CD28 shedding blockers

JP2024532970A5Pending Publication Date: 2025-09-25BIONDO BIOLOGICS LTD
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Patent Information

Application Number
JP2024538512
Authority / Receiving Office
JP · JP
Patent Type
Applications
Current Assignee / Owner
Priority Date
2022-06-01
Filing Date
2022-09-06
Publication Date
2025-09-25

AI Technical Summary

Technical Problem

There is a need for molecules that inhibit the proteolytic shedding of CD28 to enhance immunotherapy without acting as CD28 antagonists or agonists, thereby modulating immune responses effectively.

Method used

Development of single domain antibodies (sdAbs) that specifically target the CD28 cleavage site to inhibit proteolytic shedding, formulated as monomers or dimers with linker technologies to enhance their inhibitory effect.

Benefits of technology

The sdAbs effectively inhibit CD28 cleavage, maintaining membrane CD28 levels for sustained immune stimulation while avoiding indiscriminate activation or suppression, thus enhancing immunotherapy outcomes.

✦ Generated by Eureka AI based on patent content.

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Abstract

Single domain antibodies (sdAbs) that block CD28 cleavage are provided. Also provided are dimeric agents, at least two membrane CD28 binding agents, as well as methods of using the sdAbs and / or dimeric agents and pharmaceutical compositions and kits that include the sdAbs and / or dimeric agents.
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Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 241,010, filed September 6, 2021, U.S. Provisional Patent Application No. 63 / 241,011, filed September 6, 2021, and U.S. Provisional Patent Application No. 63 / 347,756, filed June 1, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] The present invention is in the field of immunomodulation and immunotherapy. [Background technology]

[0003] The adaptive immune system plays a key role in regulating and defending against pathogens and cancer cells, primarily by orchestrating the stimulation of antigen-specific helper CD4+ and cytotoxic CD8+ T cells. Persistent and sustained activation of T cells by antigen-presenting cells (APCs) requires i) engagement of the T cell receptor (TCR) with peptides presented by the major histocompatibility complex (MHC) on the APC; and ii) the costimulatory CD28 receptor on the T cell, which binds to the B7-1 (CD80) and B7-2 (CD86) ligands that are also expressed by the APC. The biological consequences of CD28 costimulation are numerous and include control of the T cell cycle, expansion, and differentiation, as well as amplification of TCR stimulation by lowering the threshold required to achieve immune effector functions.

[0004] International Patent Publication No. 2019175885 discloses that soluble CD28 (sCD28) is produced by proteolytic cleavage of the stalk domain of CD28 and active shedding of the extracellular domain of CD28 from the plasma membrane. The use of antibodies against CD28 that inhibit this shedding and thus enhance immune responses is also disclosed. The use of these antibodies in enhancing PD-1 / PD-L1-based immunotherapy is also disclosed. Importantly, these antibodies also do not have CD28 agonist or antagonist effects, and therefore do not indiscriminately enhance immune activation and do not inhibit activation by blocking the binding of CD86 to its ligand CD28. There is a need for superior molecules that inhibit proteolytic shedding of CD28, enhance immunotherapy, and do not act as CD28 antagonists. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Patent Application Publication No. 2019175885 Summary of the Invention

[0006] The present invention provides single domain antibodies (sdAbs) that block CD28 cleavage. Also provided are dimeric agents comprising the sdAbs, methods of using the sdAbs and / or the dimeric agents, and pharmaceutical compositions and kits comprising the sdAbs and / or the dimeric agents.

[0007] According to a first aspect, there is provided a single domain antibody (sdAb) comprising three CDRs, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:1 (INSMG), CDR2 comprises the amino acid sequence shown in SEQ ID NO:2 (AINEKLLIYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO:3 (DLYGSDYWD), or CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4 (INAMG), CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 (AISGGGDTYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6 (DMIEQQWWY), or CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4 (INAMG), CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 (AISGGGDTYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 7 (DTHRGVYWY), or CDR1 comprises the amino acid sequence shown in SEQ ID NO:8 (IKTMA), CDR2 comprises the amino acid sequence shown in SEQ ID NO:9 (AINYIKEIYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO:10 (DVTKEDYWY), or CDR1 comprises the amino acid sequence shown in SEQ ID NO: 11 (INSMA), CDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 (AISNAREVYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13 (DVYFQEYWY), or CDR1 comprises the amino acid sequence shown in SEQ ID NO: 14 (INTMA), CDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 (AINSISRTYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 10 (DVTKEDYWY), or CDR1 comprises the amino acid sequence shown in SEQ ID NO:8 (IKTMA), CDR2 comprises the amino acid sequence shown in SEQ ID NO:16 (AIASDNRKYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO:10 (DVTKEDYWY), or CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17 (IRTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18 (AISSGREVYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19 (DMYWQDYWW), or CDR1 comprises the amino acid sequence shown in SEQ ID NO:1 (INSMG), CDR2 comprises the amino acid sequence shown in SEQ ID NO:20 (AISDRSEKYYADSVKG), and CDR3 comprises the amino acid sequence shown in SEQ ID NO:21 (DHHHSDWWT).

[0008] According to some embodiments the sdAb is a camelid antibody or a shark antibody.

[0009] According to some embodiments the sdAb is a VHH antibody.

[0010] According to some embodiments, the sequence from the N-terminus to CDR1 consists of X1VQLVESGGGLVQX2GX3SLRLSCX4ASGSX5X6S (SEQ ID NO: X), where X1 is E or Q, X2 is A or P, X3 is E or G, X4 is A or K, X5 is I, L or T, and X6 is A or F; and the sequence between CDR1 and CDR2 consists of WYRQAPGX7X8X9EX10VX11 (SEQ ID NO: X), where X7 is S or K, X8 is Q or G, X9 is R or L, X10 is L or R, and X11 is one of A, S or T. and the sequence between CDR2 and CDR3 is RFTX11SRDNX12KX13TX14YLQMNX15LX16X17X18DX19X20VYYCVV (sequence number X), where X11 is I or V, X12 is A or S, X13 is T or N, X14 is V, M or L, X15 is S or N, X16 is R, K or E, X17 is P or A, X18 is E or R, X19 is T or A, and X20 is A or G. The sequence from the C-terminus to CDR3 is WGQGTX21VTVSS (sequence number X), where X21 is Q or L.

[0011] According to some embodiments, the sequence from the N-terminus to CDR1 consists of EVQLVESGGGLVQAGESLRLSCAASGSIAS (SEQ ID NO: 22), the sequence between CDR1 and CDR2 consists of WYRQAPGSQRELVX (SEQ ID NO: 48), the sequence between CDR2 and CDR3 consists of RFTISRDNAKTTVYLQMNSLRPEDTAVYYCVV (SEQ ID NO: 24), and the sequence to the C-terminus CDR3 consists of WGQGTQVTVSS (SEQ ID NO: 25), where X is A or T.

[0012] According to some embodiments, the sdAb comprises a sequence selected from the group consisting of: a.EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAINEKLLIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO:26); b. EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMIEQQWWYWGQGTQVTVSS (SEQ ID NO: 27); c.EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDTHRGVYWYWGQGTQVTVSS (SEQ ID NO:28); d.EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVAAINYIKEIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO:29); e.EVQLVESGGGLVQAGESLRLSCAASGSIASINSMAWYRQAPGSQRELVAAISNAREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVYFQEYWYWGQGTQVTVSS (SEQ ID NO: 30); f.EVQLVESGGGLVQAGESLRLSCAASGSIASINTMAWYRQAPGSQRELVAAINSISRTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO:31); g.EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVTAIASDNRKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO:32); h.EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKQRELVTAIASDNRKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 70); i.EVQLVESGGGLVQPGGSLRLSCKASGSIASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO:71); j.EVQLVESGGGLVQPGGSLRLSCAASGSTASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO:72); k.EVQLVESGGGLVQPGGSLRLSCKASGSTASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO:73); l.EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 74); m.EVQLVESGGGLVQAGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMYWQDYWWWGQGTQVTVSS (SEQ ID NO: 33); n.EVQLVESGGGLVQPGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTQVTVSS (SEQ ID NO: 75); o.EVQLVESGGGLVQPGGSLRLSCKASGSIASIRTMAWYRQAPGKGLELVAAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO:76); p.EVQLVESGGGLVQPGGSLRLSCKASGSTASIRTMAWYRQAPGKGLELVSAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 77); q.EVQLVESGGGLVQPGGSLRLSCAASGSIASIRTMAWYRQAPGKGLELVSAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 78); or r.EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAISDRSEKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDHHHSDWWTWGQGTQVTVSS (SEQ ID NO: 34).

[0013] According to some embodiments, the sdAb is not a CD28 agonist.

[0014] According to some embodiments, the sdAb is not a CD28 antagonist.

[0015] According to some embodiments, the sdAb is a CD28 antagonist.

[0016] According to some embodiments, the agent does not degrade mCD28 or inhibit mCD28-mediated immune cell activation.

[0017] According to some embodiments, the agent binds within the stalk region of CD28.

[0018] According to some embodiments, the stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 35) or KGKHLCPSPLFPGPS (SEQ ID NO: 36).

[0019] According to some embodiments, the stalk region consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO:37).

[0020] According to some embodiments, the agent binds at a cleavage site for at least one protease.

[0021] According to some embodiments, the agent inhibits proteolytic cleavage by at least one protease.

[0022] According to some embodiments, the at least one protease is at least one metalloprotease.

[0023] According to some embodiments, the at least one metalloprotease is MMP-2, MMP-13, or a combination thereof.

[0024] According to another aspect there is provided a dimeric agent comprising at least two membrane CD28 (mCD28) binding single domain antibodies (sdAbs), wherein a first mCD28 binding sdAb is linked to a second mCD28 binding sdAb by a linker.

[0025] According to some embodiments, the first sdAb, the second sdAb, or both, comprise a sequence selected from the group consisting of: a.EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 40); b. EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSS (SEQ ID NO: 95); and c.QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (sequence number 96).

[0026] According to some embodiments, the dimeric agent comprises an sdAb of the invention.

[0027] According to some embodiments, the first sdAb and the second sdAb comprise the same sequence.

[0028] According to some embodiments, the first sdAb and the second sdAb comprise different sequences.

[0029] According to some embodiments, the dimeric agent inhibits proteolytic cleavage of mCD28.

[0030] According to some embodiments the first sdAb, the second sdAb or both when not part of a dimeric agent are CD28 antagonists, and the dimeric agent is not a CD28 antagonist.

[0031] According to some embodiments the dimeric agent comprises a first polypeptide comprising a first sdAb and a second polypeptide comprising a second sdAb, and a linker links the first and second polypeptides.

[0032] According to some embodiments, the first polypeptide comprises a first free cysteine ​​amino acid on the outside of the first sdAb, the second polypeptide comprises a second free cysteine ​​amino acid on the outside of the second sdAb, and the linker comprises a bond between the first and second free cysteine ​​amino acids.

[0033] According to some embodiments, the first free cysteine, the second free cysteine, or both, are C-terminal amino acids.

[0034] According to some embodiments, the first polypeptide comprises a first sdAb and a first dimerization domain, the second polypeptide comprises a second sdAb and a second dimerization domain, and the linker comprises the dimerization domains, a bond between the dimerization domains, or both.

[0035] According to some embodiments, the first dimerization domain comprises a first immunoglobulin (Ig) hinge domain, the second dimerization domain comprises a second Ig hinge domain, and the linker comprises a disulfide bond between the first Ig hinge domain and the second Ig hinge domain.

[0036] According to some embodiments the first sdAb is N-terminal to the first dimerization domain, the second sdAb is N-terminal to the second dimerization domain, or both.

[0037] According to some embodiments, the Ig hinge domain is a human Ig hinge domain comprising the amino acid sequence DKTHTCPPCPAPE (SEQ ID NO: 83) or ESKYGPPCPPCPAPEFEGG (SEQ ID NO: 85).

[0038] According to some embodiments, the first sdAb is separated from the first Ig hinge domain by an amino acid linker, the second sdAb is separated from the second Ig hinge domain by an amino acid linker, or both.

[0039] According to some embodiments, the amino acid linker is a flexible linker.

[0040] According to some embodiments, the amino acid linker comprises a sequence selected from (GGGGS)n, (GS)n, (GGS)n, (GSGGS)n, (EGGGS)n, (EGGS)n, and combinations thereof, where n is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8.

[0041] According to some embodiments, the first dimerization domain, the second dimerization domain or both further comprise a CH2 domain of an Ig heavy chain.

[0042] According to some embodiments, the first dimerization domain, the second dimerization domain or both further comprise a CH3 domain of an Ig heavy chain.

[0043] According to some embodiments, the hinge domain is N-terminal to the CH2 domain, and the CH2 domain is N-terminal to the CH3 domain.

[0044] According to some embodiments, the dimerization domain comprises at least one mutation that does not induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) or that reduces ADCC or CDC.

[0045] According to some embodiments, the dimerization domain is DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:39) or ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:92).

[0046] According to some embodiments, the dimerization agent comprises EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVTAIASDNRKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 93).

[0047] According to some embodiments, the dimerization agent comprises EVQLVESGGGLVQAGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMYWQDYWWWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 94).

[0048] According to some embodiments, the dimeric agent does not inhibit or only weakly inhibits binding of a ligand to CD28, where weak inhibition includes less than 50% inhibition.

[0049] According to some embodiments, the ligand is CD86, CD80 or both.

[0050] According to some embodiments, the linker is a chemical linker.

[0051] According to some embodiments, the chemical linker comprises a biocompatible polymer.

[0052] According to some embodiments, the biocompatible polymer comprises polyethylene glycol (PEG).

[0053] According to some embodiments, the dimeric agent comprises a single polypeptide, the single polypeptide comprising the N-terminus of a first sdAb to a second sdAb, separated by an amino acid linker of less than 13 amino acids, and optionally the dimeric agent is not a CD28 antagonist, inhibits ligand binding to CD28 by less than 50%, or both.

[0054] According to some embodiments the dimeric agent comprises a single polypeptide, the single polypeptide comprising the N-terminus of a first sdAb to a second sdAb, separated by an amino acid linker of 10 or more amino acids.

[0055] According to some embodiments, the amino acid linker comprises a net neutral charge.

[0056] According to some embodiments, the amino acid linker comprises a sequence selected from (GGGGS)n, (GS)n, (GGS)n, (GSGGS)n, and combinations thereof, where n is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8.

[0057] According to some embodiments, the amino acid linker comprises a net positive charge.

[0058] According to some embodiments, the amino acid linker comprises a sequence selected from (XGGGS)n, (XGGS)n, (GGGXS)n, and combinations thereof, where X is selected from K, R and H, and n is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8.

[0059] According to some embodiments, X is K.

[0060] According to some embodiments, the amino acid linker comprises a net negative charge.

[0061] According to some embodiments, the amino acid linker comprises a sequence selected from (XGGGS)n, (XGGS)n, (GGGXS)n, and combinations thereof, where X is selected from E and D, and n is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0062] According to some embodiments, X is E.

[0063] According to some embodiments, the amino acid linker is a rigid linker.

[0064] According to some embodiments, the amino acid linker comprises GGGGSEAAAKEAAAKEAAAKAAAGSGGGGS (SEQ ID NO: 97).

[0065] According to some embodiments, the amino acid linker comprises up to 100 amino acids.

[0066] According to some embodiments, the dimeric agent is a CD28 antagonist.

[0067] According to some embodiments, the dimeric agent inhibits binding of a ligand to CD28, wherein inhibition comprises at least 50% inhibition.

[0068] According to another aspect, there is provided a method of reducing soluble CD28 (sCD28) levels in a subject in need thereof, comprising administering to the subject an sdAb of the invention or a dimeric agent of the invention, thereby reducing sCD28.

[0069] According to another aspect, there is provided a method of treating and / or preventing cancer in a subject in need thereof, comprising administering to the subject an sdAb of the invention or a dimeric agent of the invention, thereby treating and / or preventing cancer.

[0070] According to another aspect, there is provided a method of improving PD-1 and / or PD-L1 based immunotherapy in a subject in need thereof comprising administering to the subject an sdAb of the invention or a dimeric agent of the invention, thereby improving PD-1 and / or PD-L1 based immunotherapy.

[0071] According to some embodiments, the subject is suffering from cancer.

[0072] According to some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer and colorectal cancer.

[0073] According to some embodiments, the cancer comprises elevated or increased levels of sCD28.

[0074] According to some embodiments, the method does not degrade mCD28.

[0075] According to some embodiments, the method does not reduce mCD28-mediated immune cell activation.

[0076] According to another aspect there is provided a method of inhibiting ligand binding to mCD28 comprising contacting mCD28 with an sdAb of the invention or a dimeric agent of the invention, thereby inhibiting ligand binding to mCD28.

[0077] According to another aspect there is provided a method for suppressing an immune response in a subject in need thereof comprising administering to the subject an sdAb of the invention or a dimeric agent of the invention, thereby suppressing the immune response.

[0078] According to some embodiments, the dimeric agent inhibits ligand binding to mCD28, thereby suppressing the immune response.

[0079] According to some embodiments, the ligand is CD86, CD80 or both.

[0080] According to some embodiments, the subject is suffering from an autoimmune disease.

[0081] According to some embodiments, the autoimmune disease is selected from the group consisting of lupus, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, Becht's disease, colitis, ulcerative colitis, diabetes, Graves' disease and multiple sclerosis.

[0082] According to another aspect there is provided a pharmaceutical composition comprising an sdAb of the invention or a dimeric agent of the invention and a pharma- ceutically acceptable carrier, excipient or adjuvant.

[0083] According to some embodiments the pharmaceutical composition of the invention comprising an sdAb of the invention or a dimeric agent of the invention is for use in the treatment and / or prevention of cancer or for improving PD-1 and / or PD-L1 based immunotherapy.

[0084] According to some embodiments, the pharmaceutical composition of the invention comprising an sdAb of the invention or a dimeric agent of the invention is for use in inhibiting ligand binding to mCD28 or for suppressing an immune response.

[0085] According to another aspect there is provided a kit comprising at least one sdAb of the invention or a dimeric agent of the invention.

[0086] According to some embodiments, the kit further comprises at least one of the following: a. anti-PD-1 and / or PD-L1 immunotherapy; and b. Labeling that states that the agent of the invention is for use in conjunction with PD-1 and / or PD-L1 based immunotherapy.

[0087] According to another aspect, there is provided a method of producing a dimeric agent that inhibits proteolytic cleavage of mCD28 on the surface of a cell, the method comprising: ai. To obtain an agent that binds to mCD28 on the cell surface and blocks cleavage of mCD28 by proteases; ii. linking a first portion of the drug to a second portion of the drug via a linker to form a dimeric drug; iii. testing the ability of the dimeric agents to block cleavage of mCD28 on the cell surface by proteases; and iv. selecting a dimeric agent that blocks cleavage of mCD28 on the cell surface; And b. culturing a host cell comprising one or more vectors comprising one or more nucleic acid sequences encoding a dimeric agent, wherein the one or more nucleic acid sequences are i. obtaining an agent that binds to mCD28 on the cell surface and blocks cleavage of mCD28 by proteases; ii. linking a first portion of the drug to a second portion of the drug via a linker to form a dimeric drug; iii. testing the ability of the dimeric agents to block cleavage of mCD28 on the cell surface by proteases; and iv. of a dimeric agent selected by selecting an agent that blocks cleavage of mCD28 on the cell surface; At least one of Thereby, an agent is generated that inhibits the proteolytic cleavage of mCD28 on the surface of cells.

[0088] According to some embodiments, the resulting agent is an sdAb.

[0089] According to some embodiments, obtaining the medicament includes: a. immunizing a shark or camelid with the extracellular domain of CD28 or a fragment thereof and collecting antibodies from the immunized organism, or screening a library of agents for binding to the extracellular domain of CD28 or a fragment thereof and selecting agents which bind; b. testing the binding of an antibody or agent that binds to mCD28 on the cell surface and selecting an antibody or agent that binds to mCD28 on the cell surface; and c. Testing cleavage of mCD28 on cells in the presence of a protease and a selected antibody or agent, and an additional selected antibody or agent that blocks cleavage of mCD28 on cells.

[0090] According to some embodiments, the extracellular domain or fragment thereof comprises: a. It is a dimer, b. contains the CD28 stalk domain; or c. Both.

[0091] According to some embodiments, the protease is selected from MMP-2 and MMP-13.

[0092] According to some embodiments, the method further comprises assaying mCD28 downstream signaling in the presence of the obtained dimeric agents, and selecting at least one dimeric agent that neither substantially agonizes nor substantially antagonizes mCD28 signaling.

[0093] According to some embodiments, the method further comprises assaying mCD28 downstream signaling in the presence of the resulting dimeric agents, and selecting at least one dimeric agent that substantially antagonizes mCD28 signaling.

[0094] According to another aspect, there is provided a dimerizing agent produced by the method of the invention.

[0095] According to another aspect, there is provided a pharmaceutical composition comprising a dimeric agent of the invention and a pharma- ceutically acceptable carrier, excipient, or adjuvant.

[0096] According to some embodiments, the pharmaceutical compositions of the invention comprising the dimeric agents produced by the methods of the invention are for use in the treatment and / or prevention of cancer, or for improving PD-1 and / or PD-L1 based immunotherapy.

[0097] According to some embodiments, the pharmaceutical compositions of the invention comprising a dimeric agent produced by the methods of the invention are for use in inhibiting ligand binding to mCD28 or for suppressing an immune response.

[0098] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0099] [Figure 1] Figure 1: Line graph quantification of soluble CD28 from SEB-stimulated PBMCs in the presence of the parental clone (VHH#2A1) and two affinity matured variants.

