning / cell ELISA.

[0323] The following table provides a description of the 21 clones representing 21 different anti-mouse CD20 VHH genes. E. coli TG1 harboring the recombinant phagemid pMECS containing the anti-mouse CD20 VHH sequences was generated and stored at -80°C. The vector pMECS encodes ampicillin resistance. [Table 3]

[0324] In summary, 21 different VHHs were identified that belong to four different groups, as shown in the table below: The nucleotide and amino acid sequences of the 21 anti-mouse CD20 VHHs are shown in Figures 6 and 7, respectively. [Table 4]

[0325] Transformation of non-suppressor strains (e.g., WK6) with recombinant pMECS The VHH genes cloned into the pMECS vector contained a PelB signal sequence at the N-terminus and an HA tag and a His6 tag at the C-terminus (PelB leader-VHH-HA-His6). The PheB leader sequence targets the VHH to the periplasmic space of E. coli, and the HA and His6 tags were used for purification and detection of the VHH (e.g., by ELISA, Western blot, etc.).

[0326] In the pMECS vector, the His6 tag was followed by an amber stop codon (TAG), which was followed by gene III of the M13 phage. In suppressor E. coli strains (e.g., TG1), the amber stop codon was read as a glutamine and therefore the VHH was expressed as a fusion protein with protein III of the phage allowing display of the VHH on the phage coat for panning. In non-suppressor E. coli strains (e.g., WK6), the amber stop codon was read as a stop codon and therefore the resulting VHH was not fused to protein III.

[0327] To express and purify VHHs cloned into pMECS vectors, pMECS containing the genes of the VHHs of interest were prepared and transformed into a non-suppressor strain (e.g., WK6). The VHHs of the resulting clones were sequenced using MP057 primer (5'-TTATGCTTCCGGCTCGTATG-3') to confirm the identity of the clones. The antigen binding ability was retested by ELISA or any other appropriate assay. The non-suppressor strain (e.g., WK6) containing the recombinant pMECS vector with the VHH genes was used to express and purify the VHHs described herein.

[0328] Recloning of VHH genes from pMECS into pHEN6c vector In the pMECS vector, the His6 tag was cleaved during storage of the VHHs (even for short periods and at -20°C). Therefore, if the His6 tag was to be used for detection or other purposes, it was better to subclone the VHH genes from pMECS into the pHEN6c vector.

[0329] Specifically, the VHH genes were used as templates to amplify the VHH genes using E. coli containing recombinant pMECS carrying primers A6E and PMCF, which are primers for framework 1 and framework 4, respectively. The primer sequences were as follows: [ka]

[0330] The amplification protocol included approximately 30 cycles of PCR, each cycle including 30 s at 94° C., 30 s at 55° C., and 45 s at 72° C., followed by a 10 min extension at 72° C. at the end of the PCR. A fragment of approximately 400 bp was amplified.

[0331] The PCR products were purified (e.g., by Qiaquick PCR Purification Kit (Qiagen)) and digested overnight with PstI. The purified PCR products were digested overnight with BstEII (or Eco91I (Fermentas)). The temperature used for digestion was varied. For example, digestion with BstEII was performed at 50° C. or 60° C. depending on the enzyme supplier.

[0332] For ligation, the PCR product was purified. The pHEN6c vector was digested with PstI for 3 hours, purified as above, and then digested with BstEII for 2-3 hours. Alternatively, digestion was performed with Eco91I (Fermentas). The digested vector was run on a 1% agarose gel and the vector band was excised from the gel and purified (e.g., by Qiaquick Gel Extraction Kit (Qiagen)). The PCR fragment was then ligated into the vector.

[0333] Electrocompetent WK6 cells were transformed with the ligation reaction and transformants were selected using LB / agar / ampicillin (100 μg / ml) / glucose (1-2%) plates. Positive clones were screened by PCR using universal reverse and universal forward primers. If an insert was present, a fragment of approximately 550 bp was amplified. At least two clones per each VHH were sequenced using the universal reverse primer to confirm the identity of the clones. Antigen binding ability was retested by ELISA or any other appropriate assay.

