CD20-binding single-domain antibody

Abstract

Provided is a CD20-binding agent that binds to CD20-positive cells, such as cancer cells, and causes cell death. [Solution] Provided is a CD20-binding agent comprising at least one targeting moiety comprising three complementarity-determining regions (CDR1, CDR2, and CDR3), each of which comprises an amino acid sequence selected from any one of a specific group of sequences. 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 fully neutralize) CD20. In some embodiments, the CD20-binding agent binds to CD20-positive cells, resulting in the death of the CD20-positive cells.

Description

Technical Field

[0001] The present invention relates in part to binding agents that bind to CD20 (e.g., antibodies such as VHH, but not limited thereto) and their use as therapeutic agents.

Background Art

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

[0003] Antibodies directed against CD20 are used in the treatment of B-cell-derived leukemias and lymphomas. Specifically, Rituxan is a genetically engineered chimeric mouse / human monoclonal antibody directed against CD20. Rituxan is currently approved as a therapeutic agent for relapsed or refractory follicular lymphoma. It has been reported that by injecting Rituxan weekly, the overall response rate was 48% as a result. However, many patients do not respond to Rituxan treatment. Furthermore, patients who respond often develop resistance to Rituxan and ultimately relapse. Additionally, Rituxan is thought to non-specifically kill normal CD20-positive cells, resulting in significant toxicity and side effects.

[0004] Therefore, there is a need for a therapeutically effective CD20-binding agent 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 a CD20 binder 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 binders bind to CD20 but do not functionally modulate (e.g., partially or completely neutralize) CD20. In some embodiments, the CD20 binder binds to CD20-positive cells, resulting in the death of CD20-positive cells.

[0006] In various embodiments, the CD20 binder 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 binder comprises additional targeting moieties that bind to other targets of interest (e.g., antigens, receptors). In one embodiment, the other target of interest (e.g., antigen, receptor) is present on tumor cells. In another embodiment, the other target of interest (e.g., antigen, receptor) is present on immune cells.

[0007] In various embodiments, the CD20 binders of the present invention have been found to be useful in the treatment of various diseases or disorders comprising cells that express 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 various methods of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0008]

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Mode for Carrying Out the Invention

[0009] This application is based, in part, on the discovery of agents that recognize and bind to CD20 (e.g., antibodies such as VHH or nanobodies, as non-limiting examples). In various embodiments, these CD20-binding agents bind to CD20 but do not functionally modulate CD20. In various embodiments, these CD20-binding agents can bind and, directly or indirectly, recruit immune cells to sites that require a therapeutic effect (e.g., a tumor or tumor microenvironment). In various embodiments, the CD20-binding agent binds to CD20-positive cells, resulting in the death of such cells. This application provides pharmaceutical compositions comprising a CD20-binding agent and their use in the treatment of various diseases. In various embodiments, the CD20-binding agent is part of a chimeric protein having a modified signaling agent as described herein.

[0010] CD20-binding agent In various embodiments, the CD20-binding agent of the invention is a protein-based agent that can specifically bind to CD20. In various embodiments, the CD20-binding agent of the 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. This functions as a B cell-specific differentiation antigen in both mouse and human (Stashenko et al., 1980; Oettgen et al., 1983; Ishibashi et al., 2001). In particular, the human CD20 cDNA encodes a transmembrane protein consisting 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 binder of the present application includes 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 contiguous 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 binder of the present application can bind to the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other naturally occurring or synthetic analog, variant, or mutant of CD20 (e.g., human CD20). In various embodiments, the CD20 binder of the present application can bind to CD20 (e.g., human CD20) in any form, such as monomeric, dimeric, trimeric, tetrameric, heterodimeric, multimeric, and associated forms. In one embodiment, the CD20 binder binds to the monomeric form of CD20. In another embodiment, the CD20 binder binds to the dimeric form of CD20. In another embodiment, the CD20 binder binds to the tetrameric form of CD20. In a further embodiment, the CD20 binder binds to the phosphorylated form of CD20, which can be monomeric, dimeric, or trimeric.

[0013] In one embodiment, the CD20 binder of the present invention includes a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human CD20. In one embodiment, human CD20 includes the following amino acid sequence: MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP。

[0014] In various embodiments, the CD20 binder of the present invention comprises a targeting moiety capable of specific binding. In various embodiments, the CD20 binder comprises a targeting moiety having an antigen recognition domain such as an antibody or a derivative thereof. In one embodiment, the CD20 binder 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 comprises 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 binder comprises a targeting moiety that is an antibody derivative or format. In some embodiments, the CD20 binder of the invention comprises a targeting moiety, and the targeting moiety is a single domain antibody, recombinant heavy chain only antibody (VHH), single chain antibody (scFv), shark heavy chain only antibody (VNAR), microprotein (cysteine knot protein, knottin), DARPin; tetranectin; affibody; affimer, transbody; anticalin; adnectin; affilin; microbody; peptide aptamer; alterase, plastic antibody; phylomer; 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 are described in U.S. Patents, or U.S. Patent Publications No. 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 hereby incorporated by reference in their entirety. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0016] In some embodiments, the CD20 binder comprises a targeting moiety that is a single-domain antibody such as a VHH. The VHH can be derived, for example, from an organism that produces VHH antibodies such as camels or sharks, or the VHH can be a designed VHH. A VHH is a therapeutic protein derived from an antibody that includes the unique structural and functional properties of naturally occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies from camels that lack a light chain. These heavy-chain antibodies comprise a single variable domain (V H H) and two constant domains (CH2 and CH3). VHHs are commercially available under the trademark NANOBODIES. In one embodiment, the CD20 binder comprises a nanobody. In some embodiments, the single-domain antibodies described herein are immunoglobulin single variable domains (ISVDs).

[0017] In some embodiments, the CD20 binder comprises a targeting moiety that is a VHH that comprises a single amino acid chain having four "framework (FR)" regions and three "complementary determining regions (CDR)". As used herein, "framework region" or "FR" refers to the regions within the variable domain that are located between the CDRs. As used herein, "complementary determining region" or "CDR" refers to the variable regions within a VHH that comprise amino acid sequences that can specifically bind to an antigenic target.

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

[0019] In some embodiments, the CDR1 sequence is selected from: 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 (SEQ ID NO: 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 (SEQ ID NO: 121).

[0021] In some embodiments, the CDR3 sequence is selected from: 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 (SEQ ID NO: 126).

[0022] In various embodiments, the CD20 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder 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 binder comprises an amino acid sequence selected from the following sequences: 2HCD16: QVQLQESGGGLVQAGGSLRLSCAASGRTFSRQSMGWFRQAPGKEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 18) 2HCD22: QVQLQESGGGLVQAGDSLRLSCAASGRTFSRQSMGWFRQAPGKEREFVAVITWSGGSPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 19) 2HCD35: QVQLQESGGGLVQAGGSLRLSCAASGRTFSGQSMGWFRQAPGKEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 20) 2HCD42: QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 21) 2HCD73: QVQLQESGGGLVQAGGSLRLSCAASGRTFSRQSMGWFRQAPGEEREFVAVITWSGGSPYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSQWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 22) 2HCD81: QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYNMGWFRQAPGKEREFVAVISWSGGSPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPVSYGSSWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 23) R3CD105: QVQLQESGGGLVQAGGSLRLSCAASGRTFSNYNMGWFRQAPGKEREFVAAIDWSGGSPYYAASVRGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAAPLSYGSTWLADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 24) R3CD18: QVQLQESGGGLVQAGGSLRLSCAASGRTFSNSNMGWFRQAPGKEREFVAVIDWSGGSPYYTDSVRGRFTISRDNAKNTVYLQMNRLKPEDTAVYYCAGGVSFGSRWLSDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 25) R3CD7: QVQLQESGGGLVQAGGSLRLSCAASGRSFSSVNMGWFRQAPGKEREFVAVIDWSGGSPYYTDSVRGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGVSYGSRWLGDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 26) 2HCD25: QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 27) 2HCD78: QVQLQESGGGLVQPGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 28) 2HCD17: QVQLQESGGGLVQAGGSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVIWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 29) 2HCD40: QVQLQESGGGLVQAGGSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVTWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 30) 2HCD88: QVQLQESGGGLVQAGDSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVTWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 31) 2HCD59: QVQLQESGGGSEQPGGSLRLSCAASGRTVGSYSMGWFRQAPGKEREFVAAVTRSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 32) 2HCD68: QVQLQESGGGLVQPGGSLRLSCAASRFTLDYYAIGWFRQAPGKEREFVAAVTWSGASPYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYSGGWHAEYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 33) 2HCD43: QVQLQESGGGLVQPGGSLRLSCTASGFTLDYYAIGWLRQAPGKEREGVSCISSSGGSTNYADSVKGRFTISRDNAKNTVYLLMNSLKPEDTAVYYCAAERTWVSNYYCSGDGDGYDYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 34) 2MC57: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAMPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 85) R2MUC70: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 86) R3MUC17: QVQLQESGGGLVPAGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 87) R3MUC56: QVQLQESGGGLVQAGDSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 88) R3MUC57: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMAWFRQAPGEERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 89) R3MUC58: QVQLQESGGGLVQPGGSLRLSCAASGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 90) R2MUC85: QVQLQESGGGLVQPGGSLRLSCATSGRDFATYSMAWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 91) R3MUC66: QVQLQESGGGLVQAGGSLRLSCAASGRDFATYSMTWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 92) R2MUC21: QVQLQESGGGLVQAGDSLRLSCAASGRDFSTYSMGWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 93) 2MC52: QVQLQESGGGLVQAGGSLRLSCVASGRTFNTYSMGWFRQAPGKEREFVASISWSGQRSRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 94) R3MUC22: QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYSMGWFRQAPGKEREFVAIISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 95). R3MUC75: QVQLQESGGGSVQTGGSLRLSCAASGRDFSTYSMGWFRQAPGKERESVATISWSGQRTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPRTWGEFPPTQYDSWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 96). 2MC39: QVQLQESGGGLAQAGNSLRISCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH(SEQ ID NO: 97). 2MC51: QVQLQESGGGLAQAGNSLRISCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 98). 2MC38: QVQLQESGGGLVQAGESLRISCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 99). 2MC82: QVQLQESGGGLAQEGGSLRLSCVASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 100). 2MC20: QVQLQESGGGLVQTGGSLRLSCAASGNTFDTRAMGWFRQAPGKEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 101). 2MC42: QVQLQESGGGSVQTGGTLTLSCVASGNTFDTRAMGWFRQAPGEEREFVAAISRSSFNTYYSDSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGKYGMKWRDGADYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 102). R2MUC36: QVQLQESGGGLVQAEGSLRLSCAASGRTRDANAMGWFRQAPGKERELVAAISWSGSRTYYADSVKGRFTISRDNVMHTVYLSMNSLKPEDTAVYYCAADRSIEVQIADYDYWGRGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 103). R3MCD137: QVQLQESGGGSVQAGGSLRLSCAASGSTFSIKAMGWYRQAPGKQRELVAAFISGRGSTKYADSVKGRFAISRDNAKNTMYLQMDSLEPEDTAVYYCYIVLPTGGGSAMDYWGEGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 104). R3MCD22: QVQLQESGGGVVQAGGSLRLSCAASGSTFSIKAMGWYRQAPGKQRDLVAGFISGRGSAKYADSVKGRFAISRDNAKNTMYLQMDSLKPEDTAVYYCYIVLTTGGGSAMDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH (SEQ ID NO: 105).

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

[0048] In various embodiments, the CD20 binder comprises a targeting moiety comprising a sequence that is at least 60% identical to any one of SEQ ID NOs: 18-34 or 85-105. In various embodiments, the CD20 binder comprises a targeting moiety comprising a sequence that is at least 60% identical to any one of SEQ ID NOs: 18-34 or 85-105 minus a linker sequence, HA tag, and / or HIS6 tag. For example, the CD20 binder is at least 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%, at least 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 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 any one of SEQ ID NOs: 18-34 or 85-105) and may comprise a targeting moiety.

[0049] In various embodiments, the CD20 binder comprises a targeting moiety comprising an amino acid sequence having one or more amino acid mutations with respect to SEQ ID NOs: 18-34 or 85-105. In various embodiments, the CD20 binder comprises a targeting moiety comprising 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 with respect to SEQ ID NOs: 18-34 or 85-105. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0050] In some embodiments, the amino acid mutation is an amino acid substitution and can include conservative and / or non-conservative substitutions.

[0051] "Conservative substitutions" can be made, for example, based on the similarity of 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 the following 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 exchange of an amino acid by another amino acid listed within the same group of the above six standard amino acid groups. For example, the exchange of Asp by Glu retains one negative charge in the polypeptide so modified. Further, glycine and proline can be substituted for each other based on their ability to disrupt an α-helix.

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

[0054] In various embodiments, the substitution can be a non-classical amino acid (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, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, etc., designer amino acids, and generally amino acid analogs).

[0055] In various embodiments, the amino acid mutation can be present in the CDR of the targeting moiety (e.g., the CDR1, CDR2 or CDR3 region). In another embodiment, the amino acid change can be present in the framework region (FR) of the targeting moiety, e.g., the FR1, FR2, FR3, or FR4 region.

[0056] Modification of the amino acid sequence can be achieved using any known technique in the art, e.g., 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, N.Y., 1989, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

[0057] In various embodiments, the mutation does not substantially reduce the ability of the CD20 binder of the present invention to specifically bind to CD20. In various embodiments, the mutation does not substantially reduce the ability of the CD20 binder of the present invention to specifically bind to CD20 without neutralizing CD20.

[0058] In various embodiments, the binding affinity of the CD20 binder of the present application for the full-length, and / or mature form, and / or isoform, and / or splice variant, and / or fragment, and / or monomer, and / or dimer, and / or tetrameric form of human CD20, and / or any other naturally occurring or synthetic analog, variant or mutant (such as monomeric and / or dimeric and / or tetrameric forms), can be described by the equilibrium dissociation constant (K D d). In various embodiments, the CD20 binder has a K D of less than about 1 μM, 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, and comprises a targeting moiety that binds to the full-length, and / or mature form, and / or isoform, and / or splice variant, and / or fragment, and / or any other naturally occurring or synthetic analog, variant or mutant (such as monomeric, and / or dimeric, and / or tetrameric forms) of human CD20.

[0059] In various embodiments, the CD20 binder comprises a targeting moiety that binds to the antigen of interest, namely CD20, but does not functionally modulate it. For example, in various embodiments, the targeting moiety of the CD20 binder merely targets the antigen, but does not substantially functionally modulate (e.g., substantially inhibit, reduce, or neutralize) the biological effects of the antigen. In various embodiments, the targeting moiety of the CD20 binder binds to an epitope (e.g., the active site of the antigen) that is physically separated from the antigenic 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 binder of the present invention binds to CD20-positive cells and induces the death of such cells. In some embodiments, the CD20 binder induces cell death mediated by one or more of apoptosis or direct cell death, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cell phagocytosis (ADCP). In some embodiments, the CD20 binder of the present invention induces the translocation of CD20 into large lipid microdomains or "lipid rafts" within the plasma membrane upon binding of CD20. This clustering process enhances complement activation and exerts potent complement-dependent cytotoxicity (CDC). In other embodiments, the CD20 binder of the present invention induces direct cell death. In alternative embodiments, the therapeutic effect of the CD20 binder does not depend on B cell depletion.