[0100] [Diagram 2] Figure 2: Bar graph of IFN gamma secretion from isolated T cells in a mixed lymphocyte reaction (MLR, +mDC) in the presence of parental VHH or two affinity matured variants. VHH#3C04 = irrelevant VHH as negative control VHH#12B07 = known CD28 antagonist as positive control

[0101] [Diagram 3] Figure 3: Bar graph of IL-2 secretion from isolated T cells in a mixed lymphocyte reaction (MLR, +mDC) in the presence of Fc dimeric agents containing two copies of the parental VHH or two affinity matured variants. Human IgG4 was used as a negative control.

[0102] [Figure 4-1] Figures 4A-4B: (4A) Line graph quantification of binding affinity of VHH-hIgG4 constructs, 2A1-hFc, 9B03-hFc and 12A09-hFc to human CD28 using a direct CD28 EIA. (4B) Histograms of 2A1-hFc, 9B03-hFc and 12A09-hFc binding to human CD3 cells (black histograms). The background of the anti-human IgG detection antibody is shown in grey histograms. [Figure 4-2] Same as above.

[0103] [Diagram 5] Figures 5A-5B: Bar graphs showing sCD28 production by peripheral blood mononuclear cells (PBMCs) in response to stimulation with Staphylococcal enterotoxin B (SEB) using different concentrations of (5A) 12A09-25GS-hFc, (5B) 9B03-25GS-hFc compared to the corresponding isotype control and the parental 2A1-hFc clone.

[0104] [Figure 6] 6A-6B: (6A) Bar graphs of IL-2 secretion from CD3 cells stimulated with anti-CD3 antibody in the presence of 12A9-25GS-huFc chimeric molecule and (6B) bar graphs of IL-2 secretion from CD3 cells stimulated with anti-CD3 antibody in the presence of 9B3-25GS-huFc chimeric molecule. Repulsive anti-CD28 antibody, clone 28.2, was used as positive control (PC) and isotype control was used as negative control.

[0105] [Figure 7] Figure 7: Bar graph showing the effect of flexible linker size on CD28 shedding inhibition activity of VHH 12A9. Two VHH concentrations were tested: 100 nM (top) and 300 nM (bottom).

[0106] [Figure 8]8A-8B: (8A) Line graphs showing tumor volume in MC-38 syngenic tumor model in response to treatment with 12A09-hFc twice weekly starting 6 days prior to inoculation of MC38 cells (prophylactic model), (8B) Line graphs showing tumor volume in MC-38 syngenic tumor model in response to treatment with 9B03-hFc twice weekly starting 6 days after inoculation of MC38 cells (therapeutic model). Anti-PD1 (RMP1-14) was administered twice weekly starting 6 days after inoculation of MC38 cells. Human IgG4 and rat IgG2a served as negative controls. 8A: rG2a, n=10; aPD1, n=10; VHH, n=108B: rG2a, n=10; aPD1, n=10; VHH, n=10 *pVal of 0.014

[0107] [Figure 9A] Figures 9A-9B: Line graph quantification of the binding affinity of (9A) five humanized 12A09-hIgG4 constructs to human CD28 using a direct CD28 EIA, and (9B) one humanized 9B03-hIgG4 construct to human CD28 using a direct CD28 EIA. In both graphs, binding was compared to the parental variant (black line). [Figure 9B] Same as above.

[0108] [Figure 10] Figures 10A-10B: (10A) Line graph quantification of soluble CD28 from SEB-stimulated PBMCs in the presence of 12A9-25GS-huFc chimera and five 12A09 humanized variants of the same chimera, or (10B) Line graph quantification of soluble CD28 from SEB-stimulated PBMCs in the presence of 9B3-25GS-huFc chimera and humanized 9B3 variant VHH1 of the same chimera.

[0109] [Figure 11-1]Figures 11A-11H: (11A-11B) Line graphs showing antigen binding to recombinant human CD28-Fc fusion protein by serial dilutions of single chain tandem dimeric agents. Antigen was immobilized on Maxisorp ELISA plates. Serial dilutions of dimeric agents were preformed and the molar concentrations of the various dimeric agents were calculated and normalized to compare the amount of active sites in each assay. Detection of bound antibody was performed using anti-VHH-HRP and color development with TMB. (11C-11H) Bar graphs of the levels of soluble CD28 measured in the culture medium of PBMCs stimulated with SEB. Figure 1 shows the effect of various treatments on the levels of soluble CD28, including an MMP inhibitor (TMI-1, 1 μM), an irrelevant VHH negative control, or various concentrations (0.024 μM to 3 μM) of (11C)5GS, (11D)10GS, (11E)20GS, (11F)20K, (11G)20E, or (11H)Hel20 dimeric agents. Levels of soluble human CD28 in the supernatants were quantified using a standardized sandwich ELISA (R&D system). [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0110] [Figure 12-1] Figures 12A-12B: Histograms of HEK293 cells overexpressing human CD28 monitored by flow cytometry for CD86-Fc (2 μg / mL) binding using a secondary anti-human Fc antibody conjugated to AlexaFlour 647. Monitoring was performed in the presence of (12A) a neutral flexible linker or (12B) a charged flexible linker, or a rigid linker containing single chain dimeric agent. Light grey line - secondary antibody control Black line - CD86-Fc positive control Dark grey line - binding in the presence of dimeric molecule [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above.

[0111] [Figure 13-1]Figures 13A-13I: (13A-13F) Bar graphs of IL-2 secretion from human isolated CD9 cells stimulated for 24-48 hours with HEK / scOKT3 acting as artificial antigen presenting cells expressing CD80 as a ligand for CD28 costimulation in the presence of irrelevant negative control VHH, positive control (PC) VHH (PC) known to block CD80 interaction with CD28, and (13A) 5GS, (13B) 10GS, (13C) 20GS, (13D) 20K, (13E) 20E, (13F) Hel20 dimeric agents. (3G-3I) Bar graphs of IFN gamma secretion from isolated T cells in an allogeneic mixed lymphocyte reaction (MLR, +mDC) in the presence of (13G) 5GS, (13H) 10GS, (3I) 20GS dimeric agents. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 13-5] Same as above.

[0112] [Figure 14-1]Figures 14A-14J: (14A-14C): (14A) Line graph showing antigen binding to recombinant human CD28-Fc fusion protein by serial dilution of single cysteine ​​dimer molecules, (14B) Line graph showing antigen binding to recombinant human CD28-Fc fusion protein by serial dilution of PEG-linked dimer molecules, (14C) Line graph showing antigen binding to recombinant human CD28-Fc fusion protein by serial dilution of Fc-linked dimer molecules. (14D-14I) Bar graphs of soluble CD28 levels measured in culture medium of PBMC stimulated with SEB. Figure 1 shows the effect of different treatments on the levels of soluble CD28: irrelevant VHH negative control, monomeric 2A1 (3 μM), or various concentrations (0.024 μM to 3 μM) of (14D) 2A1-1C, (14E) 2A1-1C-bmP11, (14F) 2A1-Hinge, (14G) 2A1-hFC, (14H) 2A1-15GS-hFc, or (14I) 2A1-25GS-hFc dimeric agents. Levels of soluble human CD28 in the supernatants were quantified using a standardized sandwich ELISA (R&D system). (14J) Summary scatter plot of CD28-shedding inhibition provided by various non-competitive dimeric molecules. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Figure 14-5] Same as above. [Figure 14-6] Same as above.

[0113] [Figure 15] FIG. 15: Structure of the 2A1-bmp11-2A1 molecule utilizing a linker with 11 PEG repeats.

[0114] [Figure 16-1]Figures 16A-16B: Histograms of HEK293 cells overexpressing human CD28 monitored by flow cytometry for CD86-Fc (2 μg / mL) binding using a secondary anti-human Fc antibody conjugated to AlexaFlour 647. Monitoring was performed in the presence of (16A) an Ig-based linker containing C-terminal linker or (16B) a dimeric agent. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0115] [Figure 17-1] Figures 17A-17F: Bar graphs of IL-2 secretion from human isolated CD3 cells stimulated for 24 hours with HEK / scOKT3 acting as artificial antigen presenting cells expressing CD80 as a ligand for CD28 costimulation in the presence of an irrelevant negative control VHH, a positive control VHH known to block CD86, and (17A) 2A1-C, (17B) 2A1-1C-bmP11, (17C) 2A1-Hinge, (17D) 2A1-huFc, (17E) 2A1-15GS-huFc and (17F) 2A1-25GS-huFc dimeric agents. [Figure 17-2] Same as above. [Figure 17-3] Same as above.

[0116] [Figure 18-1] Figures 18A-18F: (18A-18E): Bar graphs of IFN-gamma secretion from isolated T cells in a mixed lymphocyte reaction (MLR, +mDC) in the presence of (18A) 2A1-C, (18B) 2A1-1C-bmP11, (18C) 2A1-huFc, (18D) 2A1-15GS-huFc and (18E) 2A1-25GS-huFc dimeric agents. (18F) Scatter plot of total changes in immune regulation in T cells. [Figure 18-2] Same as above. [Figure 18-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0117] The present invention provides, in some embodiments, single domain antibodies (sdAbs) that block CD28 cleavage. Also provided are dimeric agents comprising at least two membrane CD28 (mCD28)-binding single domain antibodies (sdAbs). Also provided are therapeutic methods comprising administering the sdAbs and / or dimeric agents, as well as compositions and kits comprising the sdAbs and / or dimeric agents.

[0118] sdAb

[0119] According to a first aspect there is provided an sdAb comprising three complementarity determining regions (CDRs), CDR1 comprising the amino acid sequence set out in SEQ ID NO:1 (INSMG), CDR2 comprising the amino acid sequence set out in SEQ ID NO:2 (AINEKLLIYYADSVKG) and CDR3 comprising the amino acid sequence set out in SEQ ID NO:3 (DLYGSDYWD).

[0120] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4 (INAMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:5 (AISGGGDTYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:6 (DMIEQQWWY).

[0121] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4 (INAMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:5 (AISGGGDTYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7 (DTHRGVYWY).

[0122] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8 (IKTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9 (AINYIKEIYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10 (DVTKEDYWY).

[0123] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO:11 (INSMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:12 (AISNAREVYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:13 (DVYFQEYWY).

[0124] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14 (INTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 (AINSISRTYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10 (DVTKEDYWY).

[0125] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8 (IKTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:16 (AIASDNRKYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10 (DVTKEDYWY).

[0126] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17 (IRTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18 (AISSGREVYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19 (DMYWQDYWW).

[0127] According to another aspect, there is provided an sdAb comprising three complementarity determining regions (CDRs), wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1 (INSMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:20 (AISDRSEKYYADSVKG) and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:21 (DHHHSDWWT).

[0128] As used herein, the terms "single domain antibody", "nanobody", "DARPin" and "VHH antibody" are synonymous and used interchangeably and refer to antibody fragments consisting of a single monomeric variable domain. SdAbs are able to selectively bind to a specific antigen in a manner similar to antibodies. However, they have a molecular weight of only 12-15 kDa and are therefore much smaller than full antibodies, Fab fragments or single chain antibodies. Due to their small size and the reliance of only three CDRs for antigen binding, the binding mechanism of single domain antibodies, specifically VHHs, is convex in shape and binds to its epitope from only one side and is therefore more suitable to bind epitopes characterized by limited solvent exposure, such as those found in protein clefts such as the stalk region of membrane-anchored CD28. In some embodiments, the sdAb is a camelid antibody. In some embodiments, the camelid is a camel, an alpaca or a llama. In some embodiments, the camelid is a camel. In some embodiments, the camelid is an alpaca. In some embodiments, the camelid is a llama. In some embodiments, the sdAb is a shark antibody. In some embodiments, the sdAb is the first sdAb in the molecule. In some embodiments, the sdAb is the second sdAb in the molecule. In some embodiments, the molecule is a dimeric molecule of the invention.

[0129] Also, as already indicated herein, the amino acid residues of Nanobodies are numbered or referred to herein according to the conventional numbering for VHs provided by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91) as applied to VHH domains from camel in the article by Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240(1-2): 185-195. According to this numbering, FR1 of a Nanobody comprises the amino acid residues at positions 1 to 30, CDR1 of a Nanobody comprises the amino acid residues at positions 31 to 35, FR2 of a Nanobody comprises the amino acids at positions 36 to 49, CDR2 of a Nanobody comprises the amino acid residues at positions 50 to 65, FR3 of a Nanobody comprises the amino acid residues at positions 66 to 94, CDR3 of a Nanobody comprises the amino acid residues at positions 95 to 102, and FR4 of a Nanobody comprises the amino acid residues at positions 103 to 113. In this regard, it should be noted that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e. one or more positions according to the Kabat numbering may not be occupied in the actual sequence or the actual sequence may comprise more amino acid residues than permitted by the Kabat numbering). This means that, in general, the Kabat numbering may not correspond to the actual numbering of the amino acid residues in the actual sequence.However, in general, according to the Kabat numbering, it can be said that, regardless of the number of amino acid residues in the CDR, position 1 according to the Kabat numbering corresponds to the start of FR1, and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2, and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3, and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4, and vice versa.

[0130] An alternative method for numbering the amino acid residues of VH domains, which can also be applied in a similar manner to camelid VHH domains and nanobodies, is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in this specification, embodiments and figures, unless otherwise indicated, the numbering according to Kabat, as applied to VHH domains by Riechmann and Muyldermans, is followed.

[0131] In some embodiments, the sdAb binds to CD28. In some embodiments, the agent binds to CD28. In some embodiments, the CD28 is mammalian CD28. In some embodiments, the CD28 is human CD28. According to some embodiments, human CD28 comprises or consists of the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 42). In some embodiments, mature CD28 lacks a signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 43). In some embodiments, the CD28 signal peptide comprises or consists of MLRLLLALNLFPSIQVTG (SEQ ID NO: 41).

[0132] In some embodiments, the DNA coding sequence encoding full-length human CD28 comprises the sequence: (SEQ ID NO:44).

[0133] In some embodiments, CD28 is membranal CD28 (mCD28). In some embodiments, membrane CD28 is membrane CD28. In some embodiments, mCD28 is on the cell surface. In some embodiments, mCD28 is in the membrane.

[0134] In some embodiments, the CD28 is extracellular CD28. In some embodiments, the CD28 is the extracellular domain (ECD) of CD28. In some embodiments, the ECD of CD28 comprises MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 45). In some embodiments, the ECD of CD28 consists of SEQ ID NO: 45. In some embodiments, the ECD comprises NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 46). In some embodiments, the ECD consists of SEQ ID NO: 46. In some embodiments, the ECD is dimeric. In some embodiments, the ECD comprises a stalk domain.

[0135] In some embodiments, CD28 is the stalk domain of CD28. In some embodiments, the first CD28 binding sdAb binds to the stalk domain. In some embodiments, the second CD28 binding sdAb binds to the stalk domain. In some embodiments, both the first and second CD28 binding sdAbs bind to the stalk domain. In some embodiments, the stalk domain is the stalk region. In some embodiments, the stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 35). In some embodiments, the stalk region comprises the amino acid sequence KGKHLCPSPLFPGPS (SEQ ID NO: 36). In some embodiments, the stalk region comprises the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 37). In some embodiments, the stalk region consists of SEQ ID NO: 35. In some embodiments, the stalk region consists of SEQ ID NO: 36. In some embodiments, the stalk region consists of SEQ ID NO: 37.

[0136] In some embodiments, the sdAb inhibits proteolytic cleavage of CD28. As used herein, "inhibiting proteolytic cleavage" refers to any reduction in proteolytic cleavage of mCD28. In some embodiments, inhibition is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% reduction in cleavage. Each possibility represents a separate embodiment of the invention. In some embodiments, inhibition of proteolytic cleavage maintains levels of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage increases levels of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage maintains levels of mCD28 sufficient for immune stimulation.

[0137] In some embodiments, the reduction in proteolytic cleavage is a reduction in cleavage by at least one protease. In some embodiments, the reduction in proteolytic cleavage is a reduction in cleavage by at least one metalloprotease. In some embodiments, the metalloprotease is MMP-2, ADAM10, ADAM17, or a combination thereof. In some embodiments, the metalloprotease is MMP-2, ADAM10, ADAM17, MMP-13, or a combination thereof. In some embodiments, the metalloprotease is MMP-2. In some embodiments, the metalloprotease is MMP-2 or MMP-13. In some embodiments, the metalloprotease is MMP-2. In some embodiments, the metalloprotease is MMP-2, MMP-13, or a combination thereof.

[0138] In some embodiments, the sdAb inhibits proteolytic cleavage by at least one protease. In some embodiments, the protease is a metalloprotease. In some embodiments, the protease is a matrix metalloprotease. In some embodiments, the protease is a serine protease. In some embodiments, the protease is a cysteine ​​protease. In some embodiments, the protease is a threonine protease. In some embodiments, the protease is a serine, cysteine ​​or threonine protease. In some embodiments, the protease is an aspartic acid protease. In some embodiments, the protease is a glutamic acid protease. In some embodiments, the protease is selected from aspartic acid, glutamic acid, serine, cysteine ​​and threonine proteases. In some embodiments, the protease is an aspartic acid peptide lyase. In some embodiments, the protease is a sheddase. In some embodiments, the metalloprotease is an exopeptidase. In some embodiments, the metalloprotease is an endopeptidase. In some embodiments, the metalloprotease is an exopeptidase or an endopeptidase. In some embodiments, the metalloprotease is zinc catalyzed. In some embodiments, the metalloprotease is cobalt catalyzed. In some embodiments, the metalloprotease is matrix metalloproteinase-2 (MMP-2). In some embodiments, the metalloprotease is matrix metalloproteinase-13 (MMP-13). In some embodiments, the metalloprotease is ADAM10. In some embodiments, the metalloprotease is ADAM17. In some embodiments, the metalloprotease is ADAM10, MMP-2, and / or ADAM17. In some embodiments, the metalloprotease is ADAM10, MMP-2, MMP-13, and / or ADAM17. In some embodiments, the metalloprotease is MMP-2, ADAM10, ADAM17, or a combination thereof.In some embodiments, the metalloprotease is MMP-2, MMP-13, ADAM10, ADAM17, or a combination thereof.

[0139] In some embodiments, the sdAb binds to the cleavage site. In some embodiments, the cleavage site is in the stalk region. In some embodiments, the cleavage site is a cleavage motif. In some embodiments, the MMP-2 cleavage motif is PXX / X, where the last X is a hydrophobic residue. In some embodiments, the PXX / X motif in CD28 is PSP / L. In some embodiments, the protease cleavage site is amino acids 142-145 of SEQ ID NO:42 (PSPL). In some embodiments, the protease cleavage site is amino acids 127-130 of SEQ ID NO:43 (PSPL). In some embodiments, the protease cleavage site is amino acids 9-12 of SEQ ID NO:37 (PSPL). In some embodiments, the agent blocks access of the protease to the cleavage site. In some embodiments, the agent binds to PSPL in the stalk domain of mCD28.

[0140] In some embodiments, the cleavage site is before a leucine. In some embodiments, the cleavage site is before a valine. In some embodiments, the cleavage site is before an aromatic amino acid. In some embodiments, the cleavage site is before a leucine, a valine, and / or an aromatic amino acid. In some embodiments, the aromatic amino acid is selected from phenylalanine, tryptophan, tyrosine, and histidine. In some embodiments, the cleavage site is before any one of histidine 134, valine 135, histidine 139, leucine 140, leucine 145, and phenylalanine 146 of SEQ ID NO:29. In some embodiments, the cleavage site is before histidine 134, valine 135, histidine 139, leucine 140, leucine 145, or phenylalanine 146 of SEQ ID NO:42. Each possibility represents a separate embodiment of the invention. In some embodiments, the cleavage site is before leucine 145 of SEQ ID NO:42. In some embodiments, the cleavage site is before leucine 127 of SEQ ID NO:43.

[0141] In some embodiments, the sdAb is not an antagonist. In some embodiments, the sdAb is not an antagonist of CD28. In some embodiments, the antagonist is a substantial antagonist. In some embodiments, the antagonist is a direct antagonist.

[0142] The term "antagonist" generally refers to a molecule, compound or agent that binds to the receptor at the same site as the agonist or at a different site, does not activate the receptor, and prevents or blocks activation of the receptor by a natural ligand and prevents or blocks activation of the receptor by a receptor agonist. In some embodiments, the sdAb binds to CD28 and blocks activation of the receptor. In some embodiments, the agent and / or sdAb does not block activation by CD86. In some embodiments, the sdAb blocks activation by CD86. In some embodiments, the sdAb blocks binding of a ligand to CD28. In some embodiments, the sdAb does not inhibit binding of a ligand to CD28. In some embodiments, the sdAb inhibits binding of a ligand to CD28. In some embodiments, the inhibition is substantial inhibition. In some embodiments, substantial is significant. In some embodiments, substantial antagonism is greater than low inhibition. In some embodiments, an agent that is not a substantial antagonist does not inhibit or inhibits to a low degree. In some embodiments, the sdAb poorly inhibits binding of the ligand to CD28. In some embodiments, poor inhibition includes less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 7, or 5% inhibition. Each possibility represents a separate embodiment of the invention. In some embodiments, poor inhibition includes less than 50% inhibition. In some embodiments, poor inhibition includes less than 35% inhibition. In some embodiments, poor inhibition includes less than 20% inhibition. In some embodiments, the CD28 ligand is selected from CD80, CD86, and ICOSL. In some embodiments, the CD28 ligand is CD86. In some embodiments, the CD28 ligand is CD80. In some embodiments, the CD28 ligand is ICOSL. In some embodiments, CD86 is CD86-Fc. In some embodiments, CD80 is CD80-Fc.

[0143] In some embodiments, the sdAb is not an agonist of CD28. In some embodiments, the agonist is a direct agonist. The term "agonist" generally refers to a molecule, compound, or agent that binds to a receptor and activates the receptor fully or partially. In some embodiments, the agonist binds at the same site as the natural ligand. In some embodiments, the agonist binds to an allosteric site that is different from the binding site of the natural ligand.