[0334] Following the protocol described above, the VHH genes cloned into the pHEN6c vector contained a PelB signal sequence at the N-terminus and a His6-tail at the C-terminus. The PelB leader sequence targets the VHH to the periplasmic space of E. coli, and the His tag was used for purification and detection of the VHH (e.g., by ELISA, Western blot, etc.).

[0335] Expression and purification of VHHs was performed. Specifically, on day 1, 10–20 ml of LB + ampicillin (100 μg / ml) + glucose (1%) was inoculated with a freshly transformed WK6 colony. This preculture was incubated overnight at 37 °C with shaking at 200–250 rpm. On day 2, TB medium was used for expression of VHHs. TB medium contained per liter 2.3 g KH2PO4, 16.4 g K2HPO4 3H2O, 12 g tryptone (Duchefa Biochemie), 24 g yeast (Duchefa Biochemie) and 4 ml 100% glycerol (Duchefa Biochemie).

[0336] A 1-liter baffled shake flask was filled with 330 ml of TB and autoclaved. KH2PO4 and K2HPO4·3H2O were not autoclaved. Instead, KH2PO4 and K2HPO4·3H2O were prepared, filter sterilized, and then added to the remaining medium that had already been autoclaved. Approximately 1 ml of the preculture was added to 330 ml of TB supplemented with 100 μg / ml ampicillin, 2 mM MgCl2, and 0.1% glucose, followed by incubation at OD with shaking (200–250 rpm). 600 The cells were grown at 37°C until the OD reached 0.6-0.9. VHH expression was induced by adding IPTG (final concentration 1 mM). The cultures were incubated overnight (approximately 16-18 hours) with shaking at 28°C. The OD after overnight induction was 600 The yield was usually 25-30. At least 1 liter of culture (3 bottles) was prepared per clone with an average yield of 1-15 mg / l.

[0337] Extraction of VHHs from the periplasm of E. coli was performed on day 3. The solution used contained TES: 0.2 M Tris pH 8.0, 0.5 mM EDTA, 0.5 M sucrose, and TES / 4:TES diluted 4-fold in water.

[0338] Overnight induced cultures were centrifuged at 8000 rpm for 8 min. Cell pellets from 1 liter cultures were resuspended in 12 ml TES by pipetting up and down and shaking on ice for 1 h. For each 12 ml TES used, approximately 18 ml TES / 4 was added and incubated on ice with shaking for an additional hour, followed by centrifugation at 8000 rpm for 30 min at 4°C. The supernatant containing proteins extracted from the periplasmic space was transferred to a fresh Falcon tube.

[0339] VHHs were subsequently purified by IMAC utilizing the following solutions: HIS-select (SIGMA), PBS, and 50 mM NaAcetate pH 4.6.

[0340] His-select was equilibrated with PBS. Specifically, 1 ml of resin (approximately 2 ml of His-select solution) was added to a 50 ml Falcon tube for each periplasmic extract derived from 1 liter of culture. PBS was added to a final volume of 50 ml and mixed. Centrifugation was performed at 2000 rpm for 2 min and the supernatant was discarded. The resin was washed twice with PBS as described above. The periplasmic extract was added to the resin and incubated at room temperature for 30 min to 1 h with gentle shaking. The sample was loaded onto a PD-10 column with a filter at the bottom (GE healthcare, cat.No.17-0435-01) and washed with 50-100 ml of PBS (50-100 ml of PBS was used per ml of resin). Elution was performed three times, each time with 1 ml of PBS / 0.5 M imidazole per ml of resin used (for efficient elution, the beads were suspended and left overnight at 4 °C with the bottom of the column closed). Dialysis was performed overnight at 4 °C against PBS (cutoff 3500 Daltons) to remove imidazole. For efficient dialysis, the dialysis buffer (PBS) was changed 2-3 times. Alternatively, instead of elution with imidazole, bound VHHs could be eluted with 10 ml of 50 mM sodium acetate pH 4.6. When 50 mM sodium acetate pH 4.6 was used to elute the VHHs, the eluted VHHs were immediately neutralized with 1 M Tris pH 8.0 and no dialysis was required.