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

[0062] Therapeutic agent comprising the CD20 binder of the present invention Chimeras and fusions with signaling agents In various embodiments, the CD20 binder of the present application is part of a chimera or fusion with one or more signaling agents. Accordingly, the present application provides, for example, a chimeric or fusion protein comprising a targeting moiety for CD20 and one or more signaling agents.

[0063] In various embodiments, the signaling agent is modified such that the affinity or activity of one or more of its receptors is reduced, thereby enabling attenuation of activity (such as agonism or antagonism), and / or preventing non-specific signaling or unwanted sequestration of chimeric or fusion proteins. In various embodiments, the signaling agent is an antagonist in its wild-type form and carries one or more mutations that weaken its antagonistic action. In various embodiments, the signaling agent is antagonistic due to one or more mutations, for example, an agonist signaling agent is converted to an antagonistic signaling agent, and such a converted signaling agent carries one or more mutations that weaken its antagonistic action (as described, for example, in International Publication No. WO 2015 / 007520, which is hereby incorporated by reference in its entirety).

[0064] Accordingly, in various embodiments, the signaling agent is a modified (e.g., mutated) form of a signaling agent having one or more mutations. In various embodiments, by modifying (e.g., mutating), the modified signaling agent can have one or more of attenuated activities, such as one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to the unmodified or non-mutated, i.e., wild-type form of the signaling agent (e.g., compared to the wild-type form versus the modified or mutated form of the same signaling agent). In some embodiments, mutations that weaken or reduce binding or affinity include those that substantially reduce or abolish binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity are different from mutations that substantially reduce or abolish binding or activity. As a result, in various embodiments, compared to the non-mutated, i.e., wild-type signaling agent (e.g., compared to the same signaling agent in the wild-type form versus the modified form (e.g., mutant)), the mutation enables improvement in the safety of the signaling agent, such as reduction in systemic toxicity, reduction in side effects, and reduction in off-target effects.

[0065] As described herein, the agent may have improved safety by one or more modifications, such as mutations. In various embodiments, improved safety means that the chimeric protein of the invention has lower toxicity (e.g., systemic toxicity and / or tissue / organ related toxicity), and / or reduced or substantially eliminated side effects, and / or increased tolerance, reduced or substantially eliminated adverse events, and / or decreased 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 for 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 for the receptor. In some embodiments, the activity conferred 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 being able to deliver a reduced activation signal to the target cell or eliminating the activation signal. In some embodiments, the activity conferred 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 antagonistic activity at the receptor. For example, the mutation results in the modified signaling agent being able to deliver a reduced inhibitory signal to the target cell or eliminating the inhibitory signal. In various embodiments, the signaling agent is made antagonistic by one or more mutations. For example, an agonist signaling agent is converted to an antagonist signaling agent (as described, for example, in International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference), and the signaling agent thus converted optionally also bears one or more mutations that reduce its binding affinity or activity for one or more of its receptors or substantially reduce or eliminate its binding affinity or activity for one or more of its receptors.

[0067] In some embodiments, a decrease in affinity or activity at the receptor can be restored by attachment to one or more of the targeting moieties described herein (e.g., a targeting moiety to CD20, or any other targeting moiety described herein). In other embodiments, a decreased affinity or activity at the receptor is not substantially restorable 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 that weaken or abolish binding affinity or activity at the receptor. In various embodiments, for example, such a reduction in side effects is observed compared to wild-type signaling agents. In various embodiments, the signaling agent is active on the target cell to compensate for a defective / inadequate binding (e.g., no restriction and / or binding force) although the targeting moiety(ies) is / are required for substantial activation. In various embodiments, the modified signaling agent is substantially inactive until it reaches the site of therapeutic activity and has its effect substantially on specifically targeted cell types that greatly reduce unwanted side effects.

[0069] In some embodiments, the signaling agent can include one or more mutations that weaken or reduce the binding or affinity for one receptor (i.e., the therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity in a second receptor. In such embodiments, these mutations can 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 in one receptor are different from the mutation(s) that are substantially reduced or eliminated in another receptor. In some embodiments, the mutation(s) that reduce binding and / or activity in one receptor are the same as the mutation(s) that are substantially reduced or eliminated in another receptor. In some embodiments, the chimeric protein of the invention has a modified signaling agent with a mutation that weakens binding and / or activity in the therapeutic receptor and thus can more controllably regulate a more controlled on-target therapeutic effect (e.g., relative to the wild-type signaling agent), as well as a mutation that substantially reduces or eliminates binding and / or activity in another receptor and thus reduces side effects (e.g., as compared to the wild-type signaling agent).

[0070] In some embodiments, a substantial decrease or loss of binding or activity is not substantially recoverable by the targeting moiety (e.g., a targeting moiety to CD20 or any other targeting moiety described herein). In some embodiments, a substantial decrease or loss of binding or activity is recoverable by the targeting moiety. In various embodiments, substantially decreasing or eliminating binding or activity at a second receptor can also prevent deleterious effects mediated by other receptors. Alternatively or in addition, substantially decreasing or eliminating binding or activity at other receptors reduces or eliminates sequestration of the therapeutic chimeric protein away from the site of therapeutic action, thereby improving the therapeutic effect. For example, in some embodiments, this obviates the need for high doses of the chimeric proteins of the invention that compensate for loss at other receptors. The ability to reduce the dose in this way results in a lower likelihood of side effects.

[0071] In various embodiments, the modified signaling agent comprises one or more mutations by which the signaling agent has a reduced, substantially reduced, or lost affinity, e.g., binding (e.g., K D ), and / or (e.g., if the modified signaling agent is an agonist of the receptor measurable as, e.g., K A and / or EC 50 ) activation, and / or (e.g., if the modified signaling agent is K I and / or IC 50when it is an antagonist of the receptor that can be measured) inhibition will be exerted on one or more of those receptors. In various embodiments, a decrease in affinity at the receptor of the agent enables attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling agent has an affinity for the receptor that is 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% that of 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, more than 200-fold lower than that of the wild-type signaling agent.

[0072] In embodiments where the chimeric protein has a mutation that reduces binding to one receptor and substantially reduces or eliminates binding to a second receptor, the attenuation or decrease in the binding affinity of the modified signaling agent for one receptor is less than the substantial reduction or elimination of the affinity for the other receptor. In some embodiments, the attenuation or decrease in the binding affinity of the modified signaling agent for one receptor is 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% less than the substantial reduction or elimination of the affinity for the other receptor. In various embodiments, a substantial reduction or elimination refers to a greater reduction in binding affinity and / or activity than an attenuation or decrease.

[0073] In various embodiments, the modified signaling agent comprises one or more mutations that reduce the intrinsic activity of the signaling agent, for example, by 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 a reduced affinity of the signaling agent for its target moiety(ies) for its receptor(s). In some embodiments, this difference in binding affinity is the difference between the signaling agent / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity allows the signaling agent, e.g., the mutated signaling agent, to have a localized on-target effect and minimize the off-target effects that underlie the side effects observed with the wild-type signaling agent. In some embodiments, this 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 lower.

[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 of binding data and computer fitting (e.g., Scatchard, 1949), or by reflectance interference spectroscopy under flow-through conditions as described by Brecht et al. (1993), the entire contents of which are incorporated herein by reference.

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

[0077] In some embodiments, the signaling agent is an interleukin or a 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 inhibiting 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, D.C. 1987, pages 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 interferons. 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 a tumor necrosis factor (TNF), or a modified form of a 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 well known in the art. Thus, in various embodiments, the modified signaling agent has an amino acid sequence having 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 known wild-type amino acid sequences of the signaling agents described herein.

[0081] In various embodiments, the modified signaling agent comprises an amino acid sequence having 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 any amino acid sequence of the signaling agents described herein.

[0082] In various embodiments, the modified signaling 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 mutation is an amino acid substitution and can include conservative and / or non-conservative substitutions, as described elsewhere herein.

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

[0084] As described herein, the modified signaling agent carries a mutation that affects affinity and / or activity at one or more receptors. As described herein, the receptors for any signaling agent are known in the art.

[0085] Exemplary mutations that result in decreased affinity and / or activity (e.g., agonistic activity) at the receptor are found in International Publication No. WO 2013 / 107791 (e.g., related to interferon), International Publication No. WO 2015 / 007542 (e.g., related to interleukin), and International Publication No. WO 2015 / 007903 (e.g., related to TNF), the entire contents of each of which are incorporated herein by reference. Exemplary mutations that result in decreased affinity and / or activity (e.g., antagonistic activity) at the receptor are found in International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference.

[0086] In one embodiment, the modified signaling agent is interferon alpha. In such an embodiment, the modified IFN-α agent has decreased affinity and / or activity for the IFN-α / β-1 receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFN-α agent has substantially decreased or abolished affinity and / or activity for the IFN-α / β-1 receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

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

[0088] In an exemplary embodiment, the modified signal transducer is the allelic form 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 above IFN-α2 mutants (IFN-α2a or IFN-α2b) are mutated with 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 International Publication No. 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 a 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 International Publication No. WO 2010 / 030671, the entire content of which is incorporated herein by reference.

[0091] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A, as described in International Publication No. WO 2008 / 124086, the entire content of which is 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 International Publication No. WO 2015 / 007520 and International Publication No. WO 2010 / 030671, the entire content of which is 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 a reduced affinity and / or activity for IFNAR1 and retains the affinity and / or activity for IFNR2.

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

[0094] In one embodiment, the modified signaling agent is interferon β. In such an embodiment, the modified interferon β agent has a 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 β agent has a substantially reduced or absent 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 signaling agent is interferon γ. In such an embodiment, the modified interferon γ agent has a reduced affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon γ agent has a substantially reduced or absent affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains.

[0096] In one embodiment, the modified signaling agent is TNF-α. TNF is a pleiotropic cytokine with many diverse functions such as 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, while TNFR2 is preferentially expressed in specific populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. Although very different biological pathways are activated in response to receptor activation, there is also some overlap. As a general rule, without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), and TNFR2 signaling 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, activation of TNFR2 is also associated with a wide range of activities, and it should be noted that targeting and control of TNF activity are important in the context of developing TNF-based therapies, as is the case with TNFR1.

[0097] In some embodiments, the modified signaling agent has reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling agent has substantially reduced or abolished 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 related to methods of treating cancer, the modified signaling agent has reduced affinity and / or activity for TNFR1 and / or substantially reduced or abolished affinity and / or activity for TNFR2. In these embodiments, the chimeric protein may be targeted to cells in which apoptosis is desired, such as tumor cells or tumor vascular endothelial cells. For example, in embodiments related to methods of promoting cell survival in neurogenesis for the treatment of neurodegenerative diseases, the modified signaling agent has reduced affinity and / or activity for TNFR2 and / or substantially reduced or abolished 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 a death signal or a survival signal.

[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 abolished affinity and / or activity for TNFR2. Such chimeras are, in some embodiments, more potent apoptosis inducers compared to chimeras that carry only mutations (s) that cause a reduction in affinity and / or activity for wild-type TNF and / or TNFR1. Such chimeras are, in some embodiments, found to be useful in inducing tumor cell death or tumor vasculature endothelial cell death (e.g., in the treatment of cancer). Also, in some embodiments, these chimeras, for example, T via TNFR2 regAvoid or reduce cell activation and thus, in vivo, further assist TNFR1-mediated antitumor activity.

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

[0100] In some embodiments, a chimera (e.g., a modified TNF having reduced affinity and / or activity for TNFR2 and / or substantially reduced or abolished affinity and / or activity for TNFR1) causes the death of autoreactive T cells, e.g., by activation of TNFR2 and / or avoidance of TNFR1. Without wishing to be bound by theory, these autoreactive T cells alter apoptosis / survival signals, e.g., by changes in NFkB pathway activity / signal transduction.

[0101] In some embodiments, TNFR-2-based chimeras have additional therapeutic uses in various heart diseases, especially demyelination and neurodegenerative disorders, and diseases such as infectious diseases.

[0102] In one embodiment, 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 amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147 and produces a modified TNF-α with reduced receptor binding affinity. See, for example, 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, as described in International Publication No. WO 2015 / 007903, the entire contents of which are incorporated herein by reference (numbering according to the human TNF sequence, Genbank accession number BAG70306, version BAG70306.1 GI:197692685). In some embodiments, the modified TNF-α agent has substitution mutations 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 content of which is 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 abolished affinity and / or activity for TNFR1, and / or TNFR2, and / or herpesvirus entry mediator (HEVM), and / or LT-βR.

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

[0108] In such embodiments, the modified TNF-β agent may contain a mutation in one or more amino acids at positions 106-113, thereby producing a modified TNF-β agent having 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 mutation is 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 can be a TNF family member (e.g., TNF-alpha, TNF-beta) that is a single-chain trimeric form, as described in International Publication No. WO 2015 / 007903, the entire content of which is incorporated herein by reference.

[0110] In some embodiments, the modified agent is a TNF family member (e.g., TNF-alpha, TNF-beta) having reduced affinity and / or activity at TNFR1, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity resulting from one or more mutations (see, e.g., International Publication No. WO 2015 / 007520, the entire content of which is incorporated herein by reference)). In these embodiments, the modified agent is optionally also a TNF family member (e.g., TNF-alpha, TNF-beta) having substantially reduced or abolished affinity and / or activity for TNFR2. In some embodiments, the modified agent is a TNF family member (e.g., TNF-alpha, TNF-beta) having reduced affinity and / or activity at TNFR2, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity resulting from one or more mutations (see, e.g., International Publication No. WO 2015 / 007520, the entire content of which is incorporated herein by reference)). In these embodiments, the modified agent is optionally also a TNF family member (e.g., TNF-alpha, TNF-beta) having substantially reduced or abolished affinity and / or activity for TNFR1. Constructs of such embodiments are used, for example, in methods of attenuating the TNF response in a cell-specific manner. In some embodiments, the antagonist TNF family member (e.g., TNF-alpha, TNF-beta) is the single-chain trimeric form described in International Publication No. WO 2015 / 007903.

[0111] In one embodiment, the modified signaling 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 abolished 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 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG.

[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 signal transducer is an interleukin. In one embodiment, the modified signal transducer is IL-1. In one embodiment, the modified signal transducer is IL-1α or IL-1β. In some embodiments, the modified signal transducer has a reduced affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signal transducer has a substantially reduced or abolished affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signal transducer has a reduced affinity and / or activity for IL-1R2. In some embodiments, the modified signal transducer has a substantially reduced or abolished affinity and / or activity for IL-1R2. For example, in some embodiments, the modified IL-1 agent of the present invention avoids interaction with IL-1R2 and thus substantially reduces its function as a decoy and / or sink for therapeutic agents.