[0144] As used herein, a "direct agonist / antagonist" refers to a molecule that binds to a receptor (mCD28) and increases / decreases signaling by that molecule upon binding. In the case of mCD28, an agonist binds to mCD28 and increases mCD28 signaling within the cell upon binding. In some embodiments, an agonist increases T cell activation. In some embodiments, an agonist increases T cell proliferation. In some embodiments, an agonist increases proinflammatory cytokine secretion. Proinflammatory cytokines are well known in the art and are known to be secreted by activated T cells. Examples of proinflammatory cytokines include, but are not limited to, TNFα, IFNγ, IL-1B, IL-2, and IL-6. In some embodiments, the proinflammatory cytokine is IFNγ. In some embodiments, the proinflammatory cytokine is IL-2. In the case of mCD28, an antagonist binds to mCD28 and decreases mCD28 signaling within the cell upon binding. In some embodiments, the antagonist reduces T cell activation, reduces T cell proliferation, and / or reduces proinflammatory cytokine secretion. Molecules that achieve receptor signaling by contacting it with its ligand, with an inhibitor, with a co-receptor, or with any molecule other than the receptor in question to modify receptor signaling are not considered direct agonists / antagonists. In some embodiments, the sdAb of the present invention reduces the production of soluble CD28 (sCD28) (by inhibiting the cleavage of mCD28). In some embodiments, the agent of the present invention reduces the production of soluble CD28 (sCD28) (by inhibiting the cleavage of mCD28). sCD28 can act as a decoy by binding to the CD28 ligand, thus antagonizing mCD28. Its removal allows increased signaling through mCD28 on the cell. Although the result is increased mCD28 signaling, an agent is not an mCD28 agonist or direct agonist if its binding to mCD28 does not increase receptor signaling.

[0145] In some embodiments, the sdAb does not bind to the ligand binding domain of mCD28. In some embodiments, the sdAb does not mask or inhibit access to the ligand binding domain. In some embodiments, the sdAb does not bind to, mask or block access to the IgV domain of sCD28. In some embodiments, the IgV domain is the ligand binding domain. In some embodiments, the ligand binding domain comprises amino acids 28-137 of SEQ ID NO: 42. In some embodiments, the ligand binding domain comprises or consists of the amino acid sequence MLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG (SEQ ID NO: 47).

[0146] In some embodiments the sdAb comprises from the N-terminus to the CDR1 the sequence X 1 VQLVESGGGLVQX 2 GX 3 SLRLSCX 4 ASGSX 5 X 6 S (SEQ ID NO: 79), 1 is E or Q, and X 2 is A or P, and X 3 is E or G, and X 4 is A or K, and X 5 is I, L or T, and X 6 is A or F. In some embodiments the sequence from the N-terminus to CDR1 consists of SEQ ID NO: 79. In some embodiments the sdAb comprises from the N-terminus to CDR1 the sequence EVQLVESGGGLVQAGESLRLSCAASGSIAS (SEQ ID NO: 22). In some embodiments SEQ ID NO: 79 is SEQ ID NO: 22. In some embodiments the sequence from the N-terminus to CDR1 consists of SEQ ID NO: 22. In some embodiments the sdAb comprises between CDR1 and CDR2 the sequence WYRQAPGX 7 X 8 X9 EX 10 VX 11 (SEQ ID NO: 80), 7 is S or K, and X 8 is Q or G, and X 9 is R or L, and X 10 is L or R, and X 11 is any one of A, S, or T. In some embodiments the sequence between CDR1 and CDR2 consists of SEQ ID NO:79. In some embodiments the sdAb comprises the sequence WYRQAPGSQRELVX (SEQ ID NO:48) between CDR1 and CDR2, where X is A or T. In some embodiments the sequence between CDR1 and CDR2 consists of SEQ ID NO:48. In some embodiments SEQ ID NO:80 is SEQ ID NO:48. In some embodiments SEQ ID NO:48 is WYRQAPGSQRELVA (SEQ ID NO:23). In some embodiments SEQ ID NO:48 is WYRQAPGSQRELVT (SEQ ID NO:49). In some embodiments the sdAb comprises the sequence RFTX between CDR2 and CDR3. 11 SRDNX 12 KX 13 TX 14 YLQMNX 15 LX 16 X 17 X 18 DX 19 X 20 VYYCVV (SEQ ID NO: 81), 11 is I or V, and X 12 is A or S, and X 13 is T or N, and X 14 is V, M or L, and X 15 is S or N, and X 16 is R, K or E, and X 17 is P or A, and X 18 is E or R, and X 19 is T or A, and X 20is A or G. In some embodiments the sequence between CDR2 and CDR3 consists of SEQ ID NO: 81. In some embodiments the sdAb comprises the sequence RFTISRDNAKTTVYLQMNSLRPEDTAVYYCVV (SEQ ID NO: 24) between CDR2 and CDR3. In some embodiments SEQ ID NO: 81 is SEQ ID NO: 24. In some embodiments the sequence between CDR2 and CDR3 consists of SEQ ID NO: 24. In some embodiments the sdAb comprises the sequence WGQGTX 21 VTVSS (SEQ ID NO: 82), 21 is Q or L. In some embodiments the sequence from the C-terminus to the CDR3 consists of SEQ ID NO: 82. In some embodiments the sdAb comprises the sequence from the C-terminus to the CDR3: WGQGTQVTVSS (SEQ ID NO: 25). In some embodiments SEQ ID NO: 82 is SEQ ID NO: 25. In some embodiments the sequence from the C-terminus to the CDR3 consists of SEQ ID NO: 25.

[0147] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAINEKLLIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO:26). In some embodiments, the sdAb consists of SEQ ID NO:26. In some embodiments, SEQ ID NO:26 is the amino acid sequence of VHH 5A3. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:26. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO:26.

[0148] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMIEQQWWYWGQGTQVTVSS (SEQ ID NO:27). In some embodiments, the sdAb consists of SEQ ID NO:27. In some embodiments, SEQ ID NO:27 is the amino acid sequence of VHH 6B3. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:27. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO:27.

[0149] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDTHRGVYWYWGQGTQVTVSS (SEQ ID NO:28). In some embodiments, the sdAb consists of SEQ ID NO:28. In some embodiments, SEQ ID NO:28 is the amino acid sequence of VHH 6B10. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:28. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO:28.

[0150] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVAAINYIKEIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO:29). In some embodiments, the sdAb consists of SEQ ID NO:29. In some embodiments, SEQ ID NO:29 is the amino acid sequence of VHH 10E1. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:29. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO:29.

[0151] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINSMAWYRQAPGSQRELVAAISNAREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVYFQEYWYWGQGTQVTVSS (SEQ ID NO: 30). In some embodiments, the sdAb consists of SEQ ID NO: 30. In some embodiments, SEQ ID NO: 30 is the amino acid sequence of VHH 11E11. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 30. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 30.

[0152] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINTMAWYRQAPGSQRELVAAINSISRTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO: 31). In some embodiments, the sdAb consists of SEQ ID NO: 31. In some embodiments, SEQ ID NO: 31 is the amino acid sequence of VHH 11G11. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 31. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 31.

[0153] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVTAIASDNRKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO: 32). In some embodiments, the sdAb consists of SEQ ID NO: 32. In some embodiments, SEQ ID NO: 32 is the amino acid sequence of VHH 12A9. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 32. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 32.

[0154] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKQRELVTAIASDNRKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 70). In some embodiments, SEQ ID NO: 70 is the amino acid sequence of 12A09_VHH4. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 70. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 70. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:70.

[0155] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCKASGSIASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 71). In some embodiments, SEQ ID NO: 71 is the amino acid sequence of 12A9_VHH12. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 71. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 71. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:71.

[0156] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSTASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 72). In some embodiments, SEQ ID NO: 72 is the amino acid sequence of 12A09_VHH16. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 72. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 72. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:72.

[0157] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCKASGSTASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 73). In some embodiments, SEQ ID NO: 73 is the amino acid sequence of 12A9_VHH17. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 73. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 73. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:73.

[0158] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 74). In some embodiments, SEQ ID NO: 74 is the amino acid sequence of 12A09_VHH18. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 74. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 74. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:74.

[0159] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMYWQDYWWWGQGTQVTVSS (SEQ ID NO: 33). In some embodiments, the sdAb consists of SEQ ID NO: 33. In some embodiments, SEQ ID NO: 33 is the amino acid sequence of VHH 9B3. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 33. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 33.

[0160] In some embodiments, the first sdAb, the second sdAb, or both, comprise the amino acid sequence EVQLVESGGGLVQPGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTQVTVSS (SEQ ID NO: 75). In some embodiments, SEQ ID NO: 75 is the amino acid sequence of 9B03_VHH1. In some embodiments, the first sdAb, the second sdAb, or both, consist of SEQ ID NO: 75. In some embodiments, the first sdAb, the second sdAb, or both, comprise or consist of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 75. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:75.

[0161] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCKASGSIASIRTMAWYRQAPGKGLELVAAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 76). In some embodiments, SEQ ID NO: 76 is the amino acid sequence of 9B03_VHH12. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 76. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 76. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:76.

[0162] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCKASGSTASIRTMAWYRQAPGKGLELVSAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 77). In some embodiments, SEQ ID NO: 77 is the amino acid sequence of 9B03_VHH19. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 77. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 77. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:77.

[0163] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGSIASIRTMAWYRQAPGKGLELVSAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 78). In some embodiments, SEQ ID NO: 78 is the amino acid sequence of 9B03_VHH20. In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 78. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 78. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:78.

[0164] In some embodiments, the sdAb comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAISDRSEKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDHHHSDWWTWGQGTQVTVSS (SEQ ID NO: 34). In some embodiments, the sdAb consists of SEQ ID NO: 34. In some embodiments, SEQ ID NO: 34 is the amino acid sequence of VHH 9A7. In some embodiments, the sdAb comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 34. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 34.

[0165] It will be appreciated by those skilled in the art that for immune stimulatory purposes, e.g. in the context of cancer treatment, the sdAb may be non-antagonistic or, once incorporated into the agent of the invention, may be antagonistic, so long as the antagonistic effect is lost or reduced to an acceptably low level. For immune inhibition purposes, e.g. in the context of autoimmune disease, the sdAb may be antagonistic.

[0166] Drugs

[0167] According to another aspect there is provided an agent comprising at least two CD28-binding single domain antibodies (sdAbs).

[0168] In some embodiments, the agent comprises a first sdAb and a second sdAb. In some embodiments, at least two sdAbs are sdAbs of the present invention. In some embodiments, the first sdAb is an sdAb of the present invention. In some embodiments, the second sdAb is an sdAb of the present invention.

[0169] In some embodiments, two sdAbs of the present invention are linked by a linker. In some embodiments, a first sdAb and a second sdAb of the present invention are linked by a linker. In some embodiments, a first sdAb is linked to a second sdAb by a linker. In some embodiments, this linkage produces an agent of the present invention.

[0170] In some embodiments, the agent is a dimeric agent. As used herein, the term "dimer" refers to an agent that comprises two simpler molecules, e.g., monomers. In some embodiments, the dimeric agent comprises two sdAbs. In some embodiments, the agent is monoparatopic. As used herein, the term "monoparatopic" refers to an agent that targets only one epitope. In some embodiments, the agent comprises two sdAbs. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer. In some embodiments, the agent comprises a first sdAb and a second sdAb. In some embodiments, the agent comprises two identical sdAbs. In some embodiments, the two sdAbs comprise the same sequence. In some embodiments, the sequence is an amino acid sequence. In some embodiments, the agent comprises two different sdAbs. In some embodiments, the two sdAbs comprise different sequences. In some embodiments, the two sdAbs comprise the same CDRs. In some embodiments, the two sdAbs comprise different CDRs. In some embodiments, the target epitope of the sdAb is the CD28 stalk region. In some embodiments, the target epitope of the sdAb is the CD28 cleavage site. In some embodiments, the target epitope is the site of CD28 protease mediated shedding.

[0171] In some embodiments, the agent inhibits proteolytic cleavage of CD28. In some embodiments, the agent is superior in inhibiting proteolytic cleavage compared to the sdAb as a monomer. In some embodiments, the superiority comprises increased inhibition. In some embodiments, the increase is at least a 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400, or 500% increase. Each possibility represents a separate embodiment of the present invention.

[0172] In some embodiments, the agent is not an antagonist of CD28. In some embodiments, each sdAb, when not part of a drug, is not an antagonist of CD28. In some embodiments, the first sdAb, the second sdAb, or both, when not part of a drug, are antagonists and the agent is not an antagonist of CD28. In some embodiments, each sdAb, when not part of a drug, is an antagonist and the agent is not an antagonist of CD28. In some embodiments, the agent and / or sdAb binds to CD28 but does not activate the receptor or blocks its activation. In some embodiments, the agent and / or sdAb does not block binding of a ligand to CD28. In some embodiments, the agent does not inhibit binding of a ligand to CD28. In some embodiments, the agent inhibits binding of a ligand to CD28 to a low extent.

[0173] In some embodiments, the agent is not an agonist of CD28. In some embodiments, each sdAb, when not part of an agent, is not an agonist of CD28. In some embodiments, the agent does not bind to the ligand binding domain of mCD28. In some embodiments, the agent does not mask or inhibit access to the ligand binding domain. In some embodiments, the agent does not bind to, mask or block access to the IgV domain of sCD28.

[0174] In some embodiments, the agent comprises a first sdAb and a second sdAb. In some embodiments, the agent comprises two identical sdAbs. In some embodiments, the two sdAbs comprise the same sequence. In some embodiments, the sequence is an amino acid sequence. In some embodiments, the agent comprises two different sdAbs. In some embodiments, the two sdAbs comprise different sequences. In some embodiments, the two sdAbs comprise the same CDRs. In some embodiments, the two sdAbs comprise different CDRs. In some embodiments, the target epitope of the sdAb is the CD28 stalk region. In some embodiments, the target epitope of the sdAb is a CD28 cleavage site. In some embodiments, the target epitope is a site of CD28 protease-mediated shedding.

[0175] In some embodiments, the agent comprises at least two sdAbs. In some embodiments, the agent comprises multiple sdAbs. In some embodiments, the agent comprises at least 2, 3, 4, 5, 6 or 7 sdAbs. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent comprises two sdAbs. In some embodiments, the agent comprises a first sdAb and a second sdAb. In some embodiments, the first sdAb and the second sdAb are the same sdAb. In some embodiments, the first sdAb and the second sdAb comprise the same sequence. In some embodiments, the first sdAb and the second sdAb are different sdAbs. In some embodiments, the first sdAb and the second sdAb comprise different sequences.

[0176] In some embodiments, the first sdAb and the second sdAb bind the same mCD28 molecule. In some embodiments, the first sdAb and the second sdAb bind a single mCD28 molecule. In some embodiments, the first sdAb and the second sdAb bind different CD28 molecules. In some embodiments, the first sdAb binds a first CD28 molecule and the second sdAb binds a second CD28 molecule. In some embodiments, the first CD28 molecule and the second CD28 molecule are the same molecule. In some embodiments, the first CD28 molecule and the second CD28 molecule are different molecules.

[0177] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 40). In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 40. In some embodiments, SEQ ID NO: 40 is the amino acid sequence of VHH 2A1. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 40. Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO:40.

[0178] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGSLFSINAMAWYRQAPGKQRELVAAITSSGSTNYANSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCVVDEYGSDYWIWGQGTQVTVSS (SEQ ID NO: 95). In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 95. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 95. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 95.

[0179] In some embodiments, the first sdAb, the second sdAb, or both, comprises the amino acid sequence QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRERVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNNLEPRDAGVYYCVVDLYGEDYWIWGQGTQVTVSS (SEQ ID NO: 96). In some embodiments, the first sdAb, the second sdAb, or both, consists of SEQ ID NO: 96. In some embodiments, the first sdAb, the second sdAb, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 96. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 96.

[0180] In some embodiments, the first sdAb, the second sdAb or both comprise three CDRs, CDR1 comprises the amino acid sequence INAMG (SEQ ID NO: 4), CDR2 comprises the amino acid sequence AISGGGDTYYADSVKG (SEQ ID NO: 5) and CDR3 comprises the amino acid sequence DLYGSDYWD (SEQ ID NO: 3). In some embodiments, the CDRs of SEQ ID NO: 1 are SEQ ID NOs: 3-5. In some embodiments, the first sdAb, the second sdAb or both comprise three CDRs, CDR1 comprises the amino acid sequence INAMA (SEQ ID NO: 98), CDR2 comprises the amino acid sequence AITSSGSTNYANSVKG (SEQ ID NO: 99) and CDR3 comprises the amino acid sequence DEYGSDYWI (SEQ ID NO: 100). In some embodiments, the CDRs of SEQ ID NO: 95 are SEQ ID NOs: 98-100. In some embodiments the first sdAb, the second sdAb or both comprise three CDRs, CDR1 comprises the amino acid sequence INAMG (SEQ ID NO: 4), CDR2 comprises the amino acid sequence AITSGGSTNYADSVKG (SEQ ID NO: 101) and CDR3 comprises the amino acid sequence DLYGEDYWI (SEQ ID NO: 102). In some embodiments the CDRs of SEQ ID NO: 96 are SEQ ID NOs: 4, 101 and 102.

[0181] It will be understood by those skilled in the art that any sdAb that binds to mCD28 on cells and inhibits proteolytic cleavage and sCD28 shedding can be used as the sdAb of the present invention.For immune stimulation purposes, for example in the context of cancer, the sdAb can be non-antagonistic or antagonistic, as long as it is introduced into the agent of the present invention and the antagonistic effect is lost or reduced to an acceptable low level.For immune inhibition purposes, for example in the context of autoimmune disease, the sdAb can be antagonistic or can become antagonistic once it is part of the agent of the present invention.

[0182] In some embodiments, the two CD28 binding sdAbs are linked by a linker. In some embodiments, the first sdAb and the second sdAb are linked by a linker. In some embodiments, the first sdAb is linked to the second sdAb by a linker.

[0183] Polypeptide dimer

[0184] In some embodiments, the agent comprises a first polypeptide comprising a first sdAb. In some embodiments, the agent comprises a second polypeptide comprising a second sdAb. In some embodiments, a linker connects the first polypeptide and the second polypeptide. In some embodiments, the polypeptide is a polypeptide chain. In some embodiments, the agent comprises a first polypeptide and a second polypeptide connected by a linker. In some embodiments, the agent having two polypeptides is not a CD28 antagonist.

[0185] In some embodiments, the first polypeptide comprises a signal peptide. In some embodiments, the second polypeptide comprises a signal peptide. In some embodiments, the first polypeptide lacks a signal peptide. In some embodiments, the second polypeptide lacks a signal peptide. Although the active form of the agent does not contain a signal peptide, it will be understood by those skilled in the art that in order to express the polypeptide in a cell, it may be necessary to create a polypeptide that contains a signal peptide to facilitate secretion of the polypeptide from the cell. In some embodiments, the signal peptide is an Ig signal peptide. Any signal peptide that allows the polypeptide of the present invention to be created can be used.

[0186] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass natural peptides, peptidomimetics (typically containing non-peptide bonds or other synthetic modifications) as well as peptide analogs, peptoids and semi-peptoids or any combination thereof. In another embodiment, the described peptide polypeptides and proteins have modifications that make them more stable in the body or more permeable to cells. In one embodiment, the terms "peptide", "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms "peptide", "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids.

[0187] In some embodiments, the linkage is from C-terminus to C-terminus. In some embodiments, the C-terminus is at the most C-terminal amino acid of the polypeptide. In some embodiments, the C-terminus is at the most C-terminal domain of the polypeptide. In some embodiments, the linkage is from C-terminal domain to C-terminal domain.

[0188] In some embodiments, the first polypeptide comprises a first cysteine ​​amino acid. In some embodiments, the second polypeptide comprises a second cysteine ​​amino acid. In some embodiments, the cysteine ​​is a free cysteine. In some embodiments, the cysteine ​​is on the outside of the sdAb. In some embodiments, the polypeptide comprises a cysteine ​​on the outside of the sdAb. In some embodiments, the cysteine ​​is a C-terminal cysteine. In some embodiments, the cysteine ​​is an N-terminal cysteine. In some embodiments, the cysteine ​​is a C-terminal amino acid. In some embodiments, the cysteine ​​is in the C-terminal domain. In some embodiments, the cysteine ​​is in the domain C-terminal to the sdAb. In some embodiments, the sdAb is N-terminal to the cysteine. In some embodiments, the linkage comprises a bond between the first cysteine ​​and the second cysteine. In some embodiments, the linker is a bond. In some embodiments, the bond is a disulfide bond. In some embodiments, the bond is between the most C-terminal cysteines in each polypeptide.

[0189] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSC (SEQ ID NO: 113). In some embodiments, the first polypeptide consists of SEQ ID NO: 113. In some embodiments, the second polypeptide comprises SEQ ID NO: 113. In some embodiments, the second polypeptide consists of SEQ ID NO: 113. In some embodiments, the agent comprises a dimer of SEQ ID NO: 113. In some embodiments, the agent consists of a dimer of SEQ ID NO: 113. In some embodiments, the first polypeptide, the second polypeptide, or both, comprise or consist of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 113. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 113. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 40.