[0341] The amount of protein can be estimated at this point by OD280 measurement of the eluted samples. The extinction coefficient of each clone was determined by the protParam tool under primary structure analysis in the Expasy proteomics server. Further purification of the VHHs can be achieved by different methods. Below, we present one of these methods.

[0342] The amount of protein was determined by the OD of the eluted sample. 280The extinction coefficient of each clone was estimated by OD measurements. The extinction coefficient of each clone was determined by the protParam tool under primary structure analysis of the Expasy proteomics server. Further purification of the VHHs could be achieved by different methods. For example, samples could be concentrated by centrifugation at 2000 rpm at 4°C until a suitable volume (up to 4 ml) was obtained for loading onto a Superdex 75 16 / 60 (Vivaspin 5000 MW cutoff, Vivascience). The concentrated sample was loaded onto a Superdex 75 16 / 60 column equilibrated with PBS. Peak fractions were pooled and analyzed by OD for quantification. 280 Measurements were performed. Typically, VHHs eluted after 85-95 min when run at 1 ml / min. Aliquots of concentrated VHH samples were stored at -20°C at a concentration of approximately 1 mg / ml.

[0343] Example 3. Functional evaluation of VHHs specific for human and / or mouse CD20 Specific binding of VHHs demonstrated by FACS FACS analysis was performed to determine the binding activity of VHHs specific for human or mouse CD20. Specifically, CHO-K1 cells and CHO-K1 cells stably expressing human or mouse CD20 were incubated with 5 μg / ml of VHHs produced according to the present application. Monoclonal FITC-labeled anti-His antibody (Genscript#A01620) was applied as secondary staining. FACS analysis was performed on a FacsCalibur flow cytometer (Becton Dickinson). As shown in Figure 8, all VHHs against human CD20 showed specific binding to human CD20. Similarly, all VHHs against mouse CD20 also showed specific binding to mouse CD20 (Figure 9).

[0344] Example 4. Functional analysis of chimeric proteins containing CD20 VHH Targeting interferon activity in vivo using chimeric CD20 VHHs in the A20 lymphoma model Mice were inoculated subcutaneously with A20 cells (a CD20+, and IFN-sensitive mouse lymphoma cell line) to induce tumors. To determine the in vivo antitumor efficacy of chimeric CD20 VHHs, mice were treated perilesionally (sc to the edge of the tumor) with 2MC57 VHH (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon). Mice were also injected with 2HCD25 VHH (anti-human CD20) fused to wild-type IFN or mutant IFNα2-Q124R. As shown in Figure 10, all CD20 VHHs significantly reduced tumor size, with the chimeric 2MC57 VHH being the most potent.

[0345] Targeting interferon activity in vivo with chimeric CD20 VHHs in the B16 melanoma model Mice were inoculated subcutaneously with B16-mCD20 cells (a murine melanoma cell line stably expressing murine CD20) to induce tumors. To determine the in vivo antitumor efficacy of chimeric CD20 VHHs, mice were treated perilesionally (sc to the edge of the tumor) with 2MC57 VHHs (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon). Mice were also injected with 2HCD25 VHHs (anti-human CD20) fused to wild-type IFN or mutant IFNα2-Q124R. Control mice were treated with PBS. Tumor growth was monitored. As shown in Figure 11, mCD20 VHHs significantly reduced tumor size.