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

[0116] IL1 is an inflammatory cytokine and an important immune system regulator. It is a potent activator of CD4 T cell responses, increasing the proportion of Th17 cells and the expansion of IFNγ- and IL-4-producing cells. IL-1 is also a potent regulator of CD8 + T cells and an antigen-specific CD8 +Promote the expansion, differentiation, migration to the periphery and memory of T cells. The IL-1 receptor includes IL-1R1 and IL-1R2. Binding to IL-1R1 and signal transduction via IL-1R1 constitute the mechanism by which IL-1 mediates many of its biological (and pathological) activities. IL-1R2 functions as a decoy receptor, thereby reducing the availability of IL-1 for interaction and signal transduction via IL-1R1.

[0117] In some embodiments, the modified IL-1 has a reduced affinity and / or activity (e.g., agonist activity) for IL-1R1. In some embodiments, the modified IL-1 has a substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, there is recoverable IL-1 / IL-1R1 signal transduction in IL-R2 and prevention of disappearance of the therapeutic chimera, and thus a reduction in the dose of IL-1 required (e.g., for IL-R1, compared to a chimera having only wild-type or attenuated mutations). Such constructs have found use in methods of treating cancer, including, for example, stimulating the immune system to increase an anti-cancer response.

[0118] In some embodiments, the modified IL-1 has a reduced affinity and / or activity (e.g., antagonist activity, e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference) for IL-1R1. In some embodiments, the modified IL-1 has a substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, there is non-recoverable IL-1 / IL-1R1 signal transduction in IL-R2 and prevention of disappearance of the therapeutic chimera, and thus a reduction in the dose of IL-1 required (e.g., for IL-R1, compared to a chimera having only wild-type or attenuated mutations).

[0119] In such embodiments, the modified signaling agent produces a modified IL-1β that has a deletion of amino acids 52-54 and has a reduced binding affinity for type I IL-1R and a reduced biological activity. See, for example, International Publication No. WO 1994 / 000491, the entire contents of which are incorporated herein by reference. In some embodiments, the modified IL-1β has one or more substitution mutations selected from 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 S / N224A, N224S / K225S, E244K, N245Q (where X may be any change in an amino acid, e.g., a non-conservative change) and exhibits reduced binding to IL-1R. These are as described, for example, in International Publication Nos. WO 2015 / 007542 and WO 2015 / 007536, the entire contents of which are incorporated by reference (numbering based on the human IL-1β sequence, Genbank accession number 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 signal transduction agent is IL-2. In such an embodiment, the modified signal transduction agent has a reduced affinity and / or activity for IL-2Rα, and / or IL-2Rβ, and / or IL-2Rγ. In some embodiments, the modified signal transduction agent has a reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified signal transduction agent has a substantially reduced or abolished affinity and / or activity for IL-2Rα. Such embodiments may be relevant to the treatment of cancer, for example, when the modified IL-2 is an agonist in IL-2Rβ and / or IL-2Rγ. For example, the constructs of the present invention prefer activated CD8 + T cells (which can provide an anti-tumor effect) having IL-2 receptor β and γ, and do not prefer T reg (which can provide an immunosuppressive, pre-tumor effect) having IL-2 receptor α, β, and γ. Further, in some embodiments, by selecting IL-2Rβ and / or IL-2Rγ over IL-2Rα, side effects of IL-2 such as pulmonary edema are avoided. Also, for example, in IL-2Rβ and / or IL-2Rγ, when the modified IL-2 is an antagonist (e.g., an antagonist action that is a result of a natural antagonist action or one or more mutations, see, for example, International Publication No. WO 2015 / 007520, the entire content of which is incorporated herein by reference), the IL-2-based chimeras are useful for the treatment of diseases. For example, the constructs of the present invention prefer CD8 + T cells having IL-2 receptor β and γ with weakened suppression (and thus weakening the immune response), and may not prefer T reg having IL-2 receptor α, β, and γ. Alternatively, in some embodiments, the chimeras carrying IL-2 prefer the activation of T reg , and thus immunosuppression, and do not prefer the activation of CD8 + T cells. For example, these constructs have been found to be useful in the treatment of diseases (s) that would benefit from immunosuppression.

[0121] In some embodiments, the chimeric protein is directed to a modified IL-2 agent that has reduced affinity and / or activity for CD8 + T cells, and for IL-2Rβ and / or IL-2Rγ, and / or substantially reduced or abolished affinity and / or activity for IL-2Rα, and has the targeting moiety described herein. In some embodiments, these constructs provide targeted CD8 + T cell activity, which are generally inactive (or have substantially reduced activity) against T reg cells. In some embodiments, such constructs have an improved immunostimulatory effect (e.g., without wishing to be bound by theory, by not stimulating Tregs) compared to wild-type IL-2 and eliminate or reduce the systemic toxicity associated with IL-2.

[0122] In one embodiment, 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 agent has 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, these modified IL-2 agents are thought to have a reduced affinity for the high-affinity IL-2 receptor and to retain an affinity for the intermediate-affinity IL-2 receptor as 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 a reduced affinity and / or activity for the IL-3 receptor, which is a heterodimer having a unique alpha chain that pairs with the common beta (beta c or CD131) subunit. In some embodiments, the modified signaling agent has a substantially reduced or abolished affinity and / or activity for the IL-3 receptor, which is a heterodimer having a unique alpha chain that pairs with the common beta (beta c or CD131) subunit.

[0125] In one embodiment, the modified signal transducer is IL-4. In such embodiments, the modified signal transducer has reduced affinity and / or activity for type 1 and / or type 2 IL-4 receptors. In such embodiments, the modified signal transducer has substantially reduced or abolished affinity and / or activity for type 1 and / or type 2 IL-4 receptors. The type 1 IL-4 receptor is composed of an IL-4Rα subunit having a common γ chain and specifically binds to IL-4. The type 2 IL-4 receptor includes an IL-4Rα subunit bound to a different subunit known as IL-13Rα1. In some embodiments, the modified signal transducer has substantially reduced or abolished affinity and / or activity for the type 2 IL-4 receptor.

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

[0127] In such embodiments, 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 activity mediated by the type I receptor but significantly reduce 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, such as the ligand-binding 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 thought to be involved in neurite outgrowth and neuron survival, and thus may be important in nerve regeneration by myelin reorganization. Accordingly, in some embodiments, the modified signaling agent has a reduced affinity and / or activity for IL-6R / gp130 and / or sIL-6R. In some embodiments, the modified signaling agent has a substantially reduced or abolished affinity and / or activity for IL-6R / gp130 and / or sIL-6R.

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

[0130] In such embodiments, the modified signaling agent has one or more mutations at amino acids 58, 160, 163, 171, or 177. Without wishing to be bound by theory, these modified IL-6 agents are thought to exhibit a reduced binding affinity for IL-6Rα and a 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 signal transducer is IL-10. In such an embodiment, the modified signal transducer has a reduced affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2. In some embodiments, the modified signal transducer has a substantially reduced affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2.

[0132] In one embodiment, the modified signal transducer is IL-11. In such an embodiment, the modified signal transducer has a reduced affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130. In such an embodiment, the modified signal transducer has a lost or substantially reduced affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0133] In one embodiment, the modified signal transducer is IL-12. In such an embodiment, the modified signal transducer has a reduced affinity and / or activity for IL-12R-β1 and / or IL-12R-β2. In such an embodiment, the modified signal transducer has a substantially reduced or lost affinity and / or activity for IL-12R-β1 and / or IL-12R-β2.

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

[0135] In one embodiment, 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 agent has 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, these modified IL-13 agents are thought to 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 abolished affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or abolished affinity and / or activity for IL-18RαII, an isoform of IL-18Rα that lacks the TIR domain required for signaling.

[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 contain one or more mutations in an amino acid or amino acid region selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183, and Q190-N191, as described in International Publication No. WO 2015 / 007542, the entire contents of which are incorporated herein by reference (numbering based on the human IL-18 sequence, Genbank accession number AAV38697, version AAV38697.1, GI:54696650).

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

[0142] In such embodiments, the modified IL-33 agent may contain one or more mutations in the 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 International Publication No. WO 2015 / 007542, the entire contents of which are incorporated herein by reference (numbering based on the human sequence, Genbank accession number NP_254274, version NP_254274.1, GI:15559209).

[0143] In one embodiment, the chimeric protein of the present invention has (i) a targeting moiety for CD20 and (ii) a targeting moiety directed against tumor cells, together with either a modified or mutant signaling agent as described herein. In one embodiment, the chimeric protein of the present invention has a targeting moiety for CD20 on tumor cells and a second targeting moiety for PD-L1 or PD-L2.

[0144] Multispecific chimeras and fusions with signaling agents In various embodiments, the CD20 binder of the present application is part of a chimera or fusion with one or more signaling agents as described herein and / or one or more additional targeting moieties. Accordingly, the present application provides chimeric or fusion proteins comprising 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 protein of the present application has targeting moieties that target two different cells (e.g., to form a synapse) or the same cell (e.g., to obtain a more concentrated signaling agent effect).

[0146] In various embodiments, the CD20 binding agent of the present application is multispecific, i.e., the CD20 binding agent comprises 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 agent 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 binding affinity and / or improved selectivity. In one embodiment, the CD20 binding agent of the present application comprises two targeting moieties and is bispecific, i.e., it binds to and recognizes 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, and each targeting moiety is an antibody or antibody derivative 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 immune cell.

[0148] In various embodiments, the multispecific CD20 binding agent of the present invention has two or more targeting moieties that target different antigens or receptors, and one of the targeting moieties may be attenuated with respect to that antigen or receptor, e.g., the targeting moiety binds to that antigen or receptor with a low affinity or binding strength (e.g., with an affinity or binding strength lower than that of the other targeting moieties for that antigen or receptor, 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, and e.g., at the lower affinity or binding strength, the targeting moiety has a K in the range of moderate to high nM, or low to moderate μM Dand can bind to the antigen or receptor, with a higher affinity or binding strength, the targeting moiety has a K in the range of moderate to high pM or low to moderate nM D and can bind to the antigen or receptor). For example, in some embodiments, the multispecific CD20 binder of the invention comprises a weakened targeting moiety directed to a promiscuous antigen or receptor. This can improve targeting to the cell of interest (e.g., via other targeting moieties) and prevent effects across multiple cell types, such as those that are not targeted for treatment (e.g., by binding to the promiscuous antigen or receptor with a higher affinity than provided in these embodiments).

[0149] The multispecific CD20 binders of the present application can be constructed using methods known in the art. See, for example, U.S. Patent No. 9,067,991, U.S. Patent Publication No. 20110262348, and International Publication No. 2004 / 041862, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the multispecific CD20 binder of the present application comprising two or more targeting moieties can be constructed by chemically crosslinking, for example, by reacting amino acid residues with an organic derivatizing agent 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, a multispecific CD20 binder comprising two or more targeting moieties is constructed by gene fusion, i.e., by constructing a single polypeptide comprising the polypeptides of the individual targeting moieties. For example, a single polypeptide construct can 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 a further exemplary embodiment, the multispecific CD20 binder of the present application can be constructed using a linker. For example, the carboxy terminus of a first VHH having an antigen recognition domain for CD20 can be linked (or vice versa) to the amino terminus of a second antibody or antibody derivative having a recognition domain for a tumor antigen. Exemplary linkers that can be used are described herein. In some embodiments, the components of the multispecific CD20 binder of the present application are directly linked to each other without using 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 agent specifically binds to the antigen of interest and effectively mobilizes one or more immune cells either directly or indirectly. In an exemplary embodiment, the CD20 binding agent of the present invention can directly or indirectly mobilize immune cells (e.g., dendritic cells) to the site of action (e.g., but not limited to, the tumor microenvironment, etc.).

[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 cells or tumor cells. In these embodiments, the CD20 binding agent of the present invention can have enhanced selectivity for CD20-positive cancer cells or tumor cells by binding to two or more antigens on cancer cells or tumor cells (i.e., CD20 and another cancer antigen or tumor antigen).

[0152] In some embodiments, the CD20 binding agent of the invention can be used, or its use can be found, in a method involving shifting the balance of immune cells for the immune attack of tumors. For example, the CD20 binding agent of the invention can kill and / or suppress cells such as tumors (e.g., T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, and those contrasting with cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg); tumor-associated neutrophils (TAN), M2 macrophages, tumor-associated macrophages (TAM), or subsets thereof), so as to shift the proportion of immune cells at clinically important sites. In some embodiments, the CD20 binding agent of the invention can increase the ratio of effector T cells to regulatory T cells.

[0153] In some embodiments, the 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 a receptor) associated with cancer cells or tumor cells. In some embodiments, the targeting moiety directly or indirectly mobilizes tumor cells. For example, in some embodiments, the mobilization of tumor cells is towards one or more effector cells (e.g., the immune cells described herein) that can kill and / or suppress tumor cells.

[0154] A tumor cell or cancer cell refers to uncontrollable proliferation of cells or tissues, and / or inhibition of abnormally increased cell survival rate and / or apoptosis that interferes with the normal functions of organs and systems of the body. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, 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 cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal 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; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lips, 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, and B-cell lymphoma (such as low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocyte (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; large lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD) and abnormal blood vessel proliferation associated with phakomatosis, edema (e.g., associated with brain tumors), and Meigs syndrome.

[0155] Tumor cells, or cancer cells, include, but are not limited to, carcinomas (e.g., various subtypes such as 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 and non-Hodgkin lymphomas, light chain, non-secretory, MGUS, and plasmacytoma, etc.), and cancers of the central nervous system (e.g., brain (e.g., gliomas (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningioma, pituitary adenoma, neuroma, etc.), and spinal cord tumors (e.g., meningioma and neurofibroma)).

[0156] In certain embodiments, the cancer or tumor cells refer to leukemia cells or lymphoma cells such as the following cells: B cell lymphoma, non-Hodgkin lymphoma (NHL), lymphocyte-predominant subtype of Hodgkin lymphoma, precursor B cell lymphoblastic leukemia / lymphoma, mature B cell neoplasm, B cell chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), 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 marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, multiple myeloma, and anaplastic large cell type 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, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin and gamma-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as 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-1CT-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 are included. In various embodiments, the CD20 binder comprises a targeting moiety that binds to one or more of these tumor cell antigens.

[0158] In some embodiments, the multispecific CD20 binder of the present invention recognizes and binds to CD20 and an antigen on tumor cells. In some embodiments, the multispecific CD20 binder directly or indirectly recruits immune cells to tumor cells or the tumor microenvironment.