[0190] In some embodiments, an agent comprising SEQ ID NO: 113 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO: 113 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0191] In some embodiments, the agent comprising the linked cysteines is not a CD28 antagonist. In some embodiments, the agent comprising two polypeptides, each having a C-terminal free cysteine, to which the free cysteines are linked, is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0192] In some embodiments, the linker is a chemical linker. In some embodiments, the linker is an artificial linker. In some embodiments, the linker is not an amino acid linker. In some embodiments, the linker is not just a bond. In some embodiments, the linker comprises a biocompatible polymer. In some embodiments, the biocompatible polymer is at least partially biodegradable. In some embodiments, the biocompatible polymer is or comprises a polyglycol ether, a polyester, a polyamide, or any combination thereof. In some embodiments, the polyglycol ether is or comprises a polyethylene glycol (PEG). In some embodiments, the linker of the present invention comprises a PEG. In some embodiments, the linker of the present invention comprises a PEG characterized by an Mn of 100 Da to 5000 Da (including any range therebetween). In some embodiments, the PEG linker comprises at least 10 repeats of PEG. In some embodiments, the PEG linker comprises at least one repeat of PEG. In some embodiments, the PEG linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 repeats of PEG. Each possibility represents a separate embodiment of the present invention. In some embodiments, the PEG linker comprises at least 11 repeats of PEG. In some embodiments, the PEG linker comprises 11 repeats of PEG. In some embodiments, the PEG linker is a linker provided in FIG. 5. In some embodiments, the PEG linker comprises or consists of maleimide-N-(CH2)2-CO-NH-(CH2)2-(O-CH2CH2)n-(CH2)2-NH-CO-(CH2)2-N-maleimide. In some embodiments, the PEG linker comprises or consists of N-(CH2)2-CO-NH-(CH2)2-(O-CH2CH2)n-(CH2)2-NH-CO-(CH2)2-N.

[0193] In some embodiments, an agent comprising a cysteine ​​linked by a chemical linker is not a CD28 antagonist. In some embodiments, an agent consisting of two polypeptides each comprising a cysteine ​​linked by a chemical linker is not a CD28 antagonist. In some embodiments, a CD28 antagonist is a substantial antagonist. In some embodiments, a chemical linker is linked to a cysteine. In some embodiments, a chemical linker links a first and a second cysteine. In some embodiments, a chemical linker is linked to a cysteine ​​by a maleimide reactive group. In some embodiments, the reaction is to a thiol in the polypeptide. In some embodiments, the reaction is to a thiol in the cysteine. Methods for generating chemical linkers and attaching them to polypeptides are well known in the art, and any such method can be used to produce the agent of the invention.

[0194] In some embodiments, the first polypeptide further comprises a first dimerization domain. In some embodiments, the second polypeptide further comprises a second dimerization domain. In some embodiments, the dimerization domain is C-terminal to the sdAb. In some embodiments, the dimerization domain is N-terminal to the sdAb. In some embodiments, the sdAb is N-terminal to the dimerization domain. In some embodiments, the dimerization domain is a C-terminal domain. In some embodiments, the dimerization domain comprises a cysteine. In some embodiments, the linker comprises a dimerization domain. In some embodiments, the linker comprises a bond between the dimerization domains. In some embodiments, the bond is a disulfide bond.

[0195] In some embodiments, the dimerization domains are capable of dimerizing with each other. In some embodiments, the first dimerization domain is capable of dimerizing with the second dimerization domain. In some embodiments, the first and second dimerization domains are capable of dimerizing with each other. In some embodiments, the ability to dimerize is configured to dimerize. In some embodiments, the dimerization is under physiological conditions. In some embodiments, the dimerization is in a bodily fluid. In some embodiments, the bodily fluid is blood. In some embodiments, the bodily fluid is plasma. In some embodiments, the bodily fluid is serum. In some embodiments, the dimerization is in a subject. In some embodiments, the dimerization is in vivo. In some embodiments, the dimerization is in vitro.

[0196] As used herein, the term "dimerization domain" refers to an amino acid sequence that, upon contact with another amino acid sequence (another dimerization domain), binds to it to form a dimer. Dimerization domains are well known in the art, as many protein sequences are known to bind to each other. In some embodiments, dimerization involves the formation of a covalent bond between the dimerization domains. In some embodiments, dimerization involves an electrostatic bond. In some embodiments, dimerization does not involve an electrostatic bond. In some embodiments, dimerization is reversible. In some embodiments, dimerization is irreversible. In some embodiments, dimerization involves a bond formed between the dimerization domains. In some embodiments, the bond is a chemical bond. In some embodiments, the bond is a disulfide bond. In some embodiments, the bond is a peptide bond. Examples of dimerization domains include the hinge domain of an antibody heavy chain, the CH1 / CL domain of an antibody heavy / light chain, and the ECD domain of TCR alpha / beta, to name a few.

[0197] In some embodiments, the dimerization domain comprises or consists of an immunoglobulin (Ig) hinge domain. In some embodiments, the agent comprising an Ig hinge domain is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist. In some embodiments, the first dimerization domain is a first Ig hinge domain. In some embodiments, the second dimerization domain is a second Ig hinge domain. In some embodiments, the Ig hinge domain is a heavy chain hinge domain. In some embodiments, the Ig is a human Ig. In some embodiments, the immunoglobulin is selected from IgA, IgD, IgE, IgG, and IgM. In some embodiments, the immunoglobulin is an IgG. In some embodiments, the IgG is an IgG1. In some embodiments, the IgG is an IgG2. In some embodiments, the IgG is an IgG3. In some embodiments, the IgG is selected from IgG1 and IgG3. In some embodiments, the IgG is an IgG4. In some embodiments, the IgG is a human IgG. In some embodiments, the first and second dimerization domains are both Ig hinge domains. In some embodiments, the first and second dimerization domains are identical. In some embodiments, the first and second dimerization domains are at least 95% identical. In some embodiments, the first and second dimerization domains are at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, or 100% identical. Each possibility represents a separate embodiment of the present invention.

[0198] In some embodiments, the Ig hinge domain comprises the amino acid sequence DKTHTCPPCPAPEL (SEQ ID NO:38). In some embodiments, the Ig hinge domain consists of SEQ ID NO:38. In some embodiments, the IgG1 hinge domain comprises or consists of SEQ ID NO:38. In some embodiments, the Ig hinge domain comprises the amino acid sequence DKTHTCPPCPAPE (SEQ ID NO:83). In some embodiments, the Ig hinge domain consists of SEQ ID NO:83. In some embodiments, the IgG1 hinge domain comprises or consists of SEQ ID NO:83. In some embodiments, the hinge domain comprises the amino acid sequence EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO:50). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO:50. In some embodiments, the IgG1 hinge comprises or consists of SEQ ID NO:50. In some embodiments, the hinge domain comprises the amino acid sequence EPKCCVECPPCPAPPAAA (SEQ ID NO:51). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO:51. In some embodiments, the IgG2 hinge comprises or consists of SEQ ID NO:51. In some embodiments, the hinge domain comprises the amino acid sequence EPKCCVECPPCPAPPVAGP (SEQ ID NO:84). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO:84. In some embodiments, the IgG2 hinge comprises or consists of SEQ ID NO:84. In some embodiments, the hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFLGG (SEQ ID NO:52). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO:52. In some embodiments, the IgG4 hinge comprises or consists of SEQ ID NO:52. In some embodiments, the hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFEGG (SEQ ID NO:85). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO:85. In some embodiments, the IgG4 hinge comprises or consists of SEQ ID NO:85.In some embodiments, the hinge domain comprises the amino acid sequence ESKYGPPCPSCPAPEFLGG (SEQ ID NO: 86). SEQ ID NO: 85 comprises the S228P and L235E mutations known in the art. SEQ ID NO: 85 is SEQ ID NO: 86 with the mutations. These mutations are known to reduce aggregation, increase stability, and reduce effector function. In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 86. In some embodiments, the IgG4 hinge comprises or consists of SEQ ID NO: 86. In some embodiments, the hinge domain comprises the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGP (SEQ ID NO: 53). In some embodiments, the hinge domain consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the IgG3 hinge comprises or consists of SEQ ID NO: 53. In some embodiments, the hinge domain comprises the CPXCP (SEQ ID NO: 54) motif. In some embodiments, SEQ ID NO:54 is the hinge domain core. In some embodiments, X of SEQ ID NO:54 is selected from P and R. In some embodiments, SEQ ID NO:54 is CPPCP (SEQ ID NO:55). In some embodiments, the IgG1 core consists of SEQ ID NO:55. In some embodiments, the IgG2 core comprises SEQ ID NO:55. In some embodiments, the IgG2 core consists of CCVECPPCP (SEQ ID NO:87). In some embodiments, the IgG4 core comprises or consists of SEQ ID NO:55. In some embodiments, SEQ ID NO:54 is CPRCP (SEQ ID NO:56). In some embodiments, the IgG3 core comprises or consists of SEQ ID NO:56. In some embodiments, SEQ ID NO:54 is CPSCP (SEQ ID NO:88). In some embodiments, the IgG4 core comprises or consists of SEQ ID NO:88. It will thus be understood that the hinge domain cysteines found in SEQ ID NO:54 are necessary for disulfide bonding and dimerization.

[0199] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSDKTHTCPPCPAPEL (SEQ ID NO: 114). In some embodiments, the first polypeptide consists of SEQ ID NO: 114. In some embodiments, the second polypeptide comprises SEQ ID NO: 114. In some embodiments, the second polypeptide consists of SEQ ID NO: 114. In some embodiments, the agent comprises a dimer of SEQ ID NO: 114. In some embodiments, the agent consists of a dimer of SEQ ID NO: 114. In some embodiments, the first polypeptide, the second polypeptide, or both, comprise or consist of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 114. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 114. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence having a homolog comprises the CDRs of SEQ ID NO: 40.

[0200] In some embodiments, an agent comprising SEQ ID NO: 114 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO: 114 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0201] In some embodiments, the dimerization domain further comprises a CH2 domain of an Ig. In some embodiments, the CH2 domain is of an Ig heavy chain. In some embodiments, the dimerization domain further comprises a CH3 domain of an Ig. In some embodiments, the CH3 domain is of an Ig heavy chain. In some embodiments, the first dimerization domain comprises a CH2 domain, a CH3 domain, or both. In some embodiments, the second dimerization domain comprises a CH2 domain, a CH3 domain, or both. In some embodiments, the Ig is an IgG. In some embodiments, the IgG is an IgG1. In some embodiments, the Ig is an IgG2. In some embodiments, the IgG is an IgG4. In some embodiments, the IgG is an IgG3. In some embodiments, the IgG1 is a modified IgG1. In some embodiments, the IgG3 is a modified IgG3. In some embodiments, the modification is modified to reduce effector function. In some embodiments, the modification is modified to abolish effector function. In some embodiments, the modification is a PG-LALA modification.

[0202] In some embodiments, the dimerization domain comprises an Fc domain. In some embodiments, the dimerization domain consists of an Fc domain. In some embodiments, the Fc domain comprises a hinge, a CH2 and a CH3 domain. In some embodiments, the Fc domain consists of a hinge, a CH2 and a CH3 domain. In some embodiments, the agent comprising an Fc domain is not a CD28 antagonist.

[0203] In some embodiments, the CH2 domain comprises the amino acid sequence LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 57). In some embodiments, the CH2 domain consists of SEQ ID NO: 57. In some embodiments, SEQ ID NO: 57 is an IgG1 CH2 domain. In some embodiments, the CH2 domain comprises the amino acid sequence PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 58). In some embodiments, the CH2 domain consists of SEQ ID NO: 58. In some embodiments, SEQ ID NO: 58 is an IgG1 CH2 domain. In some embodiments, the CH2 domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:57. Each possibility represents a separate embodiment of the present invention. In some embodiments, the CH2 domain comprises or consists of a dimer of a polypeptide sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:58. Each possibility represents a separate embodiment of the present invention. In some embodiments, the CH2 domain comprises the amino acid sequence PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO:89). In some embodiments, the CH2 domain consists of SEQ ID NO:89. In some embodiments, SEQ ID NO:89 is an IgG4 CH2 domain. In some embodiments, the CH2 domain consists of SEQ ID NO: 89. In some embodiments, SEQ ID NO: 89 is an IgG4 CH2 domain.In some embodiments, the CH2 domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 89. Each possibility represents a separate embodiment of the present invention. In some embodiments, the CH2 domain comprises or consists of a dimer of a polypeptide sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 89. Each possibility represents a separate embodiment of the present invention.

[0204] In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:59). In some embodiments, the CH3 domain consists of SEQ ID NO:59. In some embodiments, SEQ ID NO:59 is an IgG1 CH3 domain. In some embodiments, the CH3 domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:59. Each possibility represents a separate embodiment of the present invention. In some embodiments, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 90). In some embodiments, the CH3 domain consists of SEQ ID NO: 90. In some embodiments, SEQ ID NO: 90 is an IgG4 CH3 domain. In some embodiments, the CH3 domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 90. Each possibility represents a separate embodiment of the present invention.

[0205] In some embodiments, the Fc domain comprises DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 60). In some embodiments, the Fc domain consists of SEQ ID NO: 60. In some embodiments, the Fc domain of an IgG1 comprises or consists of SEQ ID NO: 60. In some embodiments, the Fc domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 60. Each possibility represents a separate embodiment of the present invention. In some embodiments, the Fc domain comprises ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 91). In some embodiments, the Fc domain consists of SEQ ID NO: 91. In some embodiments, the IgG4 Fc domain comprises or consists of SEQ ID NO: 91. In some embodiments, the Fc domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:91. Each possibility represents a separate embodiment of the invention. In some embodiments, SEQ ID NO:91 comprises an S10 to P mutation. In some embodiments, SEQ ID NO:91 comprises an L17 to E mutation.

[0206] In some embodiments, the dimerization domain does not induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the dimerization domain does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the dimerization domain is configured not to induce ADCC or CDC. In some embodiments, the dimerization domain is configured to reduce ADCC or CDC. In some embodiments, the dimerization domain has no effector function. In some embodiments, the dimerization domain comprises reduced effector function. In some embodiments, the dimerization domain comprises at least one mutation that reduces or abolishes effector function. In some embodiments, the dimerization domain comprises at least one mutation that reduces ADCC or CDC. In some embodiments, the dimerization domain comprises at least one mutation that reduces effector function. In some embodiments, the reduced CDC, ADCC or effector function comprises at least one mutation that reduces CDC, ADC or effector function.

[0207] It will be known to those skilled in the art that IgG2 and IgG4 have greatly reduced effector functions and are generally not cytotoxic in nature. Furthermore, mutations such as S228P and L235E in IgG4 are known to further reduce effector functions. Furthermore, mutations that reduce cytotoxicity / effector functions of IgG1 and IgG3 are well known in the art. In some embodiments, the IgG comprises at least one mutation. In some embodiments, the mutation is multiple mutations. In some embodiments, the mutation reduces cytotoxicity. In some embodiments, the mutation improves stability. In some embodiments, the mutation reduces aggregation. In some embodiments, the multiple mutations that reduce cytotoxicity comprise PG-LALA mutations. In some embodiments, the mutation is a proline 329 to glycine mutation of the IgG1 human heavy chain (P329G). In some embodiments, the P to G mutation is a P109 to G mutation of SEQ ID NO: 60. In some embodiments, the mutation is a leucine 234 to alanine mutation of the IgG1 human heavy chain (L234A). In some embodiments, the L to A mutation is a mutation of L14 to A in SEQ ID NO: 60. In some embodiments, the mutation is a mutation of Leucine 235 to Alanine in IgG1 human heavy chain (L235A). In some embodiments, the L to A mutation is a mutation of L15 to A in SEQ ID NO: 60. In some embodiments, the multiple mutations include P109G, L14A and L15A in SEQ ID NO: 60. In some embodiments, the multiple mutations include P329G, L234A and L235A in IgG1 human heavy chain. It will also be understood by those skilled in the art that parallel mutations may be performed in an IgG3 heavy chain or a non-human IgG1 heavy chain. In some embodiments, the mutation is a mutation of Leucine 235 to Glutamic Acid in IgG4 human heavy chain (L235E). In some embodiments, the mutation is a mutation of Serine 228 to Proline in IgG4 human heavy chain (S228P). It will be understood that the numbers given herein relate to full length IgG including the variable domains. The numbers can be shifted to correspond to these amino acid positions only within the Fc portion of IgG.

[0208] In some embodiments, the dimerization domain comprising reduced cytotoxicity and / or effector function comprises DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39). In some embodiments, the dimerization domain comprises SEQ ID NO: 39. In some embodiments, the dimerization domain consists of SEQ ID NO: 39. In some embodiments, the dimerization domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 39. Each possibility represents a separate embodiment of the invention. In some embodiments, sequences with homologs include a P to G mutation, an L to A mutation, and an L to A mutation. In some embodiments, the dimerization domain comprising reduced cytotoxicity and / or effector function comprises ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 92). In some embodiments, the Fc domain consists of SEQ ID NO: 92. In some embodiments, the IgG4 Fc domain comprises or consists of SEQ ID NO: 92. In some embodiments, the Fc domain comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:92.Each possibility represents a separate embodiment of the present invention. In some embodiments, the homologous sequence contains a P at position 10. In some embodiments, the homologous sequence contains an E at position 17.

[0209] In some embodiments, the sdAb and the dimerization domain are separated by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a peptide bond. In some embodiments, the first sdAb and the first dimerization domain are separated by a linker. In some embodiments, the second sdAb and the second dimerization domain are separated by a linker. In some embodiments, both polypeptides include a linker. In some embodiments, either includes a linker.

[0210] In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a hydrophilic linker. In some embodiments, the linker comprises the amino acid sequence GGGGS. In some embodiments, the linker comprises the amino acid sequence GS. In some embodiments, the linker comprises the amino acid sequence (GGGGS)n, where n is an integer. In some embodiments, the linker comprises the amino acid sequence AAA(GGGGS)n, where n is an integer. In some embodiments, the linker comprises the amino acid sequence (GS)n, where n is an integer. In some embodiments, the linker comprises the amino acid sequence (GGS)n, where n is an integer. In some embodiments, the linker comprises the amino acid sequence (GSGGS)n, where n is an integer. In some embodiments, the linker comprises the amino acid sequence (EGGGS)n, where n is an integer. In some embodiments, the linker comprises the amino acid sequence (EGGS)n, where n is an integer. In some embodiments, n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each possibility represents a separate embodiment of the present invention. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 7.

[0211] In some embodiments, n is 8. In some embodiments, the linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises at least 1 amino acid. In some embodiments, the linker comprises at least 5 amino acids. In some embodiments, the linker comprises at least 8 amino acids. In some embodiments, the linker comprises at least 10 amino acids. In some embodiments, the linker comprises at least 13 amino acids. In some embodiments, the linker comprises at least 15 amino acids. In some embodiments, the linker comprises at least 18 amino acids. In some embodiments, the linker comprises at least 25 amino acids. In some embodiments, the linker comprises at least 35 amino acids. In some embodiments, the linker comprises up to 25, 28, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, or 100 amino acids. Each possibility represents a separate embodiment of the present invention. In some embodiments, the linker comprises up to 25 amino acids. In some embodiments, the linker comprises up to 28 amino acids. In some embodiments, the linker comprises up to 35 amino acids. In some embodiments, the linker comprises up to 50 amino acids. In some embodiments, the linker comprises 1-50, 1-28, 1-25, 1-18, 1-15, 1-13, 1-10, 5-50, 5-28, 5-25, 5-18, 5-15, 5-13, 5-10, 10-50, 10-28, 10-25, 10-18, 10-15, 10-13, 15-50, 15-28, 15-25, 15-18, 25-50, and 28-50 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises 15-25 amino acids. In some embodiments, the linker comprises 15-35 amino acids. In some embodiments, the linker comprises 25-35 amino acids.In some embodiments, the linker comprises 18-28 amino acids. In some embodiments, the linker comprises 10-25 amino acids. In some embodiments, the linker comprises 13-28 amino acids. In some embodiments, the linker comprises 10-20 amino acids. In some embodiments, the linker comprises 13-23 amino acids. It will be appreciated by those skilled in the art that in addition to increasing the number of repeats in the linker, the N-terminus and C-terminus may also include additional bases, such as additional G and / or As.

[0212] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116). In some embodiments, the first polypeptide consists of SEQ ID NO: 116. In some embodiments, the second polypeptide comprises SEQ ID NO: 116. In some embodiments, the second polypeptide consists of SEQ ID NO: 116. In some embodiments, the agent comprises a dimer of SEQ ID NO: 116. In some embodiments, the agent consists of a dimer of SEQ ID NO: 116. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 116. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 116. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 1. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:116.

[0213] In some embodiments, an agent comprising SEQ ID NO: 116 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO: 116 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0214] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 117). In some embodiments, the first polypeptide consists of SEQ ID NO: 117. In some embodiments, the second polypeptide comprises SEQ ID NO: 117. In some embodiments, the second polypeptide consists of SEQ ID NO: 117. In some embodiments, the agent comprises a dimer of SEQ ID NO: 117. In some embodiments, the agent consists of a dimer of SEQ ID NO: 117. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 117. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 117. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 1. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G254, A159, and A160 of SEQ ID NO:117.

[0215] In some embodiments, an agent comprising SEQ ID NO: 117 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO: 117 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0216] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 115). In some embodiments, the first polypeptide consists of SEQ ID NO: 115. In some embodiments, the second polypeptide comprises SEQ ID NO: 115. In some embodiments, the second polypeptide consists of SEQ ID NO: 115. In some embodiments, the agent comprises a dimer of SEQ ID NO: 115. In some embodiments, the agent consists of a dimer of SEQ ID NO: 115. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 115. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 115. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 1. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G1156, A131, and A132 of SEQ ID NO:115.

[0217] In some embodiments, an agent comprising SEQ ID NO: 115 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO: 115 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0218] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAINEKLLIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 61). In some embodiments, the first polypeptide consists of SEQ ID NO:61. In some embodiments, the second polypeptide comprises SEQ ID NO:61. In some embodiments, the second polypeptide consists of SEQ ID NO:61. In some embodiments, the agent comprises a dimer of SEQ ID NO:61. In some embodiments, the agent consists of a dimer of SEQ ID NO:61. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:61. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:61. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:26. In some embodiments, the homologous sequences include the P to G, L to A, and L to A mutations in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:61.