[0346] Figure 12 shows that chimeric 2MC57 VHH fused to mutant IFNα2-Q124R (i.e., AcTaferon) targeted B16-mCD20 melanoma cells without life-threatening side effects. Specifically, mice treated with chimeric 2MC57 VHH fused to mutant IFNα2-Q124R maintained body weight and normal total blood counts, including neutrophils, red blood cells, and platelets, without signs of hematologic toxicity. Furthermore, administration of chimeric 2MC57 VHH fused to mutant IFNα2-Q124R resulted in partial B cell depletion (Figure 13).

[0347] Combination therapy with chimeric CD20 VHH and doxorubicin in a B16 melanoma model The antitumor efficacy of combination therapy with doxorubicin and chimeric 2MC57 VHH was tested. Mice were subcutaneously inoculated with B16-mCD20 cells (a murine melanoma cell line stably expressing murine CD20) to induce tumors. Mice were subsequently treated perilesionally (sc at the edge of the tumor) with 2MC57 VHH (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon) with or without doxorubicin. Doxorubicin was administered at 3 mg / kg body weight every 2 days. As shown in Figure 14, combining 2MC57 VHH with doxorubicin significantly reduced tumor size compared to treatment with 2MC57 VHH without doxorubicin.

[0348] The combination of doxorubicin with 2MC57 VHH fused to mutant IFNα2-Q124R appeared to be particularly safe and effective. Specifically, combination therapy with doxorubicin exacerbated the morbidity and mortality associated with administration of wild-type IFN, but when used in combination with 2MC57 VHH fused to mutant IFNα2-Q124R, did not cause any side effects as assessed by the evolution of body weight, survival rate and several blood parameters (Figure 15).

[0349] Combination therapy using chimeric CD20 VHH and tumor necrosis factor in a B16 melanoma model The antitumor efficacy of combination therapy with tumor necrosis factor (TNF) and chimeric 2MC57 VHH was tested. Mice were inoculated subcutaneously with B16-mCD20 cells (a murine melanoma cell line stably expressing murine CD20) to induce tumors. Mice were subsequently treated perilesionally (sc at the edge of the tumor) with 2MC57 VHH (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon) with or without TNF. TNF was administered at 0.6 μg every 2 days. As shown in Figure 16, combining 2MC57 VHH with TNF significantly reduced tumor size compared to treatment with 2MC57 VHH without TNF.

[0350] The combination of TNF with 2MC57 VHH fused to mutant IFNα2-Q124R appeared to be particularly safe and effective. Specifically, combination therapy with TNF exacerbated the thrombocytopenia and / or anemia and mortality associated with administration of wild-type IFN, but when used in combination with 2MC57 VHH fused to mutant IFNα2-Q124R, did not cause any side effects as assessed by the evolution of body weight, survival rate and several blood parameter measurements (Figure 17).

[0351] Combination therapy using chimeric CD20 VHH and anti-PD-L1 camelid VHH in a B16 melanoma model The antitumor efficacy of combination therapy with anti-PD-L1 camelid VHH and chimeric 2MC57 VHH was tested. Mice were subcutaneously inoculated with B16-mCD20 cells (a murine melanoma cell line stably expressing murine CD20) to induce tumors. Mice were subsequently treated perilesionally (sc at the edge of the tumor) with 2MC57 VHH (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon) with or without anti-PD-L1 camelid VHH. Anti-PD-L1 camelid VHH was administered at 120 μg every 2 days. As shown in Figure 18, anti-PD-L1 camelid VHH produced a long-term plateauing effect on 2MC57 VHH fused to mutant IFNα2-Q124R. Furthermore, the combination of anti-PD-L1 camelid VHH and 2MC57 VHH fused to mutant IFNα2-Q124R did not cause significant weight loss (Figure 18). As shown in Figure 24, panel A shows that combination treatment with mutant IFNα2-Q124R (i.e., AcTaferon) and 2MC57 VHH fused to anti-PD-L1 camelid VHH induced a strong anti-tumor effect with no tumor recurrence by day 38 compared to treatment with either agent alone. Panel B of Figure 24 further shows that the combination treatment was well tolerated and did not induce significant weight loss compared to treatment with wild-type IFN alone. Overall, these data suggest a synergistic effect for combination therapy using chimeric 2MC57 VHH and anti-PD-L1 antibody. The anti-tumor efficacy of anti-PD-L1 camelid VHH and chimeric 2MC57 VHH in combination with Treg depletion was tested in the B16-mCD20 melanoma model. As shown in Figure 19, the triple combination of administering 2MC57 VHH fused to mutant IFNα2-Q124R, anti-PD-L1 camelid VHH, and Treg depletion further reduced tumor size.