[0159] In some embodiments, the multispecific CD20 binder 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 T cells. In some embodiments, the targeting moiety directly or indirectly recruits T cells. In one embodiment, the antigen recognition domain specifically binds to effector T cells. In some embodiments, the antigen recognition domain directly or indirectly recruits effector T cells to, for example, in some embodiments, a treatment 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., αβ TCR, CD3 + , CD8 + , CD45RO + ); CD4 + effector T cells (e.g., αβ TCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL-7R / CD127 + ); CD8 + effector T cells (e.g., αβ TCR, 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 - )(Each, TemE and TemL) such as CD8 + 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., αβ TCR, CD3 + , CD4 + ); TH17 effector T cells (e.g., αβ TCR, CD3 + , CD4 + , IL-23R + , CCR6 + , IL-1R + ); CD4 + CD45RO + CCR7 + effector T cells, ICOS + effector T cells; CD4 + CD45RO + CCR7( - ) effector T cells; Effector T cells that secrete IL-2, IL-4 and / or IFN-γ are included.

[0160] Exemplary T cell antigens for the purpose include, for example (including the extracellular domain if applicable): CD8, CD3, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCRl / IL-SRA, CCR5, CCR6, IL-10Rα, CCR7, IL-l0Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, integrin α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common γ chain / IL-2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAILRI / TNFRSFlOA, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C,Examples include IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLPR. In various embodiments, the CD20 binder comprises a targeting moiety that binds to one or more of these exemplary T cell antigens.

[0161] By way of non-limiting example, in various embodiments, the chimeric protein of the invention has a targeting moiety directed against a checkpoint marker expressed on a T cell, 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 binder 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 a B cell. In some embodiments, the targeting moiety directly or indirectly recruits B cells to a treatment 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 binder comprises a targeting moiety that binds to one or more of these exemplary B cell antigens.

[0163] In some embodiments, the multispecific CD20 binder of the present application includes a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with natural killer cells. In some embodiments, the targeting moiety, for example in some embodiments, directly or indirectly recruits natural killer cells to a treatment 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 for purposes of illustration 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-epsilonRII, LMIR6 / CD300LE, Fc-gammaRl / CD64, MICA, Fc-gammaRIIB / CD32b, MICB, Fc-gammaRIIC / CD32c, MULT-1, Fc-gammaRIIA / CD32a, nectin-2 / CD112, Fc-gammaRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG2C, 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 binder includes a targeting moiety that binds to one or more of these exemplary NK cell antigens.

[0164] In some embodiments, the multispecific CD20 binder of the present application includes 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 to a treatment 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, lactadherin, 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-gamma RI / CD64, osteopontin, Fc-gamma RIIB / CD32b, PD-L2, Fc-gamma RIIC / CD32c, Siglec-3 / CD33, Fc-gamma RIIA / CD32a, SIGNR1 / CD209, Fc-gamma RIII / CD16, SLAM, GM-CSF R alpha, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-gamma Rl, TLR4, IFN-gammaR2, TREM-l, IL-l RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF 4, IL-10 R alpha, ALCAM, IL-10 R beta, aminopeptidase N / ANPEP, ILT2 / CD85j, common beta chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CD11b, CCR8, integrin αX / CD11c, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, 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-γRI / CD64, PSGL-1, Fc-γRIIICD16, RP105, G-CSF R, L-selectin, GM-CSF Rα, 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 binder comprises a targeting moiety that binds to one or more of these exemplary macrophage / monocyte antigens.

[0165] In some embodiments, the multispecific CD20 binder 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 dendritic cells. In some embodiments, the targeting moiety, for example in some embodiments, directly or indirectly recruits dendritic cells to a treatment site (e.g., a locus having diseased cells or 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 α4 / CD49d, Aag, Integrin β2 / 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, NCAM1, 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, Emprin / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, and Vanilloid R1. In various embodiments, the CD20 binder comprises a targeting moiety that binds to one or more of these exemplary DC antigens. In some embodiments, the CD20 binder comprises a targeting moiety that binds to Clec9A, and such CD20 binders find use, for example, in the treatment of multiple sclerosis.,

[0166] In some embodiments, the multispecific CD20 binder of the present application includes a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with immune cells selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or subsets thereof. In some embodiments, the antigen recognition domain, for example, in some embodiments, mobilizes megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or subsets thereof directly or indirectly to a treatment site (e.g., a locus having one or more diseased cells or cells whose therapeutic effect is to be modulated).

[0167] In some embodiments, the multispecific CD20 binder of the present application includes 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 binder includes a targeting moiety that binds to one or more of these exemplary megakaryocyte and / or platelet antigens.

[0168] In some embodiments, the multispecific CD20 binder of the present application includes a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with erythrocytes. 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 antigens. In various embodiments, the CD20 binder includes a targeting moiety that binds to one or more of these exemplary erythrocyte antigens.

[0169] In some embodiments, the multispecific CD20 binder 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 mast cells. Exemplary mast cell antigens of interest include, for example, SCFR / CD117, Fc ε RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMAl, FCERlA, FCER2, TPSABl. In various embodiments, the CD20 binder comprises a targeting moiety that binds to one or more of these mast cell antigens.

[0170] In some embodiments, the multispecific CD20 binder 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 basophils. Exemplary basophil antigens of interest include, for example, Fc ε RI, CD203c, CD123, CD13, CD107a, CD107b, and CD164. In various embodiments, the CD20 binder comprises a targeting moiety that binds to one or more of these basophil antigens.

[0171] In some embodiments, the multispecific CD20 binder 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 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 binder comprises a targeting moiety that binds to one or more of these neutrophil antigens.

[0172] In some embodiments, the multispecific CD20 binder 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 eosinophils. Exemplary eosinophil antigens of interest include, for example, CD35, CD44 and CD69. In various embodiments, the CD20 binder comprises a targeting moiety that binds to one or more of these eosinophil antigens.

[0173] In various embodiments, the multispecific CD20 binder of the present application comprises 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; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COLlAl, COL1A2, COL3A1, FAP, PH-4; epithelial cell surface markers such as CDlD, K6IRS2, KRTlO, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCl, TACSTDl; neovascular markers such as CD13, TFNA, alpha-v-beta-3 (αVβ3), E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the CD20 binder 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 having these antigens.

[0174] In various embodiments, the multispecific CD20 binder of the present application has one or more targeting moieties directed against one or more of the checkpoint markers, such as 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 a non-limiting example, in various embodiments, the chimeric protein of the present invention comprises (i) a targeting moiety directed against a checkpoint marker expressed on one or more of T cells, such as 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 cancer cells or tumor cells (e.g., CD20 on cancer or tumor cells) together 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 present invention is PD-1: EVQLVESGGGLVQAGKSLRLSCAASGSIFSIHAMGWFRQAPGKEREFVAAITWSGGITYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADRAESSWYDYWGQGTQVTVSS, or a targeting moiety directed to a sequence that is 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%, at least about 98%, at least about 99%, or 100% identical to the above sequence (e.g., a sequence that is 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%, about 99%, or about 100% identical to the above sequence).

[0177] As a further non-limiting example, in various embodiments, the chimeric protein of the invention is PD-L1: EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAKCWFRQAPGKEREWVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAARHGGPLTVEYFFDYWGQGTQVTVSS、 or has a targeting moiety directed against a sequence that is 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%, at least about 98%, at least about 99%, or 100% identical to the above sequence (e.g., a sequence that is 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%, about 99%, or about 100% identical to the above sequence).

[0178] Linker and functional group In various embodiments, the CD20 binder may include one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are attached or genetically fused to either a signaling agent or a targeting moiety described herein. In some embodiments, such functional groups, residues or moieties confer one or more desired properties or functionalities to the CD20 binder of the present application. Examples of such functional groups and examples of techniques for introducing them into CD20 binders 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 pharmaceutically acceptable polymer such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG). In some embodiments, attachment of the PEG moiety increases the half-life and / or decreases the immunogenicity of the CD20-binding protein. Generally, any suitable form of pegylation can be used for proteins such as antibodies and antibody fragments (including, but not limited to, single domain antibodies such as VHH), for example, as used in the art. See, e.g., 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 International Publication No. 04060965 (which are hereby incorporated by reference in their entirety). Various reagents for pegylation of proteins are also commercially available, for example, from Nektar Therapeutics (USA). In some embodiments, site-specific pegylation is used, particularly via cysteine residues (see, e.g., Yang et al., Protein Engineering, 16, 10, 761-770 (2003), which is hereby incorporated by reference in its entirety). For example, for this purpose, PEG may be attached to cysteine residues that are naturally present 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 can be fused to the amino terminus 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 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 these are known in the art and include, but are not limited to, fluorescent labels (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 (luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate esters, dioxetanes or GFP and its analogs, etc.), metals, metal chelates or metal cations, or other metals or metal cations particularly suitable for use in in vivo, in vitro or in situ diagnosis and imaging, as well as chromophores and enzymes (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) and the like. Other suitable labels include moieties that can be detected using NMR or ESR spectroscopy. Such labeled VHHs and polypeptides of the present application may be used, for example, for in vitro, in vivo or in situ assays (ELISA, RIA, EIA and other "sandwich assays" and other immunoassays known per se), as well as for in vivo diagnosis 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 binder. In some embodiments, the CD20 binder can comprise a single tag or multiple tags. The tag is, for example, a peptide, sugar, or DNA molecule that does not inhibit or prevent the binding of the CD20 binder to other target antigens such as, for example, CD20 or 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) tag. In one embodiment, the CD20 binder comprises a His tag. In one embodiment, the CD20 binder comprises an HA tag.

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

[0184] In some embodiments, a functional group, residue, or moiety includes a functional group that is part of a specific binding pair such as a biotin-(strept)avidin binding pair. Using such a functional group, the CD20 binding agent of the present application can be linked to another protein, polypeptide, or compound by binding to the other half of the binding pair, i.e., through the formation of the 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 in a diagnostic system where a detectable signal generating agent is conjugated to avidin or streptavidin. Such binding pairs may be used, for example, to bind a CD20 binding agent to a carrier such as a carrier 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 a therapeutic active agent to the CD20 binding agent of the present application.

[0185] In some embodiments, the CD20 binding agent of the present invention optionally includes one or more linkers. In some embodiments, the CD20 binding agent includes a linker that connects each binding region and / or targeting moiety. In some embodiments, the CD20 binding agent includes a linker that connects each signaling agent and targeting moiety (or, if there are two or more targeting moieties, connects the signaling agent to one of the targeting moieties). In some embodiments, linkers can be used to link the various functional groups, residues, or moieties described herein to the CD20 binding agent. 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 characteristics of the binding region and the binding protein. In various embodiments, the linker is selected from a peptide, protein, sugar, or nucleic acid.

[0186] In some embodiments, the CD20 binder of the present invention includes a linker that connects the targeting moiety and the signaling agent. In some embodiments, the chimeric protein of the present invention includes a linker within the signaling agent (e.g., in the case of single-chain TNF, two linkers can be included to form a trimer).

[0187] This application contemplates the use of various linker sequences. In various embodiments, the linker is derived from a naturally occurring multi-domain protein or is an empirical linker as described, for example, 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 computer programs such as those described in the Linker Design Database and 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 can be functional. For example, but not limited to, the linker can function to improve the folding and / or stability of the CD20 binder of the present invention, improve expression, improve pharmacokinetics, and / or improve biological activity.

[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 greater 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 another embodiment, the linker is rigid.

[0189] In some embodiments, the length of the linker allows for efficient binding of the targeting moiety and the signaling agent to their respective receptors. For example, in some embodiments, the length of the linker allows for efficient binding of one of the targeting moieties and the signaling agent to a receptor on the same cell, as well as efficient binding of another targeting moiety to another cell. Exemplary pairs of cells are provided elsewhere in this specification.

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

[0191] In some embodiments, the linker connects two targeting moieties to each other and the linker has a short length. Also, the linker connects the targeting moiety and the signaling agent, and this linker is longer than the linker that connects the two targeting moieties. For example, the difference in the amino acid length between the linker that connects the two targeting moieties and the linker that connects 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 residues 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, such as 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) nLinkers having [specific content] are exemplified, and X represents any amino acid, for example, Ala, Lys or Glu. In various embodiments, the linker is GGS.

[0194] In some embodiments, the linker is the hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE (including subclasses such as IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA2)). In various embodiments, the linker is the hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE (including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge regions found in antibodies of the IgG, IgA, IgD, and IgE classes act as flexible spacers that allow the Fab portions to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length between immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region vary between IgG subclasses. The hinge region of IgG1 contains 216-231 amino acids, and because this region has free flexibility, the Fab fragments can rotate about their axis of symmetry and move within the sphere centered on the first of the two heavy chain interchain disulfide bridges. IgG2 has a shorter hinge than IgG1 and has 12 amino acid residues and 4 disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid poly-proline double helix stabilized by additional heavy chain interchain disulfide bridges. These properties limit the flexibility of the IgG2 molecule. IgG3 differs from other subclasses by its unique extended hinge region (about 4 times the length of the IgG1 hinge). This extended hinge region contains 62 amino acids (including 21 prolines and 11 cysteines) and forms a flexible poly-proline double helix. In IgG3, the Fab fragments are relatively far from the Fc fragment, imparting great flexibility to the molecule. Also, the elongation of the IgG3 hinge results in a higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between IgG1 and IgG2. The degree of flexibility of the hinge region reportedly decreases in the order IgG3 > IgG1 > IgG4 > IgG2.

[0195] According to crystallographic studies, the immunoglobulin hinge region can be further functionally 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 contains the amino acids from the carboxyl terminus of C H1 to the first residue within the hinge that restricts movement, and the first cysteine residue generally forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of the antibody segment. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region binds to the amino terminus of the C H2 domain and contains residues up to C H2 (ibid.). The core hinge region of wild-type human IgG1 contains the Cys-Pro-Pro-Cys sequence and, when dimerized by disulfide bond formation, results in a cyclic octapeptide that acts as a pivot and is thus thought to confer flexibility. In various embodiments, the linker of the present invention comprises one or two or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., including IgG, IgA, IgD, and IgE (subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region can also include one or more glycosylation sites that include several structurally different types of sites for attaching carbohydrates. 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 considered an advantageous property of secreted immunoglobulins. In various embodiments, the linker of the present application includes one or more glycosylation sites. In various embodiments, the linker is the hinge-CH2-CH3 domain of a human IgG4 antibody.

[0196] If desired, the CD20 binder of the present invention is C H 2 domain and C HThe antibody Fc region comprising one or both of the 3 domains, and optionally the hinge region, can be linked. For example, a vector encoding the CD20 binder of the present invention linked to the Fc region as a single nucleotide sequence can be used to prepare such polypeptides.

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

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

[0199] Modification and production of CD20 binder In various embodiments, the CD20 binder 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) isolating the V H H domain of a naturally occurring heavy chain antibody; (2) expressing the nucleotide sequence encoding the naturally occurring V H H domain; (3) "humanizing" the naturally occurring V H H domain, or such humanized V Hexpressing a nucleic acid encoding an H domain; (4) "camelizing" a naturally occurring VH domain derived from any animal species, such as a mammalian species including humans, 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 VHH using nucleic acid synthesis techniques known in the art and then expressing the thus obtained nucleic acid; and / or (8) any combination of one or more of the above.