[0219] In some embodiments, an agent comprising SEQ ID NO:61 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:61 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0220] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMIEQQWWYWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62). In some embodiments, the first polypeptide consists of SEQ ID NO:62. In some embodiments, the second polypeptide comprises SEQ ID NO:62. In some embodiments, the second polypeptide consists of SEQ ID NO:62. In some embodiments, the agent comprises a dimer of SEQ ID NO:62. In some embodiments, the agent consists of a dimer of SEQ ID NO:62. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:62. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:62. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:27. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:62.

[0221] In some embodiments, an agent comprising SEQ ID NO:62 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:62 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0222] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDTHRGVYWYWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 63). In some embodiments, the first polypeptide consists of SEQ ID NO:63. In some embodiments, the second polypeptide comprises SEQ ID NO:63. In some embodiments, the second polypeptide consists of SEQ ID NO:63. In some embodiments, the agent comprises a dimer of SEQ ID NO:63. In some embodiments, the agent consists of a dimer of SEQ ID NO:63. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:63. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:63. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:28. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:63.

[0223] In some embodiments, an agent comprising SEQ ID NO:63 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:63 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0224] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVAAINYIKEIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 64). In some embodiments, the first polypeptide consists of SEQ ID NO:64. In some embodiments, the second polypeptide comprises SEQ ID NO:64. In some embodiments, the second polypeptide consists of SEQ ID NO:64. In some embodiments, the agent comprises a dimer of SEQ ID NO:64. In some embodiments, the agent consists of a dimer of SEQ ID NO:64. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:64. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:64. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:29. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:64.

[0225] In some embodiments, an agent comprising SEQ ID NO:64 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:64 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0226] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINSMAWYRQAPGSQRELVAAISNAREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVYFQEYWYWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 65). In some embodiments, the first polypeptide consists of SEQ ID NO:65. In some embodiments, the second polypeptide comprises SEQ ID NO:65. In some embodiments, the second polypeptide consists of SEQ ID NO:65. In some embodiments, the agent comprises a dimer of SEQ ID NO:65. In some embodiments, the agent consists of a dimer of SEQ ID NO:65. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:65. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:65. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:30. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:65.

[0227] In some embodiments, an agent comprising SEQ ID NO:65 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:65 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0228] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINTMAWYRQAPGSQRELVAAINSISRTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66). In some embodiments, the first polypeptide consists of SEQ ID NO:66. In some embodiments, the second polypeptide comprises SEQ ID NO:66. In some embodiments, the second polypeptide consists of SEQ ID NO:66. In some embodiments, the agent comprises a dimer of SEQ ID NO:66. In some embodiments, the agent consists of a dimer of SEQ ID NO:66. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:66. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:66. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:31. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:66.

[0229] In some embodiments, an agent comprising SEQ ID NO:66 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:66 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0230] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVTAIASDNRKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67). In some embodiments, the first polypeptide consists of SEQ ID NO:67. In some embodiments, the second polypeptide comprises SEQ ID NO:67. In some embodiments, the second polypeptide consists of SEQ ID NO:67. In some embodiments, the agent comprises a dimer of SEQ ID NO:67. In some embodiments, the agent consists of a dimer of SEQ ID NO:67. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:67. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:67. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:32. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:67.

[0231] In some embodiments, an agent comprising SEQ ID NO:67 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:67 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0232] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMYWQDYWWWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 68). In some embodiments, the first polypeptide consists of SEQ ID NO:68. In some embodiments, the second polypeptide comprises SEQ ID NO:68. In some embodiments, the second polypeptide consists of SEQ ID NO:68. In some embodiments, the agent comprises a dimer of SEQ ID NO:68. In some embodiments, the agent consists of a dimer of SEQ ID NO:68. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:68. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:68. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:33. In some embodiments, the homologous sequences include a P to G mutation, an L to A mutation, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:68.

[0233] In some embodiments, an agent comprising SEQ ID NO:68 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:68 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0234] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAISDRSEKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDHHHSDWWTWGQGTQVTVSSAAAGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 69). In some embodiments, the first polypeptide consists of SEQ ID NO:69. In some embodiments, the second polypeptide comprises SEQ ID NO:69. In some embodiments, the second polypeptide consists of SEQ ID NO:69. In some embodiments, the agent comprises a dimer of SEQ ID NO:69. In some embodiments, the agent consists of a dimer of SEQ ID NO:69. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:69. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:69. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:34. In some embodiments, the homologous sequences include a P to G, an L to A, and an L to A mutation in the Fc domain. In some embodiments, the homologous sequences include G244, A149, and A150 of SEQ ID NO:69.

[0235] In some embodiments, an agent comprising SEQ ID NO:69 or a homolog thereof is not a CD28 antagonist. In some embodiments, an agent consisting of a dimer of SEQ ID NO:69 or a homolog thereof is not a CD28 antagonist. In some embodiments, the CD28 antagonist is a substantial antagonist.

[0236] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVTAIASDNRKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 93). In some embodiments, the first polypeptide consists of SEQ ID NO:93. In some embodiments, the second polypeptide comprises SEQ ID NO:93. In some embodiments, the second polypeptide consists of SEQ ID NO:93. In some embodiments, the agent comprises a dimer of SEQ ID NO:93. In some embodiments, the agent consists of a dimer of SEQ ID NO:93. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:93. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:93. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:32. In some embodiments, the homologous sequences include the CDRs of SEQ ID NO: 8, SEQ ID NO: 16, and SEQ ID NO: 10. It will be understood by those skilled in the art that amino acids 1-115 of SEQ ID NO: X can be replaced with any of SEQ ID NOs: 70-74.

[0237] In some embodiments, the first polypeptide comprises EVQLVESGGGLVQAGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMYWQDYWWWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 94). In some embodiments, the first polypeptide consists of SEQ ID NO:94. In some embodiments, the second polypeptide comprises SEQ ID NO:94. In some embodiments, the second polypeptide consists of SEQ ID NO:94. In some embodiments, the agent comprises a dimer of SEQ ID NO:94. In some embodiments, the agent consists of a dimer of SEQ ID NO:93. In some embodiments, the first polypeptide, the second polypeptide, or both, comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:94. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent comprises or consists of a dimer of a polypeptide having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO:94. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO:33. In some embodiments, the homologous sequences include the CDRs of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. It will be understood by those skilled in the art that amino acids 1-115 of SEQ ID NO: X can be replaced with any of SEQ ID NOs: 75-78.

[0238] In some embodiments, the agent does not modulate CD28 function and / or signaling. In some embodiments, the agent does not degrade mCD28. In some embodiments, the agent does not cause or promote mCD28 degradation. In some embodiments, the signaling is mCD28-mediated immune cell activation. In some embodiments, the agent does not inhibit immune cell activation. In some embodiments, the agent does not induce internalization or recycling of the CD28 receptor. Costimulation through mCD28 is essential for immune activation of T cells. Proteolytic cleavage removed the ligand binding domain of the extracellular region of CD28 from the transmembrane and cytoplasmic portions of the protein that remain in the membrane. Thus, cleaved CD28 cannot signal and cannot contribute to T cell activation. Thus, agents that block cleavage and are also antagonists do not activate mCD28. Similarly, agents that block cleavage but are also agonists may induce aberrant T cell activation and potentially autoimmune responses. In some embodiments, the agent is not the anti-CD28 antibody MAB342. In some embodiments, the agent is not the anti-CD28 antibody clone #37407.

[0239] In some embodiments, the agent does not reduce surface levels of mCD28 on immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the agent reduces surface levels of mCD28 by less than 50, 40, 30, 25, 20, 15, 10, 7, 5, 3, 2, or 1%. Each possibility represents a separate embodiment of the present invention.

[0240] In some embodiments, binding of the agent to a cell does not kill the cell. In some embodiments, binding of the agent to a cell does not result in killing of the cell. In some embodiments, the agent does not induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the agent does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the agent does not induce ADCC and / or CDC.

[0241] Single Chain Agents

[0242] In some embodiments, the agent comprises a single polypeptide. In some embodiments, the agent is a single chain agent. In some embodiments, the agent consists of a single polypeptide. In some embodiments, the single polypeptide comprises a first sdAb and a second sdAb. In some embodiments, the first sdAb is N-terminal to the second sdAb. In some embodiments, the second sdAb is N-terminal to the first sdAb. In some embodiments, the first sdAb is C-terminal to the second sdAb. In some embodiments, the second sdAb is C-terminal to the first sdAb. In some embodiments, the N-terminus of the single polypeptide is an sdAb. In some embodiments, the C-terminus of the single polypeptide is an sdAb. In some embodiments, the N-terminal domain, the C-terminal domain or both are sdAbs.

[0243] In some embodiments, the two sdAbs are separated by an amino acid linker. In some embodiments, the first sdAb and the second sdAb are separated by a linker.

[0244] In some embodiments, the linker is a short linker. In some embodiments, the short linker comprises fewer than 10 amino acids. In some embodiments, the short linker comprises fewer than 13 amino acids. In some embodiments, the short linker comprises 10 or fewer amino acids. In some embodiments, the short linker comprises 13 or fewer amino acids. In some embodiments, the short linker comprises up to 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the short linker comprises 5 or fewer amino acids. In some embodiments, the short linker comprises 8 or fewer amino acids. In some embodiments, the short linker comprises up to 5 amino acids. In some embodiments, the short linker comprises up to 8 amino acids. In some embodiments, the short linker comprises up to 9 amino acids. In some embodiments, the short linker comprises up to 12 amino acids. In some embodiments, the short linker is a flexible linker. In some embodiments, the short linker is a GGGGS linker. In some embodiments, the short linker comprises one GGGGS. In some embodiments, the short linker comprises (GGGGS)n, where n is an integer. In some embodiments, the short linker comprises AAAGGGGS. In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, the short linker consists of AAAGGGGS.

[0245] In some embodiments, the single polypeptide comprises the sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGGSEVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 103). In some embodiments, the single polypeptide consists of SEQ ID NO: 103. In some embodiments, the agent comprises SEQ ID NO: 103. In some embodiments, the agent consists of SEQ ID NO: 103. In some embodiments, the single polypeptide or agent comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 103. Each possibility represents a separate embodiment of the invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 40. In some embodiments, the sequence having homology comprises another sdAb of the invention. In some embodiments, the other sdAb of the invention is at position 2A1.

[0246] In some embodiments, single chain agents with short linkers are not CD28 antagonists. In some embodiments, agents comprising SEQ ID NO: 103 are not CD28 antagonists. In some embodiments, agents consisting of SEQ ID NO: 103 are not CD28 antagonists. In some embodiments, the agonist is a substantial antagonist.

[0247] In some embodiments, the linker is a long linker. In some embodiments, the long linker comprises 10 or more amino acids. In some embodiments, the long linker comprises 13 or more amino acids. In some embodiments, the long linker comprises at least 10 amino acids. In some embodiments, the linker comprises at least 10 amino acids. In some embodiments, the linker comprises at least 10 amino acids. In some embodiments, the long linker comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the long linker comprises 15 or more amino acids. In some embodiments, the long linker comprises 18 or more amino acids. In some embodiments, the long linker comprises at least 15 amino acids. In some embodiments, the long linker comprises at least 18 amino acids. In some embodiments, the long linker comprises at least 20 amino acids. In some embodiments, the long linker comprises at least 23 amino acids. In some embodiments, the long linker comprises 20 or more amino acids. In some embodiments, the long linker comprises 23 or more amino acids. In some embodiments, the long linker is a flexible linker. In some embodiments, the long linker comprises (GGGGS)n, where n is an integer. In some embodiments, the long linker comprises AAA(GGGGS)n, where n is an integer. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. Each possibility represents a separate embodiment of the invention. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, the long linker comprises or consists of AAAGGGGSGGGGS (SEQ ID NO: 46). In some embodiments, the long linker comprises or consists of AAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 109).

[0248] In some embodiments, the single polypeptide comprises the sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGGSGGGGSEVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 104). In some embodiments, the single polypeptide consists of SEQ ID NO: 104. In some embodiments, the agent comprises SEQ ID NO: 104. In some embodiments, the agent consists of SEQ ID NO: 104. In some embodiments, the single polypeptide or agent comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 104. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 40. In some embodiments, the sequence having homology comprises an sdAb of the present invention. In some embodiments, the sdAb of the present invention is at position 2A1.

[0249] In some embodiments, the single chain agent with a long linker is a CD28 antagonist. In some embodiments, the agent comprising SEQ ID NO: 104 is a CD28 antagonist. In some embodiments, the agent consisting of SEQ ID NO: 104 is a CD28 antagonist. In some embodiments, the agonist is a substantial antagonist.

[0250] In some embodiments, the single polypeptide comprises the sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 105). In some embodiments, the single polypeptide consists of SEQ ID NO: 105. In some embodiments, the agent comprises SEQ ID NO: 105. In some embodiments, the agent consists of SEQ ID NO: 105. In some embodiments, the single polypeptide or agent comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 105. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 40. In some embodiments, the sequence having homology comprises an sdAb of the present invention. In some embodiments, the sdAb of the present invention is at position 2A1.

[0251] In some embodiments, the agent comprising SEQ ID NO: 105 is a CD28 antagonist. In some embodiments, the agent consisting of SEQ ID NO: 105 is a CD28 antagonist. In some embodiments, the agonist is a substantial antagonist.

[0252] In some embodiments, the linker comprises a net neutral charge. In some embodiments, the linker consists of alanine (A), glycine (G) and serine (S) residues. In some embodiments, the linker comprises only alanine, glycine and serine residues. In some embodiments, the linker lacks charged amino acids.

[0253] In some embodiments, the linker is a charged linker. In some embodiments, the linker comprises a net charge. In some embodiments, the charged linker comprises a net positive charge. In some embodiments, the charged linker comprises a net positive charge. In some embodiments, the positively charged linker comprises at least one positively charged amino acid. In some embodiments, the positively charged amino acid is lysine (K), arginine (R) or histidine (H). In some embodiments, the charged linker comprises a net negative charge. In some embodiments, the negatively charged linker comprises at least one negatively charged amino acid. In some embodiments, the negatively charged amino acid is glutamic acid (E) or aspartic acid (D). In some embodiments, the charged linker comprises at least one charged amino acid. In some embodiments, the charged amino acid is K, R, H, E or D. In some embodiments, the charged linker comprises (GGGXS)n, where n is an integer. In some embodiments, the charged linker comprises AAA(GGGXS)n, where n is an integer. In some embodiments, X is a charged amino acid. In some embodiments, X is a positively charged amino acid. In some embodiments, X is a negatively charged amino acid. In some embodiments, X is K. In some embodiments, X is E. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. Each possibility represents a separate embodiment of the invention. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, the charged linker comprises or consists of AAAGGGGKSGGGKSGGGKSGGGKS (SEQ ID NO: 110). In some embodiments, the long linker comprises or consists of AAAGGGESGGGESGGGESGGGES (SEQ ID NO: 111).

[0254] In some embodiments, the single polypeptide comprises the sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGKSGGGKSGGGKSGGGKSEVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 106). In some embodiments, the single polypeptide consists of SEQ ID NO: 106. In some embodiments, the agent comprises SEQ ID NO: 106. In some embodiments, the agent consists of SEQ ID NO: 106. In some embodiments, the single polypeptide or agent comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 106. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 40. In some embodiments, the sequence having homology comprises an sdAb of the present invention. In some embodiments, the sdAb of the present invention is at position 2A1.

[0255] In some embodiments, the single chain agent with a charged linker is a CD28 antagonist. In some embodiments, the single chain agent with a positively charged linker is a CD28 antagonist. In some embodiments, the agent comprising SEQ ID NO: 106 is a CD28 antagonist. In some embodiments, the agent consisting of SEQ ID NO: 106 is a CD28 antagonist. In some embodiments, the agonist is a substantial antagonist.

[0256] In some embodiments, the single polypeptide comprises the sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGESGGGESGGGESGGGESEVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 107). In some embodiments, the single polypeptide consists of SEQ ID NO: 107. In some embodiments, the agent comprises SEQ ID NO: 107. In some embodiments, the agent consists of SEQ ID NO: 107. In some embodiments, the single polypeptide or agent comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 107. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sequence having homology comprises the CDRs of SEQ ID NO: 40. In some embodiments, the sequence having homology comprises an sdAb of the present invention. In some embodiments, the sdAb of the present invention is at position 2A1.

[0257] In some embodiments, the single chain agent with a negatively charged linker is a CD28 antagonist. In some embodiments, the agent comprising SEQ ID NO: 107 is a CD28 antagonist. In some embodiments, the agent consisting of SEQ ID NO: 107 is a CD28 antagonist. In some embodiments, the agonist is a substantial antagonist.

[0258] In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a helical linker. In some embodiments, the rigid linker is a helical linker. In some embodiments, the rigid linker is a long linkage. In some embodiments, the rigid linker comprises (EAAAK)n, where n is an integer. In some embodiments, the rigid linker comprises or consists of GGGGSAEAAAKEAAAKEAAAKAAAGSGGGGS (SEQ ID NO: 97). In some embodiments, the rigid linker comprises or consists of AAAGGGGSAEAAAKEAAAKEAAAKAAAGSGGGGS (SEQ ID NO: 112).

[0259] In some embodiments, the single polypeptide comprises the sequence EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSSAAAGGGGSAEAAAKEAAAKEAAAKAAAGSGGGGSEEVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 108). In some embodiments, the single polypeptide consists of SEQ ID NO: 108. In some embodiments, the agent comprises SEQ ID NO: 108. In some embodiments, the agent consists of SEQ ID NO: 108. In some embodiments, the single polypeptide or agent comprises or consists of a sequence having at least 70, 75, 80, 85, 90, 93, 95, 97, or 99% homology to SEQ ID NO: 108. Each possibility represents a separate embodiment of the invention. In some embodiments, the homologous sequence comprises the CDRs of SEQ ID NO: 40.

[0260] In some embodiments, the single chain agent with a rigid linker is a CD28 antagonist. In some embodiments, the agent comprising SEQ ID NO: 108 is a CD28 antagonist. In some embodiments, the agent consisting of SEQ ID NO: 108 is a CD28 antagonist. In some embodiments, the agonist is a substantial antagonist.

[0261] nucleic acid molecule

[0262] According to another aspect there is provided a nucleic acid molecule encoding an sdAb of the invention.

[0263] According to another aspect, there is provided a nucleic acid molecule encoding an agent of the invention.

[0264] In some embodiments, the nucleic acid molecule is a plurality of nucleic acid molecules. In some embodiments, a first nucleic acid molecule encodes a first polypeptide. In some embodiments, a second nucleic acid molecule encodes a second polypeptide. In some embodiments, a single nucleic acid molecule encodes both the first and second polypeptides.

[0265] In some embodiments, the nucleic acid molecule comprises a coding region encoding an sdAb of the invention. In some embodiments, the nucleic acid molecule comprises a coding region encoding an agent of the invention. In some embodiments, encoding the agent comprises encoding a first polypeptide. In some embodiments, encoding the agent comprises encoding a second polypeptide. In some embodiments, the nucleic acid molecule encodes a first and a second polypeptide. In some embodiments, the first and second polypeptides are identical and the nucleic acid molecule comprises a single coding region encoding the polypeptides. In some embodiments, the nucleic acid molecule is a plurality of nucleic acid molecules. In some embodiments, the plurality comprises a first molecule encoding the first polypeptide and a second molecule encoding the second polypeptide.

[0266] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a mammalian expression vector. In some embodiments, the mammal is human. In some embodiments, the vector is for expression in human cells. In some embodiments, the vector is for expression in culture. In some embodiments, the vector is for in vitro expression. In some embodiments, the vector is for in vivo expression. Expression of a nucleic acid molecule encoding an agent in a cell is well known to those of skill in the art. It can be done by transfection, viral infection, or direct modification of the genome of the cell, among many other methods.

[0267] Vector nucleic acid sequences generally contain at least one origin of replication for propagation within a cell, and optionally additional elements, such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), selection markers (e.g., antibiotic resistance), polyadenine sequences.

[0268] The vector can be a DNA plasmid delivered via non-viral or viral methods.The viral vector can be a retroviral vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector or a pox virus vector.The promoter can be active in mammalian cells.The promoter can be a viral promoter.

[0269] In some embodiments, the nucleic acid sequence encoding the agent is operably linked to a promoter. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, the promoter is a mammalian promoter. In some embodiments, the promoter is configured for expression in a target cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the mammal is a human.

[0270] In some embodiments, vectors are introduced into cells by standard methods, including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with viral vectors, high velocity ballistic penetration with small particles that contain the nucleic acid either within the matrix of small beads or particles or on their surface (Klein et al., Nature 327.70-73 (1987)), and the like.

[0271] As used herein, the term "promoter" refers to a group of transcriptional control modules centered around the initiation site of RNA polymerase, i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and contain one or more recognition sites for transcriptional activator or repressor proteins.

[0272] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize the precursors of mRNA and most snRNAs and microRNAs.

[0273] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0274] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0275] In some embodiments, recombinant viral vectors are used for in vivo expression, which offer advantages such as lateral infection and target specificity. In one embodiment, lateral infection is inherent in, for example, the life cycle of retroviruses, a process in which a single infected cell generates many progeny virions that bud and infect neighboring cells. In one embodiment, this results in the rapid infection of a large area, most of which were not initially infected by the original viral particle. In one embodiment, a viral vector is created that cannot spread laterally. In one embodiment, this feature can be useful when the desired goal is to introduce a specific gene only into a localized number of target cells.

[0276] A variety of methods can be used to introduce the expression vectors of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988); and Gilboa et al. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. Additionally, see US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0277] It will be understood that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression construct of the present invention may also contain sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0278] composition

[0279] According to another aspect there is provided a composition comprising an sdAb of the invention.

[0280] Another aspect provides a composition comprising an agent of the invention.

[0281] Another aspect provides a composition comprising a nucleic acid molecule of the invention.