[0352] Systemic administration of chimeric CD20 VHHs in the B16 melanoma model The efficacy of systemically administered chimeric CD20 VHHs was tested in the B16-mCD20 melanoma model. Specifically, mice were given 2MC57 VHHs (anti-mouse CD20) fused to mutant IFNα2-Q124R (i.e., AcTaferon), or 2HCD25 VHHs (anti-human CD20) fused to mutant IFNα2-Q124R by intravenous injection. Chimeric VHHs were injected intravenously at a dose of 5,500 IU on days 8–11 after tumor inoculation. As shown in Figure 20, systemic administration of chimeric antibodies resulted in a significant reduction in tumor growth, with the chimeric 2MC57 VHH being more potent.

[0353] Role of B cell depletion in the antitumor effect of chimeric CD20 VHH Mice were implanted with B16-hCD20 cells (a murine melanoma cell line stably expressing human CD20) to induce tumors. To determine the in vivo antitumor efficacy of chimeric CD20 VHHs, mice were treated perilesionally (sc to the edge of the tumor) with 2HCD25 VHHs (anti-human CD20) fused to wild-type human IFN, murine IFN or mutant IFNα2-Q124R (i.e., AcTaferon). Mice were also given 2MC57 VHHs (anti-mouse CD20) fused to mutant IFNα2-Q124R. Tumor growth was monitored. As shown in Figure 21, chimeric 2HCD25 VHHs fused to either murine IFN or mutant IFNα2-Q124R caused significant tumor regression. However, as shown in the inset, chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R did not appear to reduce lymphocyte numbers, suggesting that B cell depletion is not required for the antitumor effect of chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R.

[0354] A similar study was performed by administering chimeric CD20 antibodies to mice systemically iv. In agreement with previous results, chimeric 2HCD25 VHH fused to either mouse IFN or mutant IFNα2-Q124R caused significant tumor regression (Figure 22). Given that chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R did not appear to reduce lymphocyte numbers (Figure 21), this result suggests that B cell depletion is not required for the antitumor effect of chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R.

[0355] Efficacy of chimeric CD20 VHHs in a mouse model of multiple sclerosis An established murine experimental autoimmune encephalomyelitis (EAE) model was used to test the efficacy of chimeric CD20 VHHs in treating multiple sclerosis. Specifically, mice were transfected with a chimeric CD20 VHH containing the immunodominant epitope of MOG (MOG 92-106 ) to induce brain inflammation and central nervous system (CNS) demyelination. Mice were also given 2MC57 VHH (anti-mouse CD20) fused to mutant IFNα2-Q124R by ip injection on days 7-25 after administration of MOG peptide. As shown in FIG. 23, 2MC57 VHH (anti-mouse CD20) fused to mutant IFNα2-Q124R significantly improved clinical scores and delayed the onset and appearance of symptoms such as disease and paralysis. Taken together, these results suggest that the chimeric CD20 VHH of the present application may have therapeutic effects in the treatment of multiple sclerosis as well as other CNS demyelinating diseases in humans.