[0200] In one embodiment, the CD20 binder comprises a VHH corresponding to the VH domain of a naturally occurring heavy chain antibody directed against human CD20. H In some embodiments, such VH sequences can generally be generated or obtained by appropriately immunizing a camelid species with the CD20 molecule (i.e., eliciting an immune response and / or heavy chain antibody directed against CD20), obtaining a biological sample (such as a blood sample, or any sample of B cells) from a suitable camelid family, and starting from the sample and generating a VH sequence directed against CD20 using any suitable known technique. H In some embodiments, the naturally occurring VH domain directed against CD20 is the VH domain of a camelid animal. H In some embodiments, such VH sequences can generally be generated or obtained by appropriately immunizing a camelid species with the CD20 molecule (i.e., eliciting an immune response and / or heavy chain antibody directed against CD20), obtaining a biological sample (such as a blood sample, or any sample of B cells) from a suitable camelid family, and starting from the sample and generating a VH sequence directed against CD20 using any suitable known technique. H In some embodiments, the naturally occurring VH domain directed against CD20 is the VH domain of a camelid animal. HFrom a naive library of H arrays, for example, using CD20 or at least one portion, fragment, antigen determinant or epitope thereof, such a library can be obtained by screening with 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, naive V H An improved synthetic or semi-synthetic library derived from the H library, for example, a library described in WO 0043507, the entire contents of which are incorporated herein by reference, by techniques such as random mutagenesis and / or CDR shuffling from the naive V H Obtained from the H library, V H An H library, etc. may be used. In some embodiments, V directed against CD20 H Another technique for obtaining the H sequence is to appropriately immunize a transgenic mammal capable of expressing a heavy chain antibody (i.e., to elicit an immune response and / or a heavy chain antibody directed against CD20), obtain a suitable biological sample (e.g., a blood sample, or any sample of B cells) from the transgenic mammal, and then use any suitable known technique to generate a V directed against CD20 starting from the sample H This involves generating the H sequence. For example, for this purpose, a heavy chain antibody-expressing mouse, and additional methods and techniques described in WO 02085945 and WO 04049794, the entire contents of which are incorporated herein by reference, can be used.

[0201] In one embodiment, the CD20 binder is a "humanized" VHH, i.e., a naturally occurring V H"Humanized" VHHs are included by substituting one or more amino acid residues within the amino acid sequence of the H array (and in particular the framework array) with one or more of the amino acid residues present at the corresponding position(s) within the VH domain from a conventional human four-chain antibody. This can be done using humanization techniques known in the art. In some embodiments, the 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 can become more "human-like" while still retaining favorable properties such as reduced immunogenicity compared to the corresponding naturally occurring V H H domain. In various embodiments, the humanized VHHs of the present application can be obtained by any suitable method known in the art and thus are not strictly limited to polypeptides obtained using a polypeptide containing a naturally occurring V H H domain as a starting material.

[0202] In one embodiment, the CD20 binder 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 conventional four-chain antibody are replaced with the V of a heavy-chain antibody from a camelid HIt includes "camelized" VHHs by substituting one or more of the amino acid residues present at the corresponding position(s) within the H domain. In some embodiments, such "camelization" substitutions are formed at and / or inserted into the amino acid positions that form the VH-VL interface and / or the characteristic residues of so-called camelids (see, e.g., WO 94 / 04678, which is hereby incorporated by reference in its entirety). In some embodiments, the VH sequence used as a starting material or starting point for generating or designing camelized VHHs is a VH sequence derived from a mammal, such as a human VH sequence, such as a VH3 sequence. In various embodiments, camelized VHHs can be obtained in any suitable manner known in the art (i.e., as shown in points (1)-(8) above), and thus, strictly speaking, it is not limited to polypeptides obtained using a polypeptide containing a naturally occurring VH domain as a starting material.

[0203] In various embodiments, both "humanization" and "camelization" provide the nucleotide sequences encoding the naturally occurring V H H domain or VH domain, respectively, and then, in a manner known in the art, one or more codons in the nucleotide sequence can be changed such that the novel nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. This nucleic acid can then be expressed in a manner known in the art, and as a result, the desired VHH of the present application can be provided. Alternatively, based on the amino acid sequence of the naturally occurring V H H domain or VH domain, respectively, 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. Also, the naturally occurring V HBased on the amino acid sequence or nucleotide sequence of the H domain or VH domain, nucleotide sequences encoding the desired humanized or camelized VHHs are designed respectively, and then synthesized de novo using nucleic acid synthesis techniques known in the art. Thereafter, the nucleic acids thus obtained can be expressed in a manner known in the art, and as a result, the desired VHHs of the present application can be provided. Starting from a naturally occurring VH sequence or V H H sequence, other suitable methods and techniques for obtaining the VHHs of the present application and / or the nucleic acids encoding them are known in the art. For example, one or more portions of one or more naturally occurring VH sequences (e.g., one or more FR sequences and / or CDR sequences), one or more portions of one or more naturally occurring V H H sequences (e.g., one or more FR sequences or CDR sequences), and / or combinations of one or more synthetic or semi-synthetic sequences in a suitable manner may be included, thereby resulting in the VHHs of the present application or the nucleotide sequences or nucleic acids encoding them.

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

[0205] The nucleic acid encoding the CD20 binder of the present application can 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 binder of the present application can be introduced into a host cell by retroviral transduction. Exemplary host cells are Escherichia 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 cells can be grown under conditions that allow the host cell to express the gene encoding the CD20 binder of the present application. Thus, in various embodiments, the present application provides an expression vector comprising a nucleic acid encoding the CD20 binder of the present application. In various embodiments, the present application further provides a host cell comprising such an expression vector.

[0206] 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, e.g., Trp or Tac, and the gene engineered downstream of a prokaryotic signal sequence. In another example, when expressing an engineered gene in a eukaryotic host cell, e.g., a CHO cell, the gene is first inserted into an expression vector containing, e.g., a suitable eukaryotic promoter, a secretion signal, an enhancer and various introns. The gene construct can be introduced into a host cell using transfection, transformation or transduction techniques.

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

[0208] Accordingly, in various embodiments, the present application provides a nucleic acid encoding the CD20 binder of the present application. In various embodiments, the present application provides a host cell comprising a nucleic acid encoding the CD20 binder of the present application.

[0209] Pharmaceutically acceptable salts and excipients The CD20 binders described herein have sufficiently basic functional groups that can react with inorganic or organic acids, or carboxyl groups 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, those 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 hereby incorporated by reference in their entirety.

[0210] By way of non-limiting example, pharmaceutically acceptable salts include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, glucuronates, saccharinates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, camphorsulfonates, pamoates, phenylacetates, trifluoroacetates, acrylamides, chlorobenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, methylbenzoates, o-acetoxybenzoates, naphthalene-2-benzoates, isobutyrates, phenylbutyrates, α-hydroxybutyrates, butyne-1,4-dicarboxylates, hexyne-1,4-dicarboxylates, caprates, caprylates, cinnamates, glycolates, heptanoates, hippurates, malates, hydroxymaleates, malonates, mandelates, mesylates, nicotinates, phthalates, terephthalates, propiolates, propionates, phenylpropionates, sebacates, suberates, p-bromobenzenesulfonates, chlorobenzenesulfonates, ethylsulfonates, 2-hydroxyethylsulfonates, methylsulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, naphthalene-1,5-sulfonates, xylenesulfonates, and salts of tartaric acid.

[0211] The term "pharmaceutically acceptable salt" also refers to salts of the compositions of the present application having acidic functional groups (such as carboxylic acid functional groups) and bases. 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 and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxy-substituted mono-, di- or trialkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis- or tris-(2-OH-lower alkylamine), for example mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amine, for example, N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine and lysine, etc.

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

[0213] Pharmaceutical Compositions and Formulations In various embodiments, the present application relates to a pharmaceutical composition comprising a CD20 binder described herein and a pharmaceutically acceptable carrier or excipient. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally further comprise a suitable amount of a pharmaceutically acceptable excipient to provide a form for appropriate administration.

[0214] In various embodiments, the pharmaceutical excipient can be a liquid such as water and oil, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., of petroleum, animal, vegetable or synthetic origin. The pharmaceutical excipient can be, for example, physiological saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Further, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. In one embodiment, the pharmaceutically acceptable excipient is sterilized when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Also, suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Any of the agents 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 hereby incorporated by reference into this specification.

[0215] This application includes the described pharmaceutical compositions (and / or additional therapeutic agents) in various formulations. Any pharmaceutical composition of the invention described herein (and / or additional therapeutic agents) can take the form of a solution, suspension, emulsion, drops, tablets, pills, pellets, capsules, capsules containing a liquid, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen suspensions, dry powders, or any other suitable form 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] Optionally, the pharmaceutical compositions of the invention (and / or additional agents) can also include solubilizing agents. Also, the agents can be delivered using suitable vehicles or delivery devices known in the art. The combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.

[0217] Formulations containing the pharmaceutical compositions of the invention (and / or additional agents) of this application are preferably provided in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. Such methods generally include the step of associating the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Typically, the formulation is prepared by uniformly and intimately bringing the therapeutic agent into contact with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form of the formulation (e.g., wet or dry granules, powder blends, etc., followed by tableting using conventional methods known in the art).

[0218] In various embodiments, any pharmaceutical composition (and / or additional agent) described herein is formulated according to routine procedures as a composition adapted to the mode 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 oral. In another embodiment, administration is by parenteral injection. The mode of administration may be at the discretion of the practitioner and depends in part on the site of the medical condition. In most cases, administration results in the release of any of the agents described herein into the bloodstream.

[0220] In one embodiment, the CD20 binders described herein are formulated according to routine procedures as a composition suitable for oral administration. Compositions for oral delivery can be, for example, in the form of tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Compositions for oral administration can contain one or more agents, such as sweetening agents like fructose, aspartame or saccharin; flavoring agents like peppermint, wintergreen oil or cherry; coloring agents; and preservatives, and can provide pharmaceutically palatable preparations. Further, in the form of tablets or pills, the composition can be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a long period of time. Selectively permeable membranes surrounding any of the CD20 binders described herein that are driven by osmotic activity are also suitable for oral administration compositions. In these latter platforms, fluid from the environment surrounding the capsule is absorbed by the driving compound, causing them to swell and move the agent or agent composition through the opening. In these delivery platforms, an essentially zero-order delivery profile can be provided, in contrast to the spike profile of an immediate-release formulation. Time-delay materials such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can contain 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 for these mixtures, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like.

[0221] Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injections and infusions) include, for example, solutions, suspensions, dispersions, emulsions, etc. These can also be manufactured 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. These may contain, for example, suspending or dispersing agents known in the art. Suitable formulation components for parenteral administration include sterile diluents such as water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffering agents such as acetate, citrate or phosphate; 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, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof.

[0223] The compositions provided herein can be made into aerosol formulations for inhalation administration (i.e., "sprayed") alone or in combination with other suitable ingredients. The aerosol formulations can be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, etc.

[0224] Any pharmaceutical composition (and / or additional agent) of the present invention described herein can be administered by controlled release or sustained release means or by delivery devices well known to those skilled in the art. Examples include, but are not limited to, 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 hereby incorporated by reference in its entirety. Such dosage forms can be used, for example, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, other polymer matrices, gels, osmotic membranes, osmotic pressure systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide controlled release or sustained release of one or more active ingredients and can be useful for providing the desired release profile at various rates. Suitable controlled release formulations or sustained release formulations known to those skilled in the art, such as those described herein, can be readily selected for use with the active ingredients of the agents described herein. Accordingly, 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 release or sustained release.

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

[0226] In another embodiment, the controlled release system can be placed in the vicinity of the target area to be treated, and thus only a small part of the total systemic dose is required (see, for example, Goodson, Medical Applications of Controlled Release, supra, Volume 2, pages 115-138 (1984)). Other controlled release systems (discussed in the review by Langer, Science 249:1527-1533, 1990) 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 before or after lyophilization and reconstitution.

[0228] Administration and Dosage It will be understood that the actual dosage of the CD20 binder administered in accordance with this application will vary according to the specific dosage form and mode of administration. Many factors that can modify the action of the CD20 binder (e.g., body weight, gender, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combination, genetic profile, and responsiveness) can be considered by those skilled in the art. Administration can be carried out continuously or in one or more separate doses within the maximum tolerated dose. The optimal rate of administration for a given set of conditions can be determined by those skilled in the art using conventional dosage studies.

[0229] In some embodiments, suitable dosages of the CD20 binder are in the range of about 0.01 mg / kg to about 10 g / kg, about 0.01 mg / kg to about 1 g / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg per subject body weight, for example, about 0.01 mg / kg body weight, about 0.02 mg / kg body weight, about 0.03 mg / kg body weight, about 0.04 mg / kg body weight, about 0.05 mg / kg body weight, about 0.06 mg / kg body weight, about 0.07 mg / kg body weight, about 0.08 mg / kg body weight, about 0.09 mg / kg body weight, about 0.1 mg / kg body weight, about 0.2 mg / kg body weight, about 0.3 mg / kg body weight, about 0.4 mg / kg body weight, about 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, and include all values and ranges therebetween.

[0230] The individual dosages of the CD20 binder can be administered, for example, in unit dosage forms containing from about 0.01 mg to about 100 g, from about 0.01 mg to about 75 g, from about 0.01 mg to about 50 g, from about 0.01 mg to about 25 g, from about 0.01 mg to about 10 g, from about 0.01 mg to about 7.5 g, from about 0.01 mg to about 5 g, from about 0.01 mg to about 2.5 g, from about 0.01 mg to about 1 g, from about 0.01 mg to about 100 mg, from about 0.1 mg to about 100 mg, from about 0.1 mg to about 90 mg, from about 0.1 mg to about 80 mg, from about 0.1 mg to about 70 mg, from about 0.1 mg to about 60 mg, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 30 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 1 mg, or from about 5 mg to about 80 mg of the active ingredient per unit dosage form. For example, the unit dosage forms can be 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, 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, and can include all values and ranges therebetween.