[0282] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition includes a therapeutically acceptable carrier, excipient, or adjuvant.

[0283] As used herein, the term "carrier", "excipient" or "adjuvant" refers to any component of a pharmaceutical composition that is not an active agent.As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium, such as physiological saline. Some examples of materials which may function as pharma- ceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate, ethyl laurate, agar; buffers such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffers, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can function as carriers herein include sugar, starch, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible materials used in other pharmaceutical preparations.Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present.Any non-toxic, inert and effective carrier can be used to formulate the compositions contemplated herein.In this regard, suitable pharma- ceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, and are described, for example, in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck&Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and "Inactive Ingredient Guide", US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (all of which are incorporated herein by reference in their entirety).Examples of pharma-ceutically acceptable carriers, carriers and diluents useful in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety.The compositions described herein may also be included in artificially created structures such as liposomes, ISCOMS, sustained release particles, and other vehicles that extend the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is generally determined by considerations such as liposome size and stability in blood. A variety of methods are available for preparing liposomes, as reviewed, for example, in Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0284] The carriers may in total constitute from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0285] In some embodiments, the composition comprises a therapeutically effective amount of sdAb. In some embodiments, the composition comprises a therapeutically effective amount of a drug. In some embodiments, the composition comprises a therapeutically effective amount of a nucleic acid molecule. As used herein, the term "therapeutically effective amount" refers to an amount of drug effective to treat a disease or disorder in a mammal. The term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen will be determined by a physician depending on the patient's condition.

[0286] In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for local administration. In some embodiments, the local administration is to a site of inflammation. In some embodiments, the local administration is intratumoral administration. In some embodiments, the local administration is to a site of an immune response. In some embodiments, the immune response is an autoimmune response.

[0287] As used herein, the terms "administer", "administration" and the like refer to any method of delivering a composition containing an active agent to a subject in a manner that produces a therapeutic effect in sound medical practice. One aspect of the subject matter provides intravenous administration of a therapeutically effective amount of an agent of the present invention to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular or intraperitoneal.

[0288] Treatment method

[0289] According to another aspect there is provided a method of reducing sCD28 levels in a subject in need thereof, comprising administering to the subject an sdAb of the invention, an agent of the invention or a pharmaceutical composition of the invention, thereby reducing sCD28 levels.

[0290] According to another aspect, there is provided a method of reducing CD28 cleavage on the surface of a cell comprising contacting the cell with an sdAb of the invention, an agent of the invention or a pharmaceutical composition of the invention, thereby reducing sCD28 levels.

[0291] According to another aspect, there is provided a method of treating and / or preventing a disease in a subject in need thereof, the method comprising administering an sdAb of the invention, an agent of the invention or a pharmaceutical composition of the invention to the subject, thereby treating and / or preventing the disease.

[0292] According to another aspect there is provided a method of improving immunotherapy in a subject in need thereof comprising administering an sdAb of the invention, an agent of the invention or a pharmaceutical composition of the invention to the subject, thereby improving immunotherapy.

[0293] The ability to reduce sCD28, and in particular block CD28 cleavage, has been demonstrated to be an effective treatment for cancer, a method for increasing immune stimulation, and an effective boost for immunotherapy, as demonstrated in International Patent Application Publication Nos. 2019 / 175885 and 2020111441, the entireties of which are incorporated herein by reference.

[0294] In some embodiments, the immunotherapy is a PD-1 and / or PD-L1 based cancer immunotherapy. In some embodiments, the PD-1 / PD-L1 based immunotherapy comprises administering an anti-PD1 or anti-PD-L1 antibody. In some embodiments, the treatment comprises blockade of the PD-1 checkpoint. In some embodiments, the immunotherapy comprises administering allogeneic, syngenic or autologous immune cells to the subject. In some embodiments, the immune cells are T cells. In some embodiments, the subject in need of immunotherapy is afflicted with cancer.

[0295] As used herein, the term "treatment" or "treating" of a disease, disorder, or condition includes alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a useful composition herein only needs to reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or improve the quality of life of the patient or subject.

[0296] In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject is suffering from a disease. In some embodiments, the subject is in need thereof. In some embodiments, the subject is in need of immunotherapy. In some embodiments, the subject is being treated with immunotherapy.

[0297] In some embodiments, the subject's blood contains elevated levels of sCD28. In some embodiments, the subject's blood contains elevated levels of sCD28 that subsequently decline. In some embodiments, the levels are higher than that of a healthy subject. In some embodiments, the subject's sCD28 levels are at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% elevated above that of a healthy subject. Each possibility represents a separate embodiment of the present invention. In some embodiments, levels are elevated to greater than 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng per ml of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, levels are elevated to greater than 5 ng / ml. In some embodiments, levels are elevated to greater than 10 ng / ml. In some embodiments, levels are elevated to greater than 20 ng / ml. In some embodiments, the subject's blood comprises at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng of sCD28 per ml of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the subject's blood contains at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng of sCD28 per ml of blood prior to depletion. Each possibility represents a separate embodiment of the present invention. In some embodiments, the subject's blood contains at least 5 ng of sCD28 per ml. In some embodiments, the subject's blood contains at least 10 ng of sCD28 per ml. In some embodiments, the subject's blood contains at least 20 ng of sCD28 per ml. In some embodiments, the subject's blood contains at least 5 ng of sCD28 per ml prior to depletion. In some embodiments, the subject's blood contains at least 10 ng of sCD28 per ml prior to depletion.In some embodiments, the subject's blood contains at least 20 ng of sCD28 per ml before being depleted.

[0298] In some embodiments, the subject comprises an increased level of sCD28. In some embodiments, the increased level is an increased level in blood. In some embodiments, the increase is from a first time point to a second time point. In some embodiments, the increase is from before to after treatment with immunotherapy. In some embodiments, the increase increases as the disease progresses. International Patent Application Publication No. 2021 / 111442 (the entire contents of which are incorporated herein by reference) shows that sCD28 levels can increase during cancer progression and immunotherapy, and that cleavage blockers can be used to treat such cancers.

[0299] In some embodiments, the subject is afflicted with cancer. In some embodiments, the cancer may be treated by immunotherapy. In some embodiments, the cancer is a cancer that may be treated with PD-1 / PD-L1 therapy. In some embodiments, the subject has undergone PD-1 / PD-L1 therapy. In some embodiments, the subject is non-responsive to PD-1 / PD-L1 therapy. In some embodiments, the subject is naïve to PD-1 / PD-L1 therapy. In some embodiments, the methods of the invention are performed in conjunction with PD-1 / PD-L1 therapy. In some embodiments, the methods of the invention are performed prior to PD-1 / PD-L1 therapy.

[0300] In some embodiments, the method further comprises administering another immunotherapy to the subject. In some embodiments, the method further comprises administering a PD-1 and / or PD-L1 based immunotherapy. In some embodiments, the another immunotherapy is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1 and / or PD-L1 inhibitor. In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the another immunotherapy is a chimeric antigen receptor (CAR) based immunotherapy. In some embodiments, the CAR is a CAR-T. In some embodiments, the CAR is a CAR-NK. In some embodiments, the another immunotherapy is a cancer vaccine.

[0301] As used herein, the terms "CAR-T cells" and "CAR-NK cells" refer to engineered receptors that have specificity for at least one protein of interest (e.g., an immunogenic protein whose expression is increased following treatment with an epigenetic modifier) ​​and are grafted onto immune effector cells (T cells or NK cells). In some embodiments, the CAR-T cells have the specificity of a monoclonal antibody grafted onto a T cell. In some embodiments, the CAR-NK cells have the specificity of a monoclonal antibody grafted onto a NK cell. In some embodiments, the T cells are selected from cytotoxic T lymphocytes and regulatory T cells.

[0302] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target and are cytotoxic to the protein that the receptor binds. In some embodiments, CAR-T or CAR-NK cells target at least one viral protein. In some embodiments, CAR-T or CAR-NK cells target multiple viral proteins. In some embodiments, CAR-T or CAR-NK cells target viral proteins that are increased in expression by contact with epigenetic modifiers.

[0303] The construction of CAR-T cells is well known in the art. In one non-limiting example, monoclonal antibodies against viral proteins can be made, and then vectors encoding the antibodies can be constructed. The vectors will also include a costimulatory signal region. In some embodiments, the costimulatory signal region includes the intracellular domain of a known T cell or NK cell stimulatory molecule. In some embodiments, the intracellular domain is selected from at least one of CD3Z, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. In some embodiments, the vector also includes a CD3Z signaling domain. This vector is then transfected into T cells, for example, by lentiviral infection.

[0304] In some embodiments, the cancer is a cancer associated with elevated sCD28 levels. In some embodiments, the cancer is present in a subject with elevated sCD28 levels. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, the cancer is present in a subject with high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are at and / or above 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 ng / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% and / or more than that of a healthy subject. Each possibility represents a separate embodiment of the invention. In some embodiments, the cancer is not breast cancer. In some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, and colorectal cancer. In some embodiments, the cancer is melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, or colorectal cancer. Each possibility represents a separate embodiment of the present invention. Examples of cancer include, but are not limited to, brain cancer, oral cancer, head and neck cancer, esophageal cancer, lung cancer, skin cancer, liver cancer, pancreatic cancer, bladder cancer, kidney cancer, blood cancer, bladder cancer, bone cancer, breast cancer, thyroid cancer, cervical cancer, ovarian cancer, testicular cancer, retinoblastoma, gastric cancer, colorectal cancer, and uterine cancer.

[0305] In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is cancer. In some embodiments, the disease is treatable by immune stimulation. In some embodiments, the disease comprises elevated sCD28 levels. In some embodiments, the disease comprises increasing levels of sCD28. In some embodiments, the increase increases over time. In some embodiments, the increase increases during the progression of the disease. In some embodiments, the disease is an infectious disease. In some embodiments, the disease is a disease treatable by immunotherapy.

[0306] In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the reduction is performed in vivo. In some embodiments, the reduction is performed in vitro. In some embodiments, the reduction includes removing blood from the subject, reducing sCD28 levels in the removed blood, and returning the blood to the subject, thereby reducing sCD28 in the subject. Methods of dialysis and blood purification are well known. The present invention can be practiced by purging sCD28 in vitro and then returning the blood to the subject.

[0307] In some embodiments, the agent reduces sCD28 levels by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, or 99%. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent reduces sCD28 levels to the level of a healthy individual. In some embodiments, the agent reduces sCD28 levels by up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ng / ml. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent reduces sCD28 blood levels by up to 5 ng / ml. In some embodiments, the agent reduces sCD28 blood levels by up to 10 ng / ml. In some embodiments, the agent reduces sCD28 blood levels to a maximum of 20 ng / ml. In some embodiments, the agent reduces sCD28 levels to the level of a healthy individual. In some embodiments, the agent reduces sCD28 levels to less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ng / ml. Each possibility represents a separate embodiment of the invention. In some embodiments, the agent reduces sCD28 levels to less than 5 ng / ml. In some embodiments, the agent reduces sCD28 levels to less than 10 ng / ml. In some embodiments, the agent reduces sCD28 levels to less than 20 ng / ml. In some embodiments, the reduction or decrease occurs in the subject's blood, peripheral blood, or TME. In some embodiments, the reduction or decrease occurs in the blood.

[0308] In some embodiments, the sCD28 level is as measured by ELISA. In some embodiments, the ELISA is a sandwich ELISA. In some embodiments, the ELISA is a standardized sandwich ELISA. In some embodiments, the ELISA is a Bender MedSystems ELISA. In some embodiments, the ELISA is Bender MedSystems ELISA kit BMS290. In some embodiments, the ELISA is performed with an agent of the invention.

[0309] In some embodiments, blocking CD28 shedding comprises blocking proteolytic cleavage. In some embodiments, blocking is inhibiting. In some embodiments, blocking is reducing.

[0310] As used herein, "inhibiting proteolytic cleavage" refers to any reduction in proteolytic cleavage of mCD28. In some embodiments, the inhibition is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, or 100% reduction in cleavage. Each possibility represents a separate embodiment of the invention. In some embodiments, inhibition of proteolytic cleavage maintains the level of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage increases the level of mCD28 on immune cells. In some embodiments, inhibition of proteolytic cleavage maintains the level of mCD28 sufficient for immune stimulation. In some embodiments, the reduction in proteolytic cleavage is a reduction in cleavage by at least one protease.

[0311] In some embodiments, the methods of the present invention do not degrade or lead to the degradation of mCD28. In some embodiments, the methods of the present invention do not reduce mCD28 levels on immune cells. In some embodiments, the methods of the present invention do not reduce mCD28-mediated immune cell activation. In some embodiments, the methods of the present invention maintain mCD28 levels on immune cells of the subject. In some embodiments, the methods of the present invention increase mCD28 levels on immune cells of the subject.

[0312] In some embodiments, the reduction is at least a 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% reduction in sCD28. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reduction is in serum sCD28. In some embodiments, the reduction is in blood levels of sCD28. In some embodiments, the reduction is in levels of sCD28 in the tumor microenvironment (TME).

[0313] In some embodiments, the methods of increasing immune activation, treating a disease, and / or improving immunotherapy include administering an agent that is not an antagonist. In some embodiments, the methods of increasing immune activation, treating a disease, and / or improving immunotherapy include administering an agent that is substantially non-antagonistic. In some embodiments, the disease is a disease treatable by increasing immune activation.

[0314] According to another aspect there is provided a method of inhibiting ligand binding to CD28 comprising contacting CD28 with an sdAb of the invention or a composition of the invention, thereby inhibiting ligand binding.

[0315] According to another aspect, there is provided a method of suppressing an immune response in a subject in need thereof, the method comprising administering to the subject an sdAb of the invention or a composition of the invention, thereby suppressing the immune response.

[0316] It is well known in the art that inhibition of CD28 can block immune stimulation and can be used to treat autoimmune diseases.Furthermore, International Patent Application Publication No. 2020 / 183471, the entirety of which is incorporated herein by reference, demonstrates that increasing sCD28 levels and inhibiting CD28 are therapeutically effective modalities.In some embodiments, inhibiting ligand binding comprises suppressing immune response.

[0317] In some embodiments, the agent reduces T cell activation. In some embodiments, the agent reduces T cell proliferation. In some embodiments, the agent reduces T cell clustering. In some embodiments, the agent increases anti-inflammatory cytokine secretion. Anti-inflammatory cytokines are well known in the art. Non-limiting examples of anti-inflammatory cytokines include, but are not limited to, IL-10 and TGFβ. In some embodiments, the agent reduces pro-inflammatory cytokine secretion. In some embodiments, the pro-inflammatory cytokine is IFNγ.

[0318] In some embodiments, the agent modulates CD28 function and / or signaling. In some embodiments, the agent reduces CD28 function and / or signaling. In some embodiments, the agent reduces CD28 activation. In some embodiments, the signaling is a CD28-mediated immune response. In some embodiments, the agent increases or promotes immunosuppression.

[0319] As used herein, the term "immune response" refers to any response that the body mounts to defend itself against a pathogen or disorder. In one embodiment, an immune response includes a response that is mediated or requires immune cells.

[0320] In one embodiment, the immune response includes any response that activates or inhibits the immune system or mediators of the immune system. In another embodiment, the activation of the immune response includes the activation of immune cells. In another embodiment, the activation of immune cells results in the proliferation of a subset of immune cells. In another embodiment, the activation of immune cells results in increased secretion of immunological mediators by the activated cells. In another embodiment, the activation of immune cells results in the phagocytosis and / or destruction of pathogens, foreign cells, diseased cells, molecules derived or secreted therefrom, or any combination thereof. In another embodiment, the activation of immune cells results in the phagocytosis and / or destruction of neighboring cells, including but not limited to cells infected with a virus. In another embodiment, the activation of immune cells results in the phagocytosis and / or destruction of host cells, molecules derived or secreted therefrom, or any combination thereof. In another embodiment, the activation of immune cells results in the activation of the secretion of antibodies against specific molecules, epitopes, pathogens, or any combination thereof.

[0321] In some embodiments, the immune response is a cytotoxic response. As used herein, a cytotoxic response refers to a response that includes the activation of the complement system, resulting in cell lysis and / or other damage. In some embodiments, the immune response is a humoral response, i.e., involving the production and secretion of antibodies. In some embodiments, the immune response is an innate immune response, i.e., involving the innate immune system. In some embodiments, the immune response is an adaptive immune response, i.e., including an adaptive immune response.

[0322] In some embodiments, the subject is a graft recipient or a candidate for engraftment. In some embodiments, the graft comprises a single cell, a cell suspension, an organ, or any combination thereof. In some embodiments, the graft is an autologous graft. In some embodiments, the graft is a syngenic graft. In some embodiments, the graft is an allogeneic graft. In some embodiments, the graft is a xenograft. In some embodiments, the graft is a hematopoietic graft. In some embodiments, the graft comprises hematopoietic stem cells. In some embodiments, the graft is a non-hematopoietic graft.

[0323] In some embodiments, the subject suffers from an allergy or allergic reaction. In some embodiments, the allergic reaction is caused by an infectious disease or disorder. In some embodiments, the allergic reaction is a symptom of an infectious disease or disorder. In some embodiments, the allergic reaction is unrelated to an infectious disease or disorder. In some embodiments, the allergic reaction is stimulated in parallel with an infectious disease or disorder.

[0324] In some embodiments, the subject suffers from cytokine release syndrome (CRS). As used herein, "cytokine release syndrome" refers to a systemic inflammatory response syndrome resulting from complications of other diseases or infections. In one embodiment, CRS is induced or caused by (e.g., adverse effects of) immunotherapy, such as monoclonal antibody drugs. In one embodiment, CRS is induced or caused by adoptive T cell therapy. As used herein, the terms "CRS" and "cytokine storm" are interchangeable.

[0325] In some embodiments, the subject suffers from an infectious disease. Non-limiting examples of infectious diseases include, but are not limited to, urinary tract infection, gastrointestinal infection, enteritis, salmonellosis, diarrhea, nontuberculous mycobacterial infection, Legionnaire's disease, hospital-acquired pneumonia, skin infection, cholera, septic shock, periodontitis, infection, sinusitis, bacteremia, neonatal infection, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, scalded skin syndrome, and food poisoning.

[0326] In some embodiments, the subject suffers from an autoimmune disease. As used herein, the term "autoimmune disease" refers to any disease or disorder resulting from an immune response against the subject's own tissues or tissue components (e.g., cells and molecules produced or secreted by them), or against an antigen that is not inherently harmful to the subject. In some embodiments, the subject suffers from a T cell-mediated autoimmune disease. Examples of autoimmune diseases include achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune ophthalmitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, and axonal neuropathy. AMAN, Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, herpes Dermatitis ulcerata, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic myositis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell inflammation (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schönlein purple Herpes gestationis or pemphigoid of gestationis (PG), Hidradenitis suppurativa (HS) (acne disease), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease,Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), seizures PNH, Parry-Romberg syndrome, pars planitis (peripheral uveitis), psoriatic Turner syndrome, pemphigus, peripheral neuropathy, perivenous myelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, prostate cancer, pulmonary edema ... Rogesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome ( In some embodiments, the autoimmune disease is selected from lupus, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, Behcet's disease, colitis, ulcerative colitis, diabetes mellitus, Graves' disease, and multiple sclerosis.

[0327] In some embodiments, the method further comprises administering at least one other immunosuppressive therapy. In some embodiments, the immunosuppressive therapy is an immunosuppressant. In some embodiments, the immunosuppressant is an immunosuppressant. In some embodiments, the immunosuppressant is sCD28 or a derivative thereof that binds to a ligand. In some embodiments, the immunosuppressant is an sCD28 stabilizer provided in International Patent Application WO2020 / 183471, which is incorporated herein by reference in its entirety. In some embodiments, the immunosuppressant is a steroid. In some embodiments, the immunosuppressant is a calcineurin inhibitor. In some embodiments, the immunosuppressant is an antiproliferative agent. In some embodiments, the immunosuppressant is an mTOR inhibitor. Immunosuppressants are well known in the art, and any such treatment can be used. Examples of immunosuppressants include, but are not limited to, prednisone, sirolimus, tacrolimus, cyclosporine, mycophenolic acid, mycophenolate sodium, azathioprine, lenalidomide, pomalidomide, methotrexate, azathioprine, and thalidomide.

[0328] In some embodiments, the autoimmune disease is an autoimmune disease associated with elevated sCD28 levels. In some embodiments, the autoimmune disease comprises high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are at and / or above 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 ng / ml. Each possibility represents a separate embodiment of the invention. In some embodiments, the autoimmune disease comprises high sCD28 levels. In some embodiments, elevated and / or high sCD28 levels are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% and / or more than the levels of a healthy subject. Each possibility represents a separate embodiment of the invention. In some embodiments, the autoimmune disease does not involve elevated levels of sCD28. In some embodiments, the autoimmune disease does not involve high levels of sCD28. In some embodiments, the high and / or elevated levels are compared to a healthy subject.

[0329] In some embodiments, the subject has elevated sCD28 levels compared to a healthy subject. In some embodiments, the subject has non-elevated sCD28 levels compared to a healthy subject. In some embodiments, the subject and the healthy subject have comparable sCD28 levels. In some embodiments, a subject with non-elevated sCD28 levels or comparable sCD28 levels to a healthy subject has 0-5% more sCD28 than a healthy subject. In some embodiments, a subject with non-elevated sCD28 levels or comparable sCD28 levels to a healthy subject comprises less than 5 ng / ml sCD28.

[0330] In some embodiments, a subject having elevated sCD28 levels comprises blood sCD28 levels that are elevated by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000%, or any value and range therebetween, above that of a healthy subject, with each possibility representing a separate embodiment of the present invention. In some embodiments, blood sCD28 levels are elevated 5-25%, 10-50%, 25-75%, 50-125%, 100-250%, 200-550%, 500-750%, or 700-1,000% above levels in healthy subjects. In some embodiments, subjects with elevated sCD28 levels include elevated levels of greater than 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng / ml of blood. Each possibility represents a separate embodiment of the invention. In some embodiments, levels are elevated above 5 ng / ml. In some embodiments, levels are elevated above 10 ng / ml.