[0356] Equivalent While the present application has been described in connection with certain embodiments thereof, it will be understood that further modifications are possible, and the present application is generally intended to cover any variations, uses, adaptations of the present application in accordance with the principles of the present invention, including such departures from the disclosure of the present invention as are within known or customary practice in the art to which the present application pertains, and which may be adapted as follows within the essential features described herein and the scope of the appended claims.

[0357] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed by the scope of the appended claims.

[0358] Incorporation by Reference All patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0359] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior filing.

[0360] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of each section may be equally applicable to all sections.

[0361] References Alduaij W, Illidge TM (2011) The future of anti-CD20 monoclonal antibodies: are we making progress? Blood 117: 2993-3001 Alduaij W, Ivanov A, Honeychurch J, Cheadle EJ, Potluri S, Lim SH, Shimada K, Chan CHT, Tutt A, Beers SA, Glennie MJ, Cragg MS, Illidge TM (2011) Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies.Blood 117: 4519-4529 Cang SD, Muhki N, Wang KM, Liu DL (2012) Novel CD20 monclonal antibodies for lymphoma therapy.Journal of Hematology & Oncology 5: 64 Cragg MS, Morgan SM, Chan HTC, Morgan BP, Filatov AV, Johnson PWM, French RR, Glennie MJ (2003) Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts.Blood 101: 1045-1052 Dolk E, van Vliet C, Perez JMJ, Vriend G, Darbon H, Ferrat G, Cambillau C, Frenken LGJ, Verrips T (2005) Induced refolding of a temperature denatured Ilama heavy-chain antibody fragment by its antigen.Proteins - Structure, Function and Bioinformatics 59: 555-564 Einfield DA, Brown JP, Valentine MA, Clark EA, Ledbetter JA (1988) Molecular cloning of the human B-cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains.EMBO Journal 7: 771 Glennie MJ, French RR, Cragg MS, Talyor RP (2007) Mechanisms of killing by anti-CD20 monoclonal antibodies.Molecular Immunology 44: 3823-3837 Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains.Nature 363, 446-448 Harmsen MM and De Haard HJ (2007) Properties, production, and applications of camelid single-domain antibody fragments.Applied Microbiology and Biotechnology 77: 13-22 Ishibashi K, Suzuki M, Sasaki S, Imai M (2001) Identification of a new multigene four-transmembrane family (MS4A) related to CD20, HTm4 and beta subunit of the high-affinity IgE receptor.Gene 264: 87-93 Lim SH, Beers SA, French RR, Johnson PW, Gelnnie MJ, Cragg MS (2010) Anti-CD20 monoclonal antibodies: historical and future perspectives.Haematologica 95: 135-143 Muyldermans S (2013) Nanobodies: natural single-domain antibodies.Annual Review of Biochemistry 82: 775-779 Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.Protein Engineering 7: 1129-1135 Niederfellner G, Lammens A, Mundigl O, Georges GJ, Schaefer W, Schwaiger M, Franke A, Wiechmann K, Jenewein S, Slootstra JW, Timmerman P, Brannstrom A, Lindstrom F, Mossner E, Umana P, Hopfner KP, Klein C (2011) Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I / II distinction of CD20 antibodies.Blood 118: 358-367 Oettgen HC, Bayard PJ, Van Ewijk W, Nadler LM, Terhorst CP (1983) Further biochemical studies of the human B-cell differentiation antigens B1 and B2.Hybridoma 2: 17-28 O’ Keefe TL, Williams GT, Davies SL, Neuberger MS (1998) Mice carrying CD20 gene disruption.Immunogenetics 48: 125-132 Robak T, Robak E. (2011) New anti-CD20 monoclonal antibodies for the treatment of B-cell lymphoid malignancies.Biodrugs 25: 13-25 Smith, MR (2003) Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance.Oncogene 22: 7359-7368 Stashenko P, Nadler LM, Hardy R, Schlossman SF (1980) Characterization of a human lymphocyte-B-specific antigen.Journal of Immunology 125: 1678-1685 Stashenko P, Nadler LM, Hardy R, Schlossman SF (1981) Expression of cell surface markers after human B lymphocyte activation.Proceedings of the National Academy of Sciences of the Unites States of America 78: 3848-3852 Townsend MJ, Monroe JG, Chan AC (2010) B-cell targeted therapies in human autoimmune diseases: an updated perspective.Immunological Reviews 237: 264-283 Uchida J, Lee Y, Hasegawa M, Liang Y, Bradney A, Oliver JA, Bowen K, Steeber DA, Haas KM, Poe JC, Tedder TF (2004) Mouse CD20 expression and function. International Immunology 16:119-129 Van Meerten T, Hagenbeek A (2010) CD20-targeted therapy: the next generation of antibodies.Seminars in Hematology 47: 199–210 Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Schueplein F, Schwarz N, Adriouch S, Boyer O, Seman M, Licea A, Serreze DV, Goldbaum FA, Haag F, Koch-Nolte F (2009) Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.Medical Microbiology and Immunology 198: 157-174