[0231] In one embodiment, the CD20 binder is administered in an amount of from about 0.01 mg / day to about 100 g / day, from about 0.01 mg / day to about 75 g / day, from about 0.01 mg / day to about 50 g / day, from about 0.01 mg / day to about 25 g / day, from about 0.01 mg / day to about 10 g / day, from about 0.01 mg / day to about 7.5 g / day, from about 0.01 mg / day to about 5 g / day, from about 0.01 mg / day to about 2.5 g / day, from about 0.01 mg / day to about 1 g / day, from about 0.01 mg / day to about 100 mg / day, from about 0.1 mg / day to about 100 mg / day, from about 0.1 mg / day to about 95 mg / day, from about 0.1 mg / day to about 90 mg / day, from about 0.1 mg / day to about 85 mg / day, from about 0.1 mg / day to about 80 mg / day, from about 0.1 mg / day to about 75 mg / day, from about 0.1 mg / day to about 70 mg / day, from about 0.1 mg / day to about 65 mg / day, from about 0.1 mg / day to about 60 mg / day, from about 0.1 mg / day to about 55 mg / day, from about 0.1 mg / day to about 50 mg / day, from about 0.1 mg / day to about 45 mg / day, from about 0.1 mg / day to about 40 mg / day, from about 0.1 mg / day to about 35 mg / day, from about 0.1 mg / day to about 30 mg / day, from about 0.1 mg / day to about 25 mg / day, from about 0.1 mg / day to about 20 mg, from about 0.1 mg / day to about 15 mg / day, from about 0.1 mg / day to about 10 mg, from about 0.1 mg / day to about 5 mg / day, from about 0.1 mg / day to about 3 mg / day, from about 0.1 mg / day to about 1 mg / day, or from about 5 mg / day to about 80 mg / day. In various embodiments, the CD20 binder is administered in an amount 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, 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, including those amounts.

[0232] According to certain embodiments of the present application, the pharmaceutical composition comprising a CD20 binder can be administered, for example, one or more times per day (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times per day), about once a day, about once every other day, about once every three days, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year.

[0233] Combination therapies and additional therapeutic agents In various embodiments, the pharmaceutical compositions of the present application are co-administered in combination with additional therapeutic agent(s). The co-administration can be simultaneous or sequential.

[0234] In one embodiment, the additional therapeutic agent and the CD20 binder 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 binder are administered at an interval of about 60 minutes or less, e.g., 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 binder may also be by simultaneously administering a single formulation (e.g., a formulation comprising the additional therapeutic agent and the CD20 binder) or separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the CD20 binder).

[0235] In co - administration, it is not necessary to administer the therapeutic agents simultaneously if the timing of those administrations is such that the pharmacological activities of the additional therapeutic agent and the CD20 binder overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the CD20 binder can be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the CD20 binder are administered more than about 60 minutes apart. For example, the time between sequential administrations of the additional therapeutic agent and the CD20 binder 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 binder being administered. Either the additional therapeutic agent or the CD20 binder cells can be administered first.

[0236] Also, in co - administration, it is not necessary to administer the therapeutic agents to the 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 binders described herein act synergistically when co - administered with another therapeutic agent. In such embodiments, the CD20 binder and the additional therapeutic agent may be administered at lower dosages than those used when the agent is used in a monotherapy setting.

[0238] In some embodiments, the present application relates to chemotherapeutic agents as additional therapeutic agents. For example, but not limited to, such combinations of the CD20 binding agents of the present invention and chemotherapeutic agents are found to be useful 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 cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (synthetic analogs, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamycin gamma II, and calicheamycin omega II (e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicin A;Bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein engyin antibiotic chromophores), aclacinomycin, actinomycin, automycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophilin, 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, mitomycin, for example mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitioestanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; dexamethasone; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;Mitansoids such as mitansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oregon); rezoxane; lysoxin; sizofuran; spirigermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitabine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., taxol paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), abraxane Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and taxotere docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs 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 inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV);Oxaliplatin; lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Further, the treatment method can further include the use of photodynamic therapy.;

[0239] In some embodiments, the present application relates to combination therapies using CD20 binders and chemotherapeutic agents. In some embodiments, the present application relates to the administration of a CD20 binder 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 exemplary embodiments, the CD20 binder acts synergistically when co-administered with doxorubicin. In exemplary embodiments, the CD20 binder acts synergistically when co-administered with doxorubicin for use in treating a tumor or cancer. For example, co-administration of a CD20 binder and doxorubicin can act synergistically to reduce or eliminate a tumor or cancer, or slow the growth, and / or progression, and / or metastasis of a tumor or cancer. In exemplary embodiments, the combination of a CD20 binder and doxorubicin can exhibit an improved safety profile compared to the agents used alone in a monotherapy situation. In exemplary embodiments, the CD20 binder and doxorubicin can be administered at lower doses than the doses used when the agents are used in a monotherapy situation. In some embodiments, the CD20 binder includes a mutant interferon such as mutant IFNα. In exemplary embodiments, the mutant IFNα includes one or more mutations at positions 148, 149 and 153 such as substitution M148A, R149A, and L153A with respect 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, such as, 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, but not limited to, nivolumab (ONO-4538 / BMS-936558, MDX1106, Opdivo, Bristol Myers Squibb), pembrolizumab (Keytruda, Merck), pidilizumab (CT-011, CureTech), MK-3475 (Merck), BMS 936559 (Bristol Myers Squibb), MPDL328OA (Roche), and other antibodies. 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, 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 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, but not limited to, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and / or tremelimumab (Pfizer) and other antibodies.In some embodiments, the chimeric protein of the present invention is combined with ipilimumab (optionally bavituximab) for the treatment of one or more of melanoma, prostate cancer, and lung cancer. In various embodiments, the immunomodulatory agent targets CD20. In various embodiments, the immunomodulatory agent is an antibody specific for CD20. For example, in some embodiments, the immunomodulatory agent is, but not limited to, antibodies such as ofatumumab ((GENMAB), obinutuzumab (GAZYVA), AME-133v (APPLIED MOLECULAR EVOLUTION), ocrelizumab (GENENTECH), TRU-015 (TRUBION / EMERGENT), belzutuzumab (IMMU-106). In one embodiment, the immunomodulatory agent is an antibody that targets OX40.

[0242] In some embodiments, the present application relates to a combination therapy using a CD20 binder and a checkpoint inhibitor. In some embodiments, the present application relates to the administration of a CD20 binder to a patient receiving 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 binder acts synergistically when co-administered with an anti-PD-L1 camelid VHH. In exemplary embodiments, the CD20 binder 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 the CD20 binder and the anti-PD-L1 camelid VHH can 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 the CD20 binder and the anti-PD-L1 camelid VHH can exhibit an improved safety profile compared to the agents used alone in a monotherapy setting. In some embodiments, the CD20 binder and the anti-PD-L1 camelid VHH can be administered at a lower dose than the dose used when the agent is used in a monotherapy setting. In some embodiments, the CD20 binder comprises a mutant interferon such as 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, with respect to SEQ ID NO: 127 or SEQ ID NO: 128.

[0244] In some embodiments, the present application relates to the administration of a CD20 binder 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 International Publication No. WO 2013 / 10779, International Publication No. WO 2015 / 007536, International Publication No. WO 2015 / 007520, International Publication No. WO 2015 / 007542, and International Publication No. WO 2015 / 007903, which are hereby incorporated by reference in their entirety.

[0246] Without limitation, in some embodiments including use in infectious diseases, the present application relates to anti-infective agents as additional therapeutic agents. In some embodiments, the anti-infective agents include, without limitation, antiviral agents such as abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, foscarnet. In some embodiments, the anti-infective agents include, without limitation, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, levaquin, floxin, tequin, avalox, and norfloxacin); 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, the anti-infective agents include antimalarial drugs (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether / lumefantrine, atovaquone / proguanil, and sulfadoxine / pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.

[0247] Without being limited thereto, in some embodiments, such as 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 non-steroidal anti-inflammatory agent (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, hydroxytriamcinolone, α-methyl dexamethasone, β-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, flucloronide acetonide, flumethasone pivalate, fluocinonide acetonide, fluocinonide, flucortine butylester, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinonide, β-methasone and the rest of its esters, chloroprednisone, clocortelone, cclescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluorometholone, flupredolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate.(NSAIDs) that may be used in the present disclosure include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin. In some embodiments, the immunosuppressive agent may be an alkylating agent, an antimetabolite (e.g., azathioprine, methotrexate), a cytotoxic antibiotic, an antibody (e.g., basiliximab, daclizumab, and muromonab), an anti-immunophilin (e.g., cyclosporine, tacrolimus, sirolimus), interferon, an opioid, a tumor necrosis factor (TNF), a TNF binding protein, mycophenolate, a small biologic agent (e.g., fingolimod, myriocin), and other cytostatic agents. 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 a combination therapy using a CD20 binder and an immunosuppressive agent. In some embodiments, the present application relates to the administration of a CD20 binder to a patient receiving treatment with an immunosuppressive agent. In one embodiment, the immunosuppressive agent is TNF.

[0249] In exemplary embodiments, the CD20 binder acts synergistically when co-administered with TNF. In exemplary embodiments, the CD20 binder acts synergistically when co-administered with TNF for use in treating a tumor or cancer. For example, co-administration of a CD20 binder and TNF can 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 binder and TNF can exhibit an improved safety profile compared to agents used alone in a monotherapy setting. In some embodiments, the CD20 binder and TNF can be administered at lower doses than the doses used when the agent is used in a monotherapy setting. In some embodiments, the CD20 binder includes a mutant interferon such as mutant IFNα. In an exemplary embodiment, the mutant IFNα includes one or more mutations at positions 148, 149 and 153, such as substitutions M148A, R149A, and L153A, with respect to SEQ ID NO: 127 or SEQ ID NO: 128.

[0250] In some embodiments, the CD20 binders described herein include derivatives modified by covalent attachment in such a way that the activity of the composition is not hindered, i.e., by covalent attachment of any type of molecule to the composition. For example, by way of non-limiting example, derivatives include, inter alia, compositions modified by glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, ligation to a cell ligand, or other proteins. Without being limited thereto, any of a number of chemical modifications can be made by known techniques, such as specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicaamycin, etc.

[0251] In yet other embodiments, the CD20 binders described herein further include a cytotoxic agent, and in exemplary embodiments, include a toxin, a chemotherapeutic agent, and an agent that causes apoptosis or cell death. Such agents can be conjugated to the compositions described herein.

[0252] Thus, the CD20 binding agents described herein may be modified after translation to add effector moieties such as chemical linkers, detectable moieties such as fluorescent dyes, enzymes, substrates, bioluminescent substances, and chemiluminescent moieties, or functional moieties such as streptavidin, avidin, biotin, cytotoxins and cytopathic agents.

[0253] Exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines such as daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, calminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics such as dactinomycin (actinomycin D), bleomycin, calicheamicin, mitramycin, anthramycin (AMC); and mitotic inhibitors such as 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, mitotane (O,P’-(DDD)), interferon, 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, platinum, 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, Arbitux, Avastin, Pertuzumab, anti-CD20 antibodies, Rituxan, Ofatumumab, Ofatumumab, DXL625, HERCEPTIN® or any combination thereof. Toxic enzymes derived from plants and bacteria, such as ricin, diphtheria toxin and Pseudomonas toxin, may be conjugated to a therapeutic agent (e.g., an antibody) to produce a cell type-specific killing reagent (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 in U.S. Patent No. 6,653,104.

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

[0256] Treatment method The methods and compositions described herein have application in 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] Furthermore, any of the agents of the present invention may be an agent for use in the treatment of various diseases and disorders (including, but not limited to, cancer, infectious diseases, immune disorders, inflammatory diseases or conditions, autoimmune diseases, and neurological disorders) involving CD20-positive cells, or for use in the manufacture of a medicament for treatment.

[0258] In some embodiments, the CD20 binding agent of the present application is used for the treatment of diseases in which depletion of CD20+ cells is therapeutically beneficial, such as Waldenström macroglobulinemia, multiple myeloma, plasma cell dyscrasia, chronic lymphocytic leukemia, transplantation therapy, hairy cell leukemia, ITP, Epstein-Barr virus lymphoma after stem cell transplantation, and kidney transplantation. In other embodiments, the CD20 binding agent of the present application is used for the treatment of diseases selected from B cell lymphoma, leukemia, myeloma, autoimmune diseases, transplantation, graft-versus-host disease, infectious diseases associated with B cells, lymphoproliferative diseases, and for 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 agent of the present application is used for the treatment of diseases selected from the group consisting of B cell lymphoma, leukemia, myeloma, transplantation, graft-versus-host disease, autoimmune diseases, lymphoproliferative states, 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 molecule of the present application is used for the treatment of B-ALL, hairy cell leukemia, multiple myeloma, Richter's syndrome, acquired factor VIII inhibitor, antiphospholipid syndrome, autoimmune hemolytic anemia, autoimmune thrombocytopenia, bullous pemphigoid, cold agglutinin disease, Evans syndrome, Goodpasture syndrome, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgM-related polyneuropathy, Kaposi's sarcoma-associated herpesvirus (KSHV)-associated multicentric Castleman disease (MCD), myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, erythroid leukemia, rheumatoid arthritis, Sjögren's syndrome, systemic immune complex vasculitis, systemic lupus erythematosus, type II mixed cryoglobulinemia, 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 a patient having cancer. As used herein, cancer refers to any uncontrolled growth of cells that can interfere with the normal functioning 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 in vital organs, it can ultimately lead to the death of the subject due to the functional deterioration of the affected organs. Metastasis is a group of cancer cells or a mass 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. Metastasis can ultimately lead to the death of the subject. For example, cancers include benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

[0260] Exemplary cancers that can be treated include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung); melanoma; multiple myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancers; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancers; vulvar cancer; lymphomas such as Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphoma (such as low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocyte (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; large lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD) and abnormal vascular proliferation associated with phakomatosis, edema (e.g., associated with brain tumors), and Meigs syndrome.

[0261] In some embodiments, the cancer is leukemia or lymphoma. Exemplary leukemias or lymphomas include, but are not limited to, non-Hodgkin lymphoma (NHL) such as B cell lymphoma, low and intermediate grade non-Hodgkin lymphoma (NHL), recurrent Hodgkin disease, high grade resistant Hodgkin disease, lymphocyte predominant subtype of Hodgkin 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), follicular lymphoma (FL) such as low, intermediate and high grade FL, cutaneous follicle center lymphoma, marginal zone B cell lymphoma, MALT type marginal zone B cell lymphoma, nodular marginal zone B cell lymphoma, splenic marginal zone B cell lymphoma, hairy cell leukemia, diffuse large cell type B cell lymphoma, Burkitt lymphoma, plasmacytoma, multiple myeloma, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, multiple myeloma, and undifferentiated large cell type lymphoma (ALCL).

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

[0263] In some embodiments, exemplary immune disorders include, but are not limited to, rheumatoid arthritis (RA), juvenile idiopathic 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, type 1 diabetes, Raynaud's syndrome, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis C - related cryoglobulinemic vasculitis, chronic encephalitis, hemophilia A, membranoproliferative glomerulonephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, neuromyelitis optica, Graves' disease, bullous skin diseases, bullous pemphigoid, pemphigus, Churg - Strauss syndrome, asthma, psoriatic arthritis, dermatitis, respiratory distress syndrome, meningitis, encephalitis, anti - NMDA receptor encephalitis, uveitis, eczema, atherosclerosis, leukocyte adhesion deficiency, type 1 diabetes, Reiter's disease, Behçet's disease, hemolytic anemia, atopic dermatitis, Wegener's granulomatosis, Ogilvie 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), erythroleukemia, Evans syndrome, factor VIII deficiency, hemophilia A, autoimmune neutropenia, Castleman's 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 (temporal) arteritis, medium - vessel vasculitis, Kawasaki disease, polyarteritis nodosa, Devic's disease, autoimmune pancreatitis, opsoclonus - myoclonus syndrome (OMS), IgG4 - related diseases, scleroderma, and chronic fatigue syndrome.