[0331] According to another aspect, there is provided a method for selecting a subject suitable for treatment with the therapeutic method of the invention, the method comprising measuring sCD28 levels in the subject and / or in the cancer, wherein a sCD28 level above a predetermined threshold indicates that the subject is suitable for treatment with the method of the invention.

[0332] In some embodiments, the method further comprises confirming an elevated level of sCD28. In some embodiments, the method further comprises measuring the level of sCD28. In some embodiments, the sCD28 level is in a subject. In some embodiments, the sCD28 level is in a cancer. In some embodiments, the sCD28 level is in a sample from the subject. In some embodiments, the sample is a bodily fluid. In some embodiments, the predetermined threshold is in a healthy subject. In some embodiments, the predetermined threshold is a threshold above which a high and / or elevated level is obtained. In some embodiments, the predetermined threshold is 5 ng / ml.

[0333] According to a further aspect there is provided an sdAb of the invention, an medicament of the invention or a pharmaceutical composition of the invention for use in the treatment and / or prevention of cancer.

[0334] According to a further aspect there is provided an sdAb of the invention, an agent of the invention or a pharmaceutical composition of the invention for use in improving immunotherapy.

[0335] According to a further aspect there is provided an sdAb of the invention, or a pharmaceutical composition of the invention, for use in suppressing an immune response.

[0336] kit

[0337] According to another aspect there is provided a kit comprising at least one sdAb of the invention or at least one agent of the invention.

[0338] In some embodiments, the kit comprises at least one composition of the present invention. In some embodiments, the kit further comprises at least one immunotherapy. In some embodiments, the kit comprises a label stating that the agent of the present invention is for use with the immunotherapy. In some embodiments, the kit comprises a label stating that the immunotherapy is for use with the agent of the present invention.

[0339] Manufacturing method

[0340] According to another aspect, there is provided a method for producing an agent of the invention, comprising the steps of: i. obtaining an sdAb that binds to CD28 and blocks cleavage; and ii. linking a first portion of an sdAb to a second portion of an sdAb via a linker to create a dimeric agent; Thereby, a method for producing a medicament is provided.

[0341] According to another aspect, there is provided a method for producing an agent of the invention, comprising culturing a host cell comprising one or more vectors comprising one or more nucleic acid sequences encoding a dimeric agent, wherein the one or more nucleic acid sequences are i. obtaining an sdAb that binds to CD28 and blocks cleavage; and ii. linking a first portion of an sdAb to a second portion of an sdAb via a linker to create a dimeric agent; and a method for producing the agent thereby.

[0342] In some embodiments, the agent is an sdAb. In some embodiments, the sdAb binds mCD28. In some embodiments, the sdAb binds mCD28 on a cell. In some embodiments, the sdAb binds mCD28 on the cell surface. In some embodiments, the cleavage is by a protease. In some embodiments, the method further comprises testing the ability of the dimeric agent to block the cleavage. In some embodiments, the method of selecting further comprises testing the ability of the dimeric agent to block the cleavage. In some embodiments, the method further comprises selecting a dimeric agent that blocks cleavage of CD28. In some embodiments, the method of selecting further comprises selecting a dimeric agent that blocks cleavage of CD28.

[0343] In some embodiments, testing the ability of the sdAb to block cleavage is by the methods described below. In some embodiments, testing the ability of the sdAb to block cleavage comprises mixing the sdAb, a protease, and the extracellular domain of CD28 or a fragment thereof comprising the cleavage site. In some embodiments, the test further comprises sequencing the extracellular domain of CD28 or a fragment thereof to check for truncation and / or cleavage. In some embodiments, the test further comprises running the extracellular domain of CD28 or a fragment thereof on a gel that is sufficiently sensitive to measure the size change due to cleavage. In some embodiments, the test further comprises measuring the production of sCD28 from cells expressing mCD28 in the presence of the agent and the protease.

[0344] In some embodiments, the resulting sdAb is a CD28 antagonist. In some embodiments, the resulting sdAb blocks or inhibits the binding of a ligand to CD28. In some embodiments, the resulting sdAb blocks or inhibits CD28-mediated immune activation. In some embodiments, the method further comprises confirming the antagonism, blocking and / or inhibition caused by the sdAb.

[0345] In some embodiments, the method further comprises isolating and / or extracting the agent from the host cell. In some embodiments, the method further comprises isolating and / or extracting the agent from the host cell culture medium. In some embodiments, the method further comprises purifying the agent from the host cell or the host cell culture medium.

[0346] In some embodiments, obtaining the agent comprises immunizing a shark or camelid with said CD28 extracellular domain or fragment thereof and collecting antibodies from said immunized organism, In some embodiments, obtaining the agent comprises screening a library of agents for binding to the CD28 extracellular domain or fragment thereof and selecting agents that bind.

[0347] In some embodiments, recovering the antibody comprises extracting B cells from the spleen of the immunized shark or camelid. In some embodiments, the B cells are fused with melanoma cells to generate hybridomas. In some embodiments, the antibody is recovered from the culture medium of the hybridoma. In some embodiments, obtaining the agent comprises immunizing an organism with the CD28 ectodomain or a fragment thereof, and collecting the antibody from the immunized organism. In some embodiments, the organism is a mouse. In some embodiments, the organism is selected from rabbit, mouse, rat, shark, camelid, chicken, goat, and phage. In some embodiments, the camelid is selected from camel and llama. In some embodiments, recovering comprises taking blood. In some embodiments, recovering comprises Extracting B cells from the spleen of an immunized organism; b. fusing the extracted B cells with myeloma cells to produce hybridomas; and c. Recovering the antibody from the hybridoma.

[0348] In some embodiments, obtaining the sdAb comprises screening a library of sdAbs for binding to the CD28 extracellular domain or a fragment thereof and selecting an sdAb that so binds. In some embodiments, obtaining the agent comprises screening a library of agents for binding to the CD28 extracellular domain or a fragment thereof and selecting an agent that so binds. In some embodiments, obtaining the sdAb comprises screening a library of sdAbs for binding to the CD28 extracellular domain or a fragment thereof and selecting an sdAb that so binds. In some embodiments, the library is a phage display library. In some embodiments, the library is an immunized library derived from splenic B cells. In some embodiments, the library is a library of VHH antibodies. In some embodiments, the library is a library of single domains or nanobodies. In some embodiments, obtaining the sdAb comprises sequencing the sdAb. In some embodiments, obtaining the sdAb comprises making a recombinant form of the sdAb. In some embodiments, the recombinant form is made from the sequence of the sdAb. In some embodiments, the method further comprises humanizing the sdAb. In some embodiments, obtaining the sdAb comprises sequencing the sdAb. In some embodiments, obtaining the sdAb comprises making a recombinant form of the sdAb. In some embodiments, the recombinant form is made from the sequence of the sdAb. In some embodiments, the method further comprises humanizing the sdAb.

[0349] In some embodiments, the method further comprises testing the binding of an agent or sdAb to mCD28. In some embodiments, mCD28 is on a cell surface. In some embodiments, the method further comprises selecting an sdAb or agent that binds to mCD28. In some embodiments, the method further comprises testing the cleavage of mCD28 on the cell surface in the presence of a protease. In some embodiments, the method further comprises selecting an agent or sdAb that blocks or inhibits the cleavage of mCD28 by a protease.

[0350] In some embodiments, a portion is a copy of an sdAb. In some embodiments, the portion is a single copy. In some embodiments, the portion is a VHH. In some embodiments, the portion is an sdAb of the invention.

[0351] In some embodiments, the method further comprises assaying mCD28 downstream signaling in the presence of the resulting dimeric agent. In some embodiments, the method further comprises selecting at least one dimeric agent that does not substantially agonize mCD28 signaling. In some embodiments, the method further comprises selecting at least one dimeric agent that does not substantially antagonize mCD28 signaling. In some embodiments, the method comprises selecting at least one dimeric agent that antagonizes mCD28 signaling. For cancer treatment, it will be understood by those skilled in the art that agonizing CD28 signaling may not be harmful, but antagonizing signaling is ineffective. It will be further understood that for treating autoimmune diseases or other conditions, it is advantageous to benefit from immunosuppressive antagonism, and agonizing CD28 signaling is counterproductive. Methods for measuring agonism and antagonism are well known in the art and are further provided below.

[0352] According to another aspect, there is provided a dimerizing agent produced by the method of the invention.

[0353] As used herein, the term "about" when combined with a value refers to ±10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ±100 nm.

[0354] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "polynucleotide" includes a plurality of such polynucleotides, a reference to a "polypeptide" includes a reference to one or more polypeptides known to those of skill in the art and equivalents thereof, and so forth. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for using exclusive language such as "exclusively," "only," and the like in connection with the recitation of claim elements, or for using a "negative" limitation.

[0355] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B and C together, etc.). It will be further understood by one of ordinary skill in the art that substantially any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to consider the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0356] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0357] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0358] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0359] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley&Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series" Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); the methodology set forth in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), Strategies for Protein Purification and Characterization-A Laboratory Course Manual, CSHL Press (1996), all of which are incorporated by reference. Other general references are provided throughout this document.

[0360] material and method Generation of recombinant 2A1 constructs - Synthetic codon-optimized genes were subcloned into the relevant pcDNA3.1 expression vector. 2A1 constructs were generated from transiently transfected ExpiCHO cells and purified by immobilized metal affinity chromatography (IMAC) for tandem constructs, MabSelect Sure Protein A for Fc chimeras, or Amsphere A3 for di-VHH2 2A1 constructs. Protein preparations in 1x PBS pH 7.4 were analyzed by SDS-PAGE for the presence of the correct chain under non-reducing conditions and by analytical size exclusion chromatography (aSEC) for quantification of monomeric forms within the preparations.

[0361] Generation of recombinant nanobodies - Synthetic codon-optimized genes were subcloned into the relevant pcDNA3.1 expression vector. Nanobody constructs were generated from transiently transfected ExpiCHO cells and purified by immobilized metal affinity chromatography (IMAC) for tandem constructs and MabSelect Sure Protein A for Fc chimeras. Protein preparations in 1x PBS pH 7.4 were analyzed by SDS-PAGE for the presence of the correct chains under non-reducing conditions and by analytical size exclusion chromatography (aSEC) for quantification of monomeric forms within the preparations.

[0362] Chemical modification of the parent 2A1 molecule - 2A1 construct with a C-terminal cysteine ​​(2A1-1C) was incubated with Bis-Mal-PEG11 chemical moiety (Broadpharm, Cat. No. BP-22151). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma Aldrich, Cat. No. 75259) was added prior to the reaction to break up the dimer content. After completion of the reaction, the mixture was loaded onto a SP cation exchange column to remove excess reagents and unreacted materials. The preparation was PBS desalted using a Viva-spin concentrator and analyzed by mass spectrometry for conjugation verification.

[0363] Cytokine ELISA - Commercially available ELISA kits were used to quantify the amount of human interferon gamma (Biolegend, Cat. No. 430103), human interleukin 2 (Biolegend, Cat. No. 431802) and human CD28 (R&D system, Cat. No. DY342). Cell proliferation and viability (MTT assay) were performed to ensure the validity of the results and were performed according to the manufacturer's instructions (Roche, Cat. No. 11465007001).

[0364] Direct CD28 EIA - Corning high binding plates or equivalent were used for screening unless otherwise stated. Each well was coated with 300ng of human CD28-Fc chimera (R&D, Cat. No. 342-CD). Plates were blocked with 1% casein in PBS for 1 hour at room temperature (RT). Plates were washed 4 times with PBST and incubated with the investigated nanobody / drugs after detection with donkey anti-human IgG FC HRP (Jackson immuno research, Cat. No. 709-035-098) for Fc chimeras, HRP-conjugated rabbit anti-camel VHH cocktail (GenScript, Cat. No. A02016) for tandem dimeric VHH hinge constructs, or mouse anti-human IgG4 pFc' HRP (abcam, Cat. No. ab99817).

[0365] Cell lines and human immune cell isolation - PBMCs were isolated from fresh blood samples of healthy donors using standard lymphocyte separation medium (MBP, Cat. No. 850494). CD3 cells were isolated from fresh blood samples of healthy donors by negative selection using the RossetteSEP™ Human T Cell Enrichment Kit (STEMCELL, Cat. No. 15061). Monocytes were isolated from fresh blood samples of healthy donors by negative selection using the EasySep™ Human Monocyte Enrichment Kit (STEMCELL, Cat. No. 17952). All cells were grown in complete RPMI-1640 medium supplemented with 10% HI-FCS and pen / strep mixture.

[0366] Transfection - CD28-FL (encoding full-length CD28 transcript), CD80-FL (encoding full-length CD80 transcript) and scOKT3-CD14 (encoding single-chain FV portion of mouse anti-CD3 OKT3 clone fused to CD14 extracellular domain) plasmids were generated by cloning DNA sequences into PCDNA3.1 vector. Transfections were performed using JetPei transfection reagent (PolyPlus Transfections). Stable transfectants were selected in G418 and / or hygromycin-containing medium.

[0367] Dendritic cell differentiation - Monocytes were cultured at a density of 1x10^6 / mL in RPMI medium with growth factors renewed on days 3 and 6. Immature dendritic cells (iDCs) were induced for 6 days with 50ng / mL GM-CSF (R&D systems, Cat. No. 215-GM) and 20ng / mL IL-4 (R&D systems, Cat. No. 204-IL). When required, iDCs were further differentiated into mature dendritic cells by adding 100ng / mL LPS (Sigma, Cat. No. L4391) and 20ng / mL interferon-gamma (R&D systems, Cat. No. 285-IF) for 48 hours. The generated cell populations were tested for the indicated phenotypes by FACS analysis of relevant markers and analysis of secretion of characteristic cytokines.

[0368] SEB activation of PBMCs to generate soluble CD28 - 0.1x10^6 PBMCs were stimulated with 10-50ng / mL SEB (Sigma, Cat. No. S4881) in 96-well plates with / without various treatments and protease inhibitors at the indicated concentrations for 7 days at 37°C. Assays were performed in complete RPMI-1640 medium supplemented with FCS and pen / strep mixture. The protease inhibitor used is TMI-1 (Sigma, Cat. No. PZ0336). Where inhibition of CD28 shedding is indicated, it has been calculated using the following formula: 100-(sCD283μM / sCD28basal*100).

[0369] Allogeneic mixed lymphocyte reaction - 0.1x10^6 T cells were mixed with 0.2x10^4 mature dendritic cells from different donors with or without treatment at the indicated concentrations for 24-96 h at 37°C. Assays were performed in complete RPMI-1640 medium supplemented with 10% FCS and pen / strep mixture. Where stated, VHH#3C04 (grown against human HER2) was used as an "irrelevant control" and VHH#12B09 was used as a "positive control" for nanobodies with CD28 antagonistic effects.

[0370] T cell stimulation with HEK / CD80 / scOKT3 artificial antigen presenting cells (aAPC-CD80) - 1x10^5 isolated CD3 T cells (from healthy donors) were stimulated with 0.5x10^4 mitomycin-treated aAPC-CD80 (HEK293 cells stably transfected with CD80 and scOKT3-CD14 chimeric plasmids) for 24-72 h at 37°C. CD28-targeting VHH or control treatments were added at the indicated concentrations in soluble form. Where stated, VHH#3C04 (grown against human HER2) was used as an "irrelevant control" and VHH#12B09 was used as a "positive control" for nanobodies with CD28 antagonistic effects. Assays were performed in complete RPMI-1640 medium supplemented with 10% FCS and pen / strep mixture.

[0371] 0.25x10^6 HEK293 cells stably transfected with FACS-human CD28 blocking CD86 were incubated with 2μg / ml biotinylated CD86-Fc (R&D systems, Cat#141-B2) without or with anti-CD28 shedding clone #2A1 at a fixed concentration of 3μM in various formats for 30 minutes at room temperature. Cells were washed and harvested for secondary binding with streptavidin conjugated to a fluorophore (Jackson immuno research, Cat#016-130-084) at 1:500 dilution for 20 minutes on ice. Incubation was performed in a volume of 100μL in 96-well U-bottom plates. Cells were washed twice with 200μL FACS buffer and transferred to FACS tubes with 150μL FACS buffer for analysis. Cells were analyzed on a Gallios Flow Cytometer (Beckman Coulter) using Kaluza for Gallios Flow Cytometry Acquisition Software.

[0372] Flow cytometry on CD3 cells - 2.5 x 10 from healthy donors 5CD3 cells were incubated for 30 min at 37 °C with / without the indicated constructs at the concentrations stated. Incubations were performed in a volume of 100 μL in 96-well U-bottom plates. Cells were washed twice with 200 μL of FACS buffer (PBS with 0.05% BSA). Binding of the tested constructs to the cells was assessed by incubation (30 min on ice) with Nano-Secondary Alpaca Anti-Human IgG Recombinant VHH Alexa Flour 647 antibody (Chromotek, Cat. No. srbAF647-1). Incubations were performed in the dark in a volume of 100 μL applying the concentrations recommended by the manufacturer. After incubation, cells were washed three times with 200 μL of FACS buffer and analyzed on a CytoFLEX Flow Cytometer (Beckman Coulter) using CytExpert Acquisition and Analysis Software.

[0373] MC-38 syngenic tumor model - MC38 tumor cells were cultured in DMEM and implanted subcutaneously (0.5x10^6 cells) into humanized CD28 transgenic C57 / Bl6 mice (genOway) (5 groups of n=10). Six days after inoculation, each group of mice was treated with anti-PD1 (RMP1-14) with or without shedding blocker twice a week (200μg / mouse) for a total of 6 injections. To set up a preventive model, one group of mice was also injected with shedding blocker (200μg / mouse, 2 times) before MC-38 inoculation. As isotype controls, InVivoMAb human IgG4 (BE0349-BioXcell) or rat IgG2a (2A3) were used. Tumor volumes were measured 3 times a week by calibrated calipers.

[0374] VHH affinity maturation - To affinity mature the parent monoclonal anti-CD28 VHH#2A01 llama antibody, complementarity determining region (CDR) residues were randomized by rational mutagenesis using trimer / primer mixes. Library design was based on human and llama natural diversity of the amino acids represented (Kim et al., "Generation, diversity determination, and application to antibody selection of a human naive Fab library", Mol. Cells, 2017, 40(9):655-666; Zemlin et al., "Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures", J. Mol. Biol., 2003, 334:733-749; and Tiller et al., "A fully synthetic human Fab antibody library based on fixed VH / VL framework pairings with favorable biophysical properties", mAbs 20135:445-470, which are incorporated herein by reference in their entireties). A PCR gene assembly protocol was performed using multiple overlapping oligonucleotides to introduce CDR diversity. The PCR products were cloned into pDCL1 phagemid to generate four different phage libraries containing mutations for CDR1, CDR2, CDR1+CDR2 and CDR3 with final sizes ranging from 8.0E+7 to 1.0E+09, respectively. The libraries were QCe'd by sequencing. Screening for affinity maturation variants, solution selection was performed against biotinylated recombinant human CD28-Fc or human CD28 stalk region dimer peptide captured on Dynabeads™ MyOne™ streptavidin T1 magnetic beads.Four cycles of selection were performed with decreasing antigen concentration and increasing free antigen in solution to achieve variants with improved off-rate kinetics. Counterselection with human IgG was performed to reduce background. Enriched CDR1-3 variants were validated by sequencing, biacore, antigen binding in ELISA, and efficiency in CD28 shedding blocking activity. Beneficial residue mutations at specific positions in CDR1-3 were picked and randomized with each other to generate a lead panel of combinatorial libraries. The combinatorial library was subjected to four cycles of in-solution selection against human antigen with increasing stringency. Twenty variants were isolated and characterized in detail for CD28 immunomodulation and CD28 shedding blocking activity. The six top performing clones were further characterized for affinity determination and generated as Fc chimeras.

[0375] Protein models of VHH humanized-VHH clones are analyzed to identify residues important for antibody conformation and binding. Using this information together with in silico tools to assess MHC class II binding and a database containing antibody segments previously screened using ex vivo T cell immunogenicity assays, a series of humanized heavy chain VHH region sequences are designed from segments of human V region sequences with the goal of avoiding T cell epitopes.

[0376] Affinity clones derived from the Biacore-VHH#2A01 affinity maturation library were determined using CD28-Fc immobilized on a CM5 Series S sensor chip. Multi-cycle kinetic measurements were performed using five-step dilutions of analyte (VHH clone) from 200 nM to 12.5 nM. Kinetic parameters and affinity values ​​were calculated using a 1:1 binding model in the Biacore T 200 E evaluation software 3.1 by introducing a twofold (blank channel and blank sample) subtraction.

[0377] Example 1: Affinity maturation of single domain antibodies Three single domain VHH antibodies were previously generated against the stalk domain of the CD28 extracellular domain and found to be effective in blocking cleavage of membrane CD28 from the surface of cells (see PCT / IL2020 / 050297, incorporated herein by reference in its entirety). All three VHHs were found to neither substantially agonize nor substantially antagonize CD28 signaling. VHH 2A1 was selected for affinity maturation of the complementarity determining regions (CDRs) as it is the most highly studied and most effective molecule.

[0378] The complete sequence of 2A1 is EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 40), which includes CDR1 of INAMG (SEQ ID NO: 4), CDR2 of AISGGGDTYYADSVKG (SEQ ID NO: 5), and CDR3 of DLYGSDYWD (SEQ ID NO: 3).