Claims

1. A CD20 binding molecule comprising at least one targeting moiety consisting of a recombinant heavy chain only antibody (VHH) or a humanized VHH that binds to CD20 and has three complementarity determining regions (CDR1, CDR2, and CDR3), CDR1 consists of the amino acid sequence of SEQ ID NO: 35, CDR2 consists of the amino acid sequence of SEQ ID NO: 46, and CDR3 consists of the amino acid sequence of SEQ ID NO: 56; CDR1 consists of the amino acid sequence of SEQ ID NO: 39, CDR2 consists of the amino acid sequence of SEQ ID NO: 49, and CDR3 consists of the amino acid sequence of SEQ ID NO: 58; CDR1 consists of the amino acid sequence of SEQ ID NO: 40, CDR2 consists of the amino acid sequence of SEQ ID NO: 50, and CDR3 consists of the amino acid sequence of SEQ ID NO: 59; CDR1 consists of the amino acid sequence of SEQ ID NO: 41, CDR2 consists of the amino acid sequence of SEQ ID NO: 50, and CDR3 consists of the amino acid sequence of SEQ ID NO: 60; CDR1 consists of the amino acid sequence of SEQ ID NO:42, CDR2 consists of the amino acid sequence of SEQ ID NO:51, and CDR3 consists of the amino acid sequence of SEQ ID NO:61; or CDR1 consists of the amino acid sequence of SEQ ID NO: 43, CDR2 consists of the amino acid sequence of SEQ ID NO: 53, and CDR3 consists of the amino acid sequence of SEQ ID NO: 62; The CD20 binding molecule further comprises a signaling agent which is optionally modified IFNα2. CD20 binding molecule.

2. Linker sequence, HA tag and / or HIS ６ The CD20 binding molecule of claim 1, comprising an amino acid sequence having at least 95% similarity to any one of SEQ ID NOs: 18, 24, 25, 26, 27, and 30 minus the tag.

3. Linker sequence, HA tag and / or HIS ６ The CD20 binding molecule of claim 1 or 2, comprising one or more of the amino acid sequences of SEQ ID NOs: 18, 24, 25, 26, 27, and 30 minus the tag.

4. The CD20 binding molecule of any one of claims 1 to 3, wherein the CD20 binding molecule comprises one or more additional targeting moieties.

5. A recombinant nucleic acid encoding a CD20 binding molecule according to any one of claims 1 to 4.

6. A host cell comprising the nucleic acid of claim 5.

7. A pharmaceutical composition comprising a CD20 binding molecule according to any one of claims 1 to 4 for use in the treatment of a disease or disorder involving cells expressing CD20.

8. 8. The pharmaceutical composition of claim 7, wherein the disease or disorder is one or more of cancer, an infectious disease, and / or an immune disorder.

9. 13. Use of a CD20 binding molecule according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of a disease or disorder involving cells expressing CD20.

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