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

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

[0266] In some embodiments, the CD20 binding agent of the present application can be used to treat immune-mediated cytopenias, such as autoimmune neutropenia and erythroleukemia.

[0267] In some embodiments, the CD20 binding agent of the present application can 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, antiphospholipid antibody syndrome, secondary hemophilia, relapsing polychondritis, sarcoidosis, stiff man syndrome, rheumatic fever, etc. A further example is eosinophilic fasciitis.

[0268] In some embodiments, the CD20 binding agent of the present application can be utilized for treating arthritis such as ankylosing spondylitis, juvenile rheumatoid 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 agent of the present application can be utilized for treating 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 Waldenstrom hyperglobulinemic purpura. Further examples are granulocytopenia, autoimmune neutropenia, Franklin's disease, Seeligman's disease, μ-chain disease, paraneoplastic syndromes secondary to thymoma and lymphoma, and formation of factor VIII inhibitor.

[0270] In some embodiments, the CD20 binding agent of the present application can be utilized for treating endocrine disorders, for example, polyendocrine disorders 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 agent of the present application may be utilized for treating liver-gastrointestinal disorders such as celiac disease, Whipple's disease, primary biliary cirrhosis, chronic active hepatitis, and primary sclerosing cholangitis. Further example is autoimmune gastritis.

[0272] In some embodiments, the CD20 binding agent of the present application can be used for treating nephropathies such as rapidly progressive glomerulonephritis, poststreptococcal glomerulonephritis, Goodpasture syndrome, membranous glomerulonephritis and cryoglobulinemic nephritis. Further example is minimal change disease.

[0273] In some embodiments, the present application relates to the treatment of neuropathy or the treatment of patients with neuropathy. Exemplary neuropathies 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, juvenile Parkinson's disease)), Huntington's disease, amyotrophic lateral sclerosis (ALS; including, but not limited to, sporadic ALS, familial ALS, West Pacific ALS, juvenile ALS, Hirayama disease), autoimmune neuropathy, multiple mononeuritis, Lambert-Eaton myasthenic syndrome, Sydenham chorea, tabes dorsalis, Guillain-Barré syndrome, and further examples include myelopathy / tropical spastic paraparesis, myasthenia gravis, acute inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy.

[0274] In some embodiments, the CD20 binder of the present application can be utilized to treat heart and lung disorders such as fibrotic alveolitis, bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis, Reiter's syndrome, myocarditis and pericarditis. Further examples are hypersensitivity pneumonitis, and paraneoplastic syndromes secondary to lung cancer.

[0275] In some embodiments, the CD20 binder of the present application can be utilized to treat allergic diseases such as bronchial asthma and the hyper-IgE syndrome. A further example is transient cataract.

[0276] In some embodiments, the CD20 binder of the present application can be utilized to treat eye diseases such as idiopathic choroiditis.

[0277] In some embodiments, the CD20 binding agent of the present application can be utilized to treat transplantation-derived disorders such as allograft and xenograft rejection and graft-versus-host disease. In some embodiments, the CD20 binding agent of the present application can be utilized after transplantation.

[0278] In some embodiments, the present application relates to the treatment of microbial infections and / or chronic infections (e.g., infections of B cells) or patients thereof. Exemplary infectious diseases 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 a kit (e.g., with or without additional therapeutic agents) for the administration of any CD20 binding agent described herein. A kit is an assembly of materials or components that includes at least one of the pharmaceutical compositions of the 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 that make up the kit will vary depending on its intended purpose. In one embodiment, the kit is configured for the purpose of treating a human subject.

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

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

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

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

[0285] "Effective amount" as used in connection with medical use is an amount effective to bring about a measurable treatment, prevention, or reduction in the incidence of the disease in question.

[0286] As used herein, when the readings of activity and / or effect are "decreased" in the presence of an agent or stimulus as compared to the case without such modulation, 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 100% or less. As will be understood by those skilled in the art, in some embodiments, the activity decreases and some downstream readings decrease, while others may increase.

[0287] Conversely, if the readings of activity and / or effect are significantly greater in the presence of the agent or stimulant, for example, 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, greater than in the absence of the agent or stimulant, the activity is "increased".

[0288] As used herein, all composition percentages are by weight of the total composition, unless otherwise specified. As used herein, the terms "include" and its variations are non-limiting, and thus the listing of items in a list is not intended to exclude other similar items that may be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variations are non-limiting, and thus a reference to an embodiment being able to include or may include a particular element or feature is not intended to exclude other embodiments of this technology that do not include those elements or features.

[0289] The open-ended term "comprising", which is synonymous with terms such as "including", "containing", or "having", is used herein to describe and claim the present invention, but the present invention or its embodiments 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 a technique that provide certain benefits under certain circumstances. However, in the same or other circumstances, other embodiments may be preferred. Furthermore, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technique.

[0291] The amount of the composition described herein required to achieve a therapeutic effect can be determined empirically according to conventional procedures for a particular purpose. Generally, for the administration of 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" refer to an amount sufficient to produce the desired physiological effect, or an amount capable of achieving the desired result, particularly for treating a disorder or disease. The effective amount as used herein includes, for example, an amount sufficient to delay the onset of symptoms of a disorder or disease, alter the course of symptoms of a disorder or disease (e.g., slow the progression of symptoms of a disease), reduce or eliminate one or more symptoms or signs of a disorder or disease, or reverse the symptoms of a disorder or disease. Therapeutic benefits also include arresting or delaying the progression of the underlying disease or disorder, whether or not an improvement is realized.

[0292] An effective amount, 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 approximately 50% of the population) and the ED50 (the dose therapeutically effective in approximately 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dosage ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. In some embodiments, compositions and methods presenting a large therapeutic index are preferred. A therapeutically effective dosage can be initially estimated from in vitro assays such as cell culture assays. Also, when determined in cell culture or in a suitable animal model, the dosage can be formulated in the animal model to achieve a circulating plasma concentration range, such as the IC50. The plasma levels of the described compositions can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by suitable bioassays. The dosage is determined by the physician and can be adjusted as needed 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 delaying the progression of the underlying disease or disorder, whether or not an improvement is realized.

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

[0295] Examples 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 the reverse primer 5’-GGTTTTTTCTCTAGATCAAGGAGAGCTGTCATTTTCTATTGG (O-7956). The amplification product was digested with BglII and XbaI and ligated into the mammalian expression vector pMet7. This plasmid was used for transient transfection of Hek293T cells and for the generation of CHO-K1 clones that stably express 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. The enrichment of antigen-specific phages was evaluated by comparing the number of phagemid particles eluted from the transfected cells to the number of phagemid particles eluted from the parental cells after each round of panning. These experiments suggested that the phage population was enriched approximately 2-fold, 8-fold, and 4-fold after the first, second, and third rounds of panning, respectively (for antigen-specific phages). A total of 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. Sixty-two out of the 95 colonies were positive in this assay. Based on the sequence data, the 62 positive colonies represented 14 different VHHs (Figure 1, upper panel).

[0297] A VHH library was constructed and panned (in solution) three times in a row 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 panning. Approximately 142 colonies randomly picked from the third panning were tested by cell ELISA as described above, and 8 colonies showed positive results. Based on the sequence data, the 8 positive colonies represented three different VHHs (Figure 1, lower panel). The three different VHHs belong to the same group. The group identified here is the same as one of the previously identified groups.

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

Table 1

[0299] In summary, as shown in the following table, 17 different VHHs belonging to four different groups were identified. The nucleotide and amino acid sequences of the 17 anti-human CD20 VHHs are shown in Figure 2 and Figure 3, respectively.

Table 2

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

[0301] In the pMECS vector, an amber stop codon (TAG) followed the His6 tag, and after this amber stop codon, gene III of the M13 phage followed. In suppressor E. coli strains (e.g., TG1), the amber stop codon was read as glutamine, and thus the VHH was expressed as a fusion protein with phage protein III, which enabled 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 thus the resulting VHH did not fuse to protein III.

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

[0303] Subcloning of the VHH gene from pMECS into the pHEN6c vector In the pMECS vector, the His6 tag was cleaved during storage of the VHH (even for a short period or at -20°C). Thus, the VHH gene was subcloned from pMECS into the pHEN6c vector, such as when the His6 tag was used for detection.

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

Chemical formula

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

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

[0307] For ligation, the pHEN6c vector was digested with PstI for 3 hours, purified as described above, and then digested with BstEII for 2 - 3 hours. Alternatively, digestion was performed using 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 the Qiaquick Gel Extraction Kit (Qiagen)). Subsequently, the PCR fragment was ligated to the vector.

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

[0309] Following the above protocol, a VHH gene cloned into the pHEN6c vector was generated. This contained a PelB signal sequence at the N-terminus and a His6-tail at the C-terminus. The PelB leader sequence directed the VHH into the periplasmic space of E. coli, and the His-tag was used for purification and detection of the VHH (e.g., in ELISA, Western blot, etc.).

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

[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, and then grown at 37 °C with shaking (200 - 250 rpm) until the OD 600 reached 0.6 - 0.9. IPTG (final concentration 1 mM) was added to induce VHH expression. The culture was incubated at 28 °C with shaking overnight (about 16 - 18 hours). The OD 600 after overnight induction was usually 25 - 30. At least 1 liter of culture (3 bottles) per clone was prepared at an average yield of 1 - 15 mg / l.

[0312] Extraction of VHH from the periplasm of E. coli was performed on the third day. The solutions 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] The overnight-induced culture was centrifuged at 8000 rpm for 8 minutes. The cell pellet from 1 liter of culture was pipetted up and down and resuspended in 12 ml of TES, and shaken on ice for 1 hour. For each 12 ml of TES used, approximately 18 ml of TES / 4 was added, incubated on ice for an additional 1 hour with shaking, and then centrifuged at 4 °C, 8000 rpm for 30 minutes. The supernatant containing the protein extracted from the periplasmic space was transferred to a fresh Falcon tube.

[0314] Subsequently, VHH was purified by IMAC using the following solutions: HIS-select (SIGMA), PBS, and 50 mM NaAcetate pH 4.6.

[0315] His-select was equilibrated with PBS. Specifically, for each periplasmic extract derived from 1 liter of culture solution, 1 ml of resin (about 2 ml of His-select solution) was added to a 50 ml Falcon tube. PBS was added to a final volume of 50 ml and mixed. Centrifugation was performed at 2000 rpm for 2 minutes, 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 minutes to 1 hour with gentle shaking. The sample was loaded onto a PD-10 column (GE healthcare, cat. No. 17-0435-01) having a filter at the bottom and washed with 50 - 100 ml of PBS (50 - 100 ml of PBS was used per 1 ml of resin). Elution was performed three times, each time with 1 ml of PBS / 0.5 M imidazole per 1 ml of resin used (to elute efficiently, the beads were resuspended and left at 4 °C overnight with the bottom of the column closed). Dialysis was performed against PBS at 4 °C overnight (cut-off 3500 daltons) to remove imidazole. For efficient dialysis, the dialysis buffer (PBS) was exchanged 2 - 3 times. Alternatively, instead of elution with imidazole, the bound VHH 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 VHH, the eluted VHH was immediately neutralized with 1 M Tris pH 8.0 and dialysis was not required.

[0316] The amount of protein was estimated by OD measurement of the eluted sample. The extinction coefficient of each clone was determined by the protParam tool under the primary structure analysis of the Expasy proteomics server. Further purification of VHH could be achieved by different methods. For example, the sample could be concentrated by centrifugation at 4 °C, 2000 rpm until an appropriate volume (up to 4 ml) for loading onto Superdex 75 16 / 60 was obtained (Vivaspin 5000 MW cut-off, Vivascience). The concentrated sample was loaded onto a Superdex 75 16 / 60 column equilibrated with PBS. The peak fractions were pooled and OD 280 measured for quantification. 280The measurement was carried out. Generally, the VHH eluted after 85 - 95 minutes when run at 1 ml / min. An aliquot of the concentrated VHH sample was 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 the forward primer 5’-gataagatctcaGGCGGATCCAGTGGACCTTTCCCAGCAGAGC (O - 7962) and the reverse primer 5’-GGTTTTTTCTCTAGATCAAGGAGCGATCTCATTTTCCACTG (O - 7964). The amplification product was digested with BglII and XbaI and ligated into the mammalian expression vector pMet7. This plasmid was used for transient transfection of Hek293T cells and for the generation of CHO - K1 clones that stably express mouse CD20.

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

[0319] Similarly, a VHH library with the size of approximately 10 8 independent transformants was obtained from peripheral blood lymphocytes (PBL). Approximately 65% of the transformants retained vectors with the correct insert size.

[0320] The library was panned three times in succession (in solution) on stably transfected CHO-K1 cells expressing mouse CD20. To serve as a negative control for calculating the enrichment of CD20-specific phage, parallel panning was performed on parental (non-transfected CHO-K1) cells. The enrichment of antigen-specific phage was evaluated by comparing the number of phagemid particles eluted from the transfected cells with the number of phagemid particles eluted from the parental cells after each panning. These experiments suggested that the phage population was enriched approximately 2-fold, 2-fold, and 10 3 -fold after the first, second, and third rounds of panning, respectively (for antigen-specific phage). A total of 285 colonies (95 from each panning) were randomly selected and their crude periplasmic extracts (such as soluble VHH) were analyzed by cell ELISA for specific binding to transfected CHO-K1, comparing them 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, upper panel). The 11 different VHHs belonged to two different groups.

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

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

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

Table 3

[0324] In summary, as shown in the following table, 21 different VHHs belonging to four different groups were identified. 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) using recombinant pMECS The VHH gene cloned into the pMECS vector contained a PelB signal sequence at the N-terminus, an HA tag and a His6 tag at the C-terminus (PelB leader-VHH-HA-His6). The PheB leader sequence directed the VHH into the periplasmic space of E. coli, and the HA and His6 tags were used for the purification and detection of VHH (e.g., in ELISA, Western blot, etc.).

[0326] In the pMECS vector, an amber stop codon (TAG) followed the His6 tag, and the gene III of M13 phage followed this amber stop codon. In suppressor E. coli strains (e.g., TG1), the amber stop codon was read as glutamine, and thus the VHH was expressed as a fusion protein with phage protein III, which enabled the display of 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 thus the resulting VHH did not fuse to protein III.

[0327] To express and purify the VHH cloned into the pMECS vector, pMECS containing the gene of the desired VHH was prepared and transformed into non-suppressor strains (e.g., WK6). The VHH of the resulting clones was sequenced using the 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. Non-suppressor strains (e.g., WK6) containing the recombinant pMECS vector with the VHH gene were used to express and purify the VHH described herein.

[0328] Sub-cloning of the VHH gene from pMECS into the pHEN6c vector In the pMECS vector, the His6 tag was cleaved during the storage of VHH (even for a short period and even at -20°C). Therefore, when using the His6 tag for detection etc., it was better to subclone the VHH gene from pMECS into the pHEN6c vector.

[0329] Specifically, using Escherichia coli containing recombinant pMECS carrying primers A6E and PMCF with the VHH gene as a template, the VHH gene was amplified. Primers A6E and PMCF are primers for framework 1 and framework 4 respectively. The primer sequences were as follows:

Chemical formula

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

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

[0332] For ligation, the PCR product was purified. The pHEN6c vector was digested with PstI for 3 hours, purified as described above, and then digested with BstEII for 2 - 3 hours. Alternatively, digestion was performed using 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 the Qiaquick Gel Extraction Kit (Qiagen)). Subsequently, the PCR fragment was ligated to the vector.

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

[0334] Following the above protocol, the VHH gene 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 directed the VHH into the periplasmic space of E. coli, and the His tag was used for purification and detection of the VHH (e.g., in ELISA, Western blot, etc.).

[0335] Expression and purification of the VHH were performed. Specifically, on the first day, freshly transformed WK6 colonies were inoculated into 10 - 20 ml of LB + ampicillin (100 μg / ml) + glucose (1%). This preculture was incubated at 37°C overnight with shaking at 200 - 250 rpm. On the second day, TB medium was used to express the VHH. The TB medium contained 2.3 g of KH2PO4, 16.4 g of K2HPO4·3H2O, 12 g of tryptone (Duchefa Biochemie), 24 g of yeast (Duchefa Biochemie), and 4 ml of 100% glycerol (Duchefa Biochemie) per liter.

[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 of ampicillin, 2 mM of MgCl2, and 0.1% glucose, and then grown at 37 °C with shaking (200 - 250 rpm) until the OD 600 reached 0.6 - 0.9. IPTG (final concentration 1 mM) was added to induce VHH expression. The culture was incubated at 28 °C overnight (about 16 - 18 hours) with shaking. The OD 600 after overnight induction was usually 25 - 30. At least 1 liter of culture solution (3 bottles) per clone was prepared at an average yield of 1 - 15 mg / l.

[0337] Extraction of VHH from the periplasm of E. coli was performed on the third day. The solutions used included 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] The overnight-induced culture was centrifuged at 8000 rpm for 8 minutes. The cell pellet from 1 liter of culture was pipetted up and down and resuspended in 12 ml of TES, and shaken on ice for 1 hour. For each 12 ml of TES used, approximately 18 ml of TES / 4 was added, incubated on ice for an additional 1 hour with shaking, and then centrifuged at 4 °C, 8000 rpm for 30 minutes. The supernatant containing the protein extracted from the periplasmic space was transferred to a fresh Falcon tube.

[0339] Subsequently, VHH was purified by IMAC using the following solutions: HIS-select (SIGMA), PBS, and 50 mM NaAcetate pH 4.6.

[0340] His-select was equilibrated with PBS. Specifically, for one periplasmic extract derived from 1 liter of culture medium, 1 ml of resin (about 2 ml of His-select solution) was added to a 50 ml Falcon tube. PBS was added to a final volume of 50 ml and mixed. Centrifugation was performed at 2000 rpm for 2 minutes, 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 minutes to 1 hour with gentle shaking. The sample was loaded onto a PD-10 column (GE healthcare, cat. No. 17-0435-01) having a filter at the bottom and washed with 50 to 100 ml of PBS (50 to 100 ml of PBS was used per 1 ml of resin). Elution was performed three times, each time with 1 ml of PBS / 0.5 M imidazole per 1 ml of resin used (to elute efficiently, the beads were suspended and left at 4 °C overnight with the bottom of the column closed). Dialysis was performed against PBS at 4 °C overnight (cutoff 3500 daltons) to remove imidazole. For efficient dialysis, the dialysis buffer (PBS) was changed 2 to 3 times. Alternatively, instead of elution with imidazole, the bound VHH 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 VHH, the eluted VHH was immediately neutralized with 1 M Tris pH 8.0 and dialysis was not required.

[0341] The amount of protein can be estimated at this point by measuring the OD280 of the eluted sample. The extinction coefficient of each clone was determined by the protParam tool under the primary structure analysis of the Expasy proteomics server. Further purification of the VHH can be achieved by different methods. One of these methods is shown below.

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

[0343] Example 3. Functional evaluation of VHH specific to human and / or mouse CD20 Specific binding of VHH shown by FACS To determine the binding activity of VHH specific to human or mouse CD20, FACS analysis was performed. Specifically, CHO-K1 cells and CHO-K1 cells stably expressing human or mouse CD20 were incubated with 5 μg / ml of the VHH produced according to this application. Monoclonal FITC-labeled anti-His antibody (Genscript #A01620) was applied as the secondary stain. 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 Targeted in vivo interferon activity using chimeric CD20 VHH with the A20 lymphoma model A20 cells (CD20+ and IFN-sensitive mouse lymphoma cell line) were subcutaneously inoculated into mice to induce tumors. To determine the in vivo antitumor effect of chimeric CD20 VHH, mice were treated in the vicinity of the lesion (s.c. at 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, and chimeric 2MC57 VHH was the most potent.

[0345] Targeting of interferon activity in vivo by chimeric CD20 VHH using the B16 melanoma model Mice were subcutaneously inoculated with B16-mCD20 cells (mouse melanoma cell line stably expressing mouse CD20) to induce tumors. To determine the in vivo antitumor effect of chimeric CD20 VHH, mice were treated in the vicinity of the lesion (s.c. at 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 given 2HCD25 VHH (anti-human CD20) fused to wild-type IFN or mutant IFNα2-Q124R by injection. Control mice were treated with PBS. Tumor growth was monitored. As shown in Figure 11, mCD20VHH 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 normal blood cell counts without signs of hematotoxicity, such as body weight and neutrophil, erythrocyte, and platelet counts. 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 effect of combination therapy with doxorubicin and chimeric 2MC57 VHH was tested. Mice were subcutaneously inoculated with B16-mCD20 cells (a mouse melanoma cell line stably expressing mouse CD20) to induce tumors. Subsequently, regardless of the presence or absence of doxorubicin, mice were treated in the vicinity of the lesion (s.c. at the edge of the tumor) with 2MC57 VHH (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon). Doxorubicin was administered at 3 mg / kg body weight every two days. As shown in Figure 14, when 2MC57 VHH was combined with doxorubicin, the tumor size decreased significantly compared to treatment with 2MC57 VHH alone without doxorubicin.

[0348] The combination of doxorubicin with 2MC57 VHH fused to mutant IFNα2-Q124R was considered to be particularly safe and effective. Specifically, combination therapy with doxorubicin worsened the morbidity and mortality associated with administration of wild-type IFN, but when used in combination with 2MC57 VHH fused to mutant IFNα2-Q124R, it caused no side effects as evaluated by the progression of body weight, survival rate, and measurement results of several blood parameters (Figure 15).

[0349] Combination therapy using chimeric CD20 VHH and tumor necrosis factor in a B16 melanoma model The antitumor effect of combination therapy using tumor necrosis factor (TNF) and chimeric 2MC57 VHH was tested. Mice were subcutaneously inoculated with B16-mCD20 cells (a mouse melanoma cell line stably expressing mouse CD20) to induce tumors. Subsequently, regardless of the presence or absence of TNF, mice were treated in the vicinity of the lesions (s.c. at the edge of the tumor) with 2MC57 VHH (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon). TNF was administered at 0.6 μg every two days. As shown in Figure 16, when 2MC57 VHH was combined with TNF, the tumor size significantly decreased compared to treatment with 2MC57 VHH without TNF.

[0350] The combination of TNF with 2MC57 VHH fused to mutant IFNα2-Q124R was considered to be particularly safe and effective. Specifically, combination therapy with TNF exacerbated thrombocytopenia and / or anemia and the mortality associated with the administration of wild-type IFN, but when used in combination with 2MC57 VHH fused to mutant IFNα2-Q124R, it caused no side effects when evaluated by the progression of body weight, survival rate, and the measurement results of some blood parameters (Figure 17).

[0351] Combination therapy using chimeric CD20 VHH and anti-PD-L1 camelid VHH in a B16 melanoma model The antitumor effects of combination therapies using anti-PD-L1 camelid VHH and chimeric 2MC57 VHH were tested. Mice were subcutaneously inoculated with B16-mCD20 cells (a mouse melanoma cell line stably expressing mouse CD20) to induce tumors. Subsequently, mice were treated in the vicinity of the lesion (s.c. at the edge of the tumor) with 2MC57 VHH (anti-mouse CD20) fused to wild-type IFN or mutant IFNα2-Q124R (i.e., AcTaferon), regardless of the presence or absence of anti-PD-L1 camelid VHH. Anti-PD-L1 camelid VHH was administered at 120 μg every two days. As shown in Figure 18, anti-PD-L1 camelid VHH produced a long-term stasis effect against 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 therapy using mutant IFNα2-Q124R (i.e., AcTaferon) and 2MC57 VHH fused to anti-PD-L1 camelid VHH induced a strong antitumor effect without tumor recurrence by day 38, compared to treatment with either agent alone. Panel B of Figure 24 further shows that the combination therapy had good tolerance 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. In the B16-mCD20 melanoma model, the antitumor effects of anti-PD-L1 camelid VHH and chimeric 2MC57 VHH combined with Treg depletion were tested. 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 VHH in a B16 melanoma model In the B16-mCD20 melanoma model, the efficacy of systemically administered chimeric CD20 VHHs was tested. Specifically, mice were given 2MC57 VHH (anti-mouse CD20) fused to mutant IFNα2-Q124R (i.e., AcTaferon), or 2HCD25 VHH (anti-human CD20) fused to mutant IFNα2-Q124R by intravenous injection. The chimeric VHHs were intravenously injected at a dose of 5,500 IU on days 8 - 11 after tumor inoculation. As shown in Figure 20, systemic administration of the chimeric antibody showed that chimeric 2MC57 VHH was more potent and resulted in a significant decrease in tumor growth.

[0353] Role of B cell depletion in the antitumor effect of chimeric CD20 VHHs Mice were transplanted with B16-hCD20 cells (a mouse melanoma cell line stably expressing human CD20) to induce tumors. To determine the in vivo antitumor effect of chimeric CD20 VHHs, mice were treated in the vicinity of the lesion (s.c. at the edge of the tumor) with 2HCD25 VHH (anti-human CD20) fused to wild-type human IFN, mouse IFN, or mutant IFNα2-Q124R (i.e., AcTaferon). Mice were also given 2MC57 VHH (anti-mouse CD20) fused to mutant IFNα2-Q124R. Tumor growth was monitored. As shown in Figure 21, chimeric 2HCD25 VHH fused to either mouse IFN or mutant IFNα2-Q124R resulted in significant tumor shrinkage. However, as shown in the inset, chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R was not thought to reduce the number of lymphocytes, suggesting that B cell depletion is not necessary for the antitumor effect of chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R.

[0354] Similar studies were conducted by systemic iv administration of chimeric CD20 antibodies to mice. Consistent with previous results, chimeric 2HCD25 VHH fused to either mouse IFN or mutant IFNα2-Q124R resulted in significant tumor regression (Figure 22). When it is considered that the chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R did not reduce lymphocyte counts (Figure 21), this result suggests that B cell depletion is not necessary for the antitumor effect of chimeric 2HCD25 VHH fused to mutant IFNα2-Q124R.

[0355] Efficacy of chimeric CD20 VHH in a mouse model of multiple sclerosis Using an established mouse experimental autoimmune encephalomyelitis (EAE) model, the efficacy of chimeric CD20 VHH in the treatment of multiple sclerosis was tested. Specifically, mice were administered a peptide corresponding to 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 the MOG peptide. As shown in Figure 23, 2MC57 VHH (anti-mouse CD20) fused to mutant IFNα2-Q124R significantly improved the clinical score and delayed the onset and appearance of symptoms such as disease and paralysis. Summarizing these results, it was suggested that the chimeric CD20 VHH of the present application may have a therapeutic effect in the treatment of multiple sclerosis as well as other CNS demyelinating diseases in humans.

[0356] Equivalents Although this application is described in connection with its specific embodiments, further modifications are possible, and this application is generally intended to cover modifications, uses, adaptations, of this application in accordance with the principles of the present invention, including departures from the disclosure of this invention such as would be within the scope of known or customary practice in the technical field to which this application pertains, and may be applied as follows within the essential features described herein and the scope of the appended claims.

[0357] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments specifically described herein. Such equivalents are intended to be encompassed by the appended claims.

[0358] Incorporation by Reference All patents and publications referred to herein are incorporated herein 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. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior invention.

[0360] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual 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 chimeric protein comprising a targeting moiety against CD20 and a signaling agent, wherein the targeting moiety against CD20 comprises a single-domain antibody (VHH) of a recombinant heavy chain having three complementarity-determining regions (CDR1, CDR2, and CDR3), (a) CDR1 comprises the amino acid sequence of SEQ ID NO: 42, (b) CDR2 comprises the amino acid sequence of SEQ ID NO: 51, (c) CDR3 comprises the amino acid sequence of SEQ ID NO: 61, the signaling agent is a modified interferon α2 (IFNα2), the modified interferon α2 (IFNα2) comprises an amino acid sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 127 or SEQ ID NO: 128 and has an R149A mutation that results in a reduced agonist affinity or activity for the IFNα receptor as compared to wild-type IFNα2, the reduced affinity or activity in the receptor is recoverable by linkage with the targeting moiety against CD20, a chimeric protein.

2. The chimeric protein according to claim 1, wherein the VHH comprises an amino acid sequence having at least 90% similarity with amino acids 1 to 118 of SEQ ID NO:

27.

3. The chimeric protein according to claim 1, wherein the VHH comprises an amino acid sequence having at least 95% similarity with amino acids 1 to 118 of SEQ ID NO:

27.

4. The chimeric protein according to claim 1, wherein the VHH comprises amino acids 1 to 118 of SEQ ID NO:

27.

5. The chimeric protein according to any one of claims 1 to 4, further comprising one or more additional targeting moieties.

6. The one or more additional targeting moieties recognize an antigen on a tumor antigen or immune cell, the one or more additional targeting moieties may modulate an antigen on a tumor antigen or immune cell, the immune cell may be selected from T cells, B cells, dendritic cells, macrophages, and NK cells, the chimeric protein according to claim 5.

7. The chimeric protein according to claim 5, wherein the one or more additional targeting moieties recognize Clec9A and recruit dendritic cells.

8. A recombinant nucleic acid encoding the chimeric protein according to any one of claims 1 to 7.

9. A host cell comprising the nucleic acid according to claim 8.

10. A composition for use in the treatment of a disease or disorder involving cells expressing CD20, the composition comprising the chimeric protein according to any one of claims 1 to 7.

11. The composition according to claim 10, wherein the disease or disorder is one or more of cancer, infectious disease, and / or immune disorder.

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