[0379] To affinity mature the parental VHH#2A1 llama antibody, CDR residues were randomized by rational mutagenesis using trimer / primer mixes. Library design was based on human and llama natural diversity of the represented amino acids. A PCR gene assembly protocol was performed using multiple overlapping oligonucleotides to introduce CDR diversity. PCR products were cloned into pDCL1 phagemid to generate four different phage libraries, containing mutations in CDR1, CDR2, CDR1+CDR2 and CDR3, with final sizes ranging from 8.0E+7 to 1.0E+09, respectively. Libraries were quality controlled by sequencing. Screening for in-solution selection of affinity matured variants was performed against biotinylated recombinant human CD28-Fc or human CD28 stalk region dimeric peptide captured on Dynabeads™ MyOne™ streptavidin T1 magnetic beads. Four cycles of selection were performed with decreasing antigen concentration on the beads and increasing free antigen in solution to achieve variants with improved off-rate kinetics. Counterselection with human IgG was performed to reduce background. Enriched CDR1-3 variants were validated by sequencing, Biacore, antigen binding in ELISA, and efficiency in CD28 shedding blocking activity. Beneficial residue mutations at specific positions in CDR1-3 were picked and randomized with each other to generate a lead panel of combinatorial libraries. The combinatorial libraries were subjected to four cycles of in-solution selection against human antigens with increasing stringency. The sequences of the generated variants are shown in Table 1, the CDRs of each variant are shown in Table 2, and the affinity constants of seven of the nine VHH clones are summarized in Table 3.

[0380] [Table 1]

[0381] [Table 2]

[0382] [Table 3]

[0383] Three affinity matured variants were selected from the first round of mutagenesis, one with CDR1+CDR2 mutations each (5A3) and two with CDR3 mutations (6B3 and 6B10). Twenty variants were isolated from the combinatorial library and characterized in detail for CD28 immunomodulatory and CD28 shedding blocking activity. The six best candidates based on CD28 binding and blocking CD28 shedding were selected. All six were found to be superior to the parental VHHs in terms of blocking (Figure 1, two representative variants, 5A3 and 6B10 are shown). However, when downstream signaling was tested in a mixed lymphocyte reaction (MLR), the affinity matured VHHs provided a strong antagonistic effect, especially at high concentrations, reducing pro-inflammatory cytokine secretion (Figure 2, two representative variants, 5A3 and 6B10 are shown).

[0384] Blocking cleavage reduces the levels of immune-inhibitory soluble CD28 (sCD28), thus increasing immune cell activation. However, if the cleavage blocker also inhibits CD28 binding to activating ligands (e.g., CD86, CD80), this antagonistic effect has the opposite effect, inhibiting immune cell activation. Thus, these molecules that produce an antagonistic effect may not be useful for immune stimulation, but may be used in conditions of immune hyperactivation, such as autoimmune diseases.

[0385] Example 2: Dimeric binders reduce CD28 antagonism effects Surprisingly, it was discovered that inclusion of monomeric sdAbs within dimeric binders could not only increase their binding efficiency but also reduce their antagonistic effect. Therefore, affinity matured variants were included in Fc chimeric molecules and assayed for their effect on CD28 signaling.

[0386] Fc chimeric molecules were generated by cloning the sdAb upstream of the Fc chain of human IgG1 (SEQ ID NO: 39, Fc with reduced effector function). A flexible amino acid linker (15 amino acid GGGGS linker; for brevity, the GGGGS repeat linker is hereafter referred to as GS linker) was inserted between the C-terminus of the sdAb and the N-terminus of the Fc. These dimeric agents were tested in MLR experiments. Surprisingly, the inclusion of the Fc domain abolished the antagonistic effect seen with the monomeric sdAb (Figure 3, two representative variants, 5A3 and 6B10 are shown). Thus, these dimeric agents are highly effective immunostimulants, since they reduce sCD28 production and do not interfere with membrane CD28 (mCD28) activation.

[0387] Example 3: Affinity matured clones 12A09-Fc and 9B03-Fc inhibit mCD28 cleavage Next, the ability of affinity matured clones fused to Fc to bind CD28 was tested. The 12A9 and 9B3 clones were cloned upstream of the Fc chain of human IgG4. IgG4 was chosen because it is a generally non-cytotoxic Fc. To further reduce cytotoxicity and enhance stability / reduce aggregation, two point mutations (S228P and L235E) were generated in the IgG4 chain. A 25 amino acid linker (GGGGSx5) was inserted between the VHH and the Fc. The binding of these two molecules to CD28 was compared to that of the parental VHH construct. As expected, the construct containing the two affinity matured VHHs bound significantly better to CD28 in solution (Figure 4A) and on the cell surface (Figure 4B).

[0388] The ability of the dimeric fusion proteins to indeed bind mCD28 on the cell surface and block cleavage was tested. Staphylococcal enterotoxin B (SEB) activates peripheral blood mononuclear cells (PBMCs) and induces CD28 cleavage and sCD28 shedding. Addition of dimeric molecules containing the parental VHH (2A1, 100 nM concentration) reduced sCD28 production, indicating true surface binding and blockade of cleavage (Figure 5A-B). Both 12A9-25GS-hIgG4 (Figure 5A) and 9B3-25GS-hIgG4 (Figure 5B) outperformed the parental molecules and resulted in a potent inhibition of CD28 cleavage in a dose-dependent manner.

[0389] Example 4: Dimeric binders are not repulsive Having confirmed that the dimeric binders bound CD28 with higher affinity than the monomeric VHHs, provided improved CD28 cleavage blockade, and did not result in antagonistic effects against CD28, we tested the possibility that these agents might act repulsively. Isolated human CD3 cells were stimulated for 2 days with cells of the A375 cell line overexpressing scOKT3 (anti-CD3). This stimulation was performed in the presence of an anti-CD28 agonist antibody, clone 28.2, which served as a positive control, an unrelated human IgG5, which served as a negative control, or an Fc-chimeric affinity-matured clone VHH (25 amino acid linker). The concentration of human IL-2 secreted in the supernatant was quantified using a standardized sandwich ELISA (Biolegend). As expected, clone 28.2 led to potent IL-2 secretion, whereas the Fc-chimeras did not show a significant repulsive effect (Figure 6A-6B, clones 12A9 and 9B3 are shown).

[0390] Example 5: Examination of linker length Since both 15 and 25 amino acid GGGGS linkers (referred to as GS linkers throughout this specification) provided effective cleavage blockage, the optimal size of the linker was investigated. Fc chimeric variants of VHH12A9 were generated with flexible linkers of different lengths between the Fc and VHH modules. Linkers from 10 amino acids to 35 were investigated. SEB-activated PBMCs were again used to measure CD28 cleavage from the cell surface. Chimeric molecules were introduced into PBMCs at two different concentrations (100 nM and 300 nM) and sCD28 levels were measured. At both concentrations, it was clearly observed that increasing linker length improved the level of shedding inhibition, with linkers of 25 amino acids or more providing more than 90% shedding inhibition (Figure 7). Since maximum shedding inhibition was achieved with the 35 amino acid linker, there is no reason to use longer linkers.

[0391] Example 6: In vivo testing The therapeutic potential of these dimeric VHH constructs was further investigated in vivo in the MC-38 syngenic model, where MC38 tumor cells were subcutaneously implanted in humanized CD28 transgenic mice. Anti-PD1 (RMP1-14) treatment, starting 6 days after implantation, slowed the rate of tumor growth compared to the corresponding isotype control (rG2a is the rat isotype control of the PD-1 antibody) (Figure 8A-B). Surprisingly, prophylactic treatment with 12A9-25GS-hIgG4 5 days before implantation of cancer cells, combined with anti-PD1 treatment, led to a complete prevention of cancer growth (Figure 8A). All cancer cells were killed by the mouse immune system, and even cancer was not detectable until day 30. Combination treatment after implantation (both on day 6) significantly slowed tumor growth, superior to treatment with anti-PD1 alone (Figure 8B). These in vivo results further support that the dimeric agents do not have an antagonistic effect on CD28. Such an effect would result in tumor growth and not shrinkage / prevention. Thus, it is entirely clear that these agents block CD28 cleavage from the cell surface and do so without antagonizing CD28 function, which would lead to robust cancer therapy.

[0392] Example 7: VHH humanization Two of the VHH clones, 12A9 and 9B3, were further humanized such that the camelid backbone of the VHH was removed. To this end, protein models of the heavy chain VHH regions were analyzed to identify residues important for antibody conformation and binding. Using this information together with in silico techniques (tool 1: evaluation of MHC class II binding, tool 2: database containing antibody segments previously screened using ex vivo T cell immunogenicity assays), a series of humanized heavy chain VHH region sequences were designed from segments of human V region sequences with the aim that T cell epitopes were avoided. Five clones were given to 12A9 and four to 9B3. The sequences of these clones are shown in Table 4.

[0393] [Table 4]

[0394] Dimeric binders were also produced using humanized VHH variants. Five 12A9 humanized variants and one 9B3 humanized variant were linked to human IgG4 Fc via a 25 amino acid GS flexible linker. Binding of the humanized variant chimeras to human CD28 was confirmed using a direct CD28 EIA as before. All five 12A9 humanized variants tested and one 9B3 humanized variant tested bound to CD28 at least as well as their parental (camelid) variant chimeras (Figures 9A-9B). Indeed, both 12A9-VHH18-25GS-huFc and 12A9-VHH4-25GS-huFc were measurably better than the parental 12A9 VHH chimera. The affinity constants of the six dimeric agents containing humanized variants and the agents containing the parental VHH are summarized in Table 5.

[0395] [Table 5]

[0396] Binding to CD28 on the cell surface was also evaluated as before. CD3 cells were analyzed by FACS in the presence of isotype control, humanized VHH chimeras or parental VHH chimeras (300 nM). The mean fluorescence intensities measured for these various agents are summarized in Tables 6 and 7. All of the humanized VHH chimeras were at least as good binders as the parental VHH chimeras, and indeed 12A09-VHH12-25GS-huFc, 12A09-VHH16-25GS-huFc (humanized), and 12A09-VHH17-25GS-huFc generated three times the fluorescence of the parental constructs.

[0397] [Table 6]

[0398] [Table 7]

[0399] The ability of the humanized variant chimeras to block CD28 shedding was also examined. PMBCs were stimulated with SEB as before in the presence of the humanized variants. All five tested 12A9 humanized variants of the Fc chimera and one tested 9B3 humanized variant blocked CD28 shedding from the cell surface at least as well as the parental variant chimeras (Figure 10A-B). Indeed, the humanized variants slightly improved shedding blockade, especially at intermediate concentrations, with 12A9-VHH12, 12A9-VHH16 and 12A9-VHH17 showing improvement over the parental VHH (Figure 10A).

[0400] Example 8: Generation of dimeric single chain sdAb molecules Three single domain VHH antibodies were previously generated against the stalk domain of the CD28 extracellular domain and were found to be effective in blocking cleavage of membrane CD28 from the surface of cells (see PCT / IL2020 / 050297, incorporated herein by reference in its entirety). All three VHHs were found to neither substantially agonize nor substantially antagonize CD28 signaling. The sequences of these single domain antibodies (sdAbs) are provided in Table 8 and the CDRs of these sdAbs are provided in Table 9.

[0401] [Table 8]

[0402] [Table 9]

[0403] To improve the specificity and efficacy of the VHH in blocking cleavage, we decided to generate a dimeric agent containing two copies of the VHH. VHH 2A1 proved to be the most effective cleavage blocker and was therefore selected for inclusion in the dimeric molecule.

[0404] We first generated a single-chain tandem dimer construct in which the C-terminus of the first 2A1 was connected to the N-terminus of the second 2A1 via a peptide linker. 4 S) linkers were tested. Dimeric single chain molecules were made with one, two or four linker repeats. The sequences of the various single chain molecules are summarized in Table 10. These dimeric constructs were tested for binding to recombinant CD28 fused to an Fc backbone and compared to monomeric 2A1 as a control. The very short 5 amino acid linker did not increase affinity for CD28 and was comparable to monomeric 2A1 (Figure 11A). In contrast, 10 or 20 amino acid linkers resulted in increased binding compared to the monomer (Figure 11A). The EC50 differences are summarized in Table 11.

[0405] [Table 10]

[0406] [Table 11]

[0407] The effect on cleavage was measured directly in cells. Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors and stimulated with Staphylococcal Enterotoxin B (SEB) to activate immune cells and produce potent levels of sCD28. Addition of the pan-metalloprotease inhibitor TMI or monomeric 2A1 (3 μM) both significantly reduced sCD28 levels, whereas an unrelated VHH had no effect (Figure 11C-11E). The 20GS construct was as potent as the TMI inhibitor and monomeric 2A1 at the lowest concentration tested, and at higher concentrations nearly abolished sCD28 production, indicating that it is a more potent cleavage inhibitor than the monomer (Figure 11E). The 10GS construct was less effective at the lowest concentration, but at increasing concentrations it still essentially abolished CD28 cleavage, still significantly better than the monomer (Figure 11D). As expected based on the binding data, the 5GS construct was the least effective, but at higher concentrations was slightly better than the TMI inhibitor and the monomer (Figure 11C). These results indicate that the single-chain dimeric molecule is more effective at blocking sCD28 production than the monomeric form of the VHH.

[0408] Next, these single-chain dimeric agents were tested to see if they produced an antagonistic effect against CD28. Blocking cleavage would reduce the levels of immunoinhibitory soluble CD28 (sCD28) and thus increase immune cell activation. However, if the cleavage blocker also inhibits CD28 binding to an activating ligand (e.g., CD86), this antagonistic effect would have the opposite result, inhibiting immune cell activation. VHH 2A1, known not to produce an antagonistic effect, did not block the binding of recombinant CD86 to HEK cells overexpressing human CD28 (Figure 12A). Unexpectedly, single-chain dimeric molecules with 10 or 20 amino acid linkers potently inhibited CD86 binding, inhibiting binding by more than 75% (Figure 12A). Short linkers did not substantially inhibit CD86 binding and appeared nearly as similar to monomeric 2A1 (Figure 12A).

[0409] It was hypothesized that the antagonistic effect was caused by an interaction between the linker and the ligand-binding region of CD28. To circumvent this interaction, two additional single-chain dimeric agents were generated. In these molecules, four G 4 A 20 amino acid long linker with S repeats has every fourth G substituted with a charged amino acid. In one construct the linker was made positive with a glycine amino acid substituted with a lysine amino acid (20K) and in the other the linker was made negative with a glycine amino acid substituted with a glutamic acid amino acid (20E). It was hypothesized that the charged amino acid would result in electrostatic repulsion from the CD28 extracellular domain and thus abolish the antagonistic effect. A third new single chain dimeric agent was also made but using a rigid helical linker instead of a flexible linker (Hel20). The sequences of these dimeric agents are shown in Table 10.

[0410] The same binding assay was repeated with these molecules, using the 20GS agent and the monomeric 2A1 molecule as controls (Figure 11B). Regardless of the charge or rigidity of the linker used, all long linker constructs behaved similarly and resulted in superior target binding compared to the monomeric 2A1. In fact, the two charged linker and rigid linker constructs were actually slightly better than the 20GS molecule (Figure 11B). The EC50 values ​​of these constructs are summarized in Table 12. All new agents also inhibited sCD28 production as expected (Figures 11F-11H).

[0411] [Table 12]

[0412] As all new linker molecules still demonstrated enhanced cleavage blockade, their antagonistic effect on CD28-CD86 binding was tested as before. In contrast to what was expected, all three new linker molecules resulted in robust CD86 blockade, with inhibition levels comparable to the flexible 20 amino acid linker (Figure 12B). These results indicate that, regardless of the linker structure, longer linkers used in single-chain VHH dimers with tandem VHHs linked C-terminus to N-terminus always result in blockade of CD86 binding.

[0413] Example 9: Functional analysis of single chain dimer tandem agents The observed functional effects of CD86 blockade were further investigated. Isolated T cells were stimulated with artificial antigen presenting cells (aAPCs, HEK cells expressing CD80 and scOKT3) and IL-2 secretion was measured. This was done in the presence of irrelevant VHHs, VHHs known to block CD86 binding, and various concentrations of single-chain dimeric agents (Figures 13A-13F). As expected, based on the blocking results, the 5GS agent had no effect on IL-2 secretion (Figure 13A). However, the 20GS construct almost completely abolished the induction of IL-2 secretion triggered by CD86 even at low concentrations (Figure 13C), and the 10GS construct produced an intermediate effect, decreasing IL-2 secretion by about 50% at low concentrations, with the reduction increasing to about 80% at higher concentrations (Figure 13B). The other three 20 amino acid linker constructs also produced robust inhibition of IL-2 secretion (Figures 13D-13F), although the rigid linker construct required higher concentrations for maximum inhibition to occur (Figure 13F). Similar results were observed when interferon gamma (IFNg) secretion was measured in a mixed lymphocyte reaction (Figures 13G-13I). These molecules that produce antagonistic effects may not be useful for immune stimulation, but may be used in conditions of immune hyperactivation, such as autoimmune diseases.

[0414] Example 10: Generation of dimeric agents with only C-terminal linkages Since all of the single chain agents in which the sdAb was linked C-terminus to N-terminus, except for the 5 amino acid linker, produced antagonistic effects, new dimeric agents were designed. In this next set of agents, a linkage was always generated between the C-terminal regions to both sdAbs. First, a C-terminal cysteine ​​residue was added to the sequence of 2A1 (2A1-C, SEQ ID NO: 113), which can dimerize by forming a disulfide bond between the two free cysteines. As desired, dimeric 2A1-C bound to recombinant CD28-Fc at lower concentrations than monomeric 2A1 (Figure 14A). The EC50 values ​​are summarized in Table 13. Similar molecules were also generated using longer linkers. A short discrete PEG chain (PEG 2A1-C) with reactivity towards the free thiol (maleimide) of cysteine ​​was added to the sequence of 2A1 (2A1-C, SEQ ID NO: 113), which can dimerize by forming a disulfide bond between the two free cysteines. As desired, dimeric 2A1-C bound to recombinant CD28-Fc at lower concentrations than monomeric 2A1 (Figure 14A). The EC50 values ​​are summarized in Table 13. Similar molecules were also generated using longer linkers. 11A chemical linker based on bmp11 was used to link the two cysteines (Figure 15, 2A1-1C-bmp11). This molecule also showed excellent binding to CD28 (Figure 14B). The EC50 values ​​are summarized in Table 14.

[0415] [Table 13]

[0416] [Table 14]

[0417] An alternative approach to C-terminal cysteines is to use C-terminal dimerization domains. The hinge region of immunoglobulin (Ig) heavy chains has several cysteine ​​residues that are primarily responsible for heavy chain dimerization. Therefore, instead of adding a single cysteine, a human IgG1 hinge region (DKTHTCPPCPAPEL, SEQ ID NO: 38) was inserted downstream of VHH 2A1 (2A1-hinge, SEQ ID NO: 114). Similarly, more C-terminal regions of the heavy chain (CH2 and CH3 domains, SEQ ID NO: 39) were also added downstream of the hinge (2A1-huFC, SEQ ID NO: 115). A PG-LALA mutation was incorporated into the heavy chain to reduce effector function. To ensure that the Fc does not interfere with VHH binding, two other agents were made with a flexible linker between the VHH and the hinge (2A1-15GS-huFC, SEQ ID NO: 116; 2A1-25GS-huFC, SEQ ID NO: 117). All these molecules were found to improve binding to CD28, with the longer linkers giving the best results (Figure 14C). The EC50 values ​​are summarized in Table 15.

[0418] [Table 15]

[0419] As before, the ability of dimeric agents to inhibit sCD28 production was also tested. The 2A1-C construct did not inhibit cleavage at its lowest concentration, but was found to be superior to monomeric 2A1 at the same concentration (Figure 14D). Similarly, 2A1 with a PEG-based linker was also superior to monomeric 2A1 when applied at the same concentration (Figure 14E). The 2A1-hinge agent inhibited sCD28 production, but only at a level comparable to the 2A1 monomer (Figure 14F). In contrast, dimeric molecules with a full Fc provided sCD28 inhibition comparable to the TMI inhibitor and superior to monomeric 2A1 (Figure 14G). Both agents containing flexible linkers performed comparably to the Fc molecule without a linker (Figure 14H-Figure 14I). The improved shedding block provided by these molecules is summarized in FIG. 14J, which shows results from three different PBMC donors treated with 3 μM of the dimeric agents.

[0420] Example 11: Dimeric agents with only C-terminal bonds are less antagonistic Since the first batch of dimeric molecules was unexpectedly a CD28 antagonist, a new batch of dimeric molecules was also tested. The C-terminal molecules, whether connected by disulfide bonds or with a PEG-based linker, both showed minimal CD86 blockade, but at a level far superior to the previously tested NC-linked dimeric molecules (Figure 16A). Molecules utilizing the hinge region for dimerization gave even better results (Figure 16B). All of these molecules did not significantly inhibit CD86 binding, were comparable to their monomeric counterparts, and therefore did not appear to be CD28 antagonists at all. They were further tested for repulsive effects on CD28, but none were noted (data not shown). All this together makes them ideal CD28 shedding blockers and therefore immune stimulatory molecules.

[0421] The lack of antagonistic effects was further confirmed in more physiologically relevant contexts. As before, the secretion of proinflammatory cytokines from T cells was examined using direct aAPC stimulation (Figures 17A-17F) and in the context of an MLR (Figures 18A-18E). The 2A1-C dimeric agent led to a modest dose-dependent reduction in IL-2 levels (Figure 17A). Similar results were observed in the MLR (Figure 18A). The use of a PEG-based linker showed similar results, with mild inhibition only at high concentrations (Figures 17B and 18B). Again, the Fc-based agent showed no antagonistic effect. The 2A1-hinge dimeric agent showed no effect on IL-2 secretion (Figure 17C). The human Fc molecule without a linker also did not show any effect on IL-2 secretion (Figure 17D) nor did it inhibit IFNg secretion (Figure 18C). Fc dimer agents with linkers were just fine with no antagonistic effects at all (Figures 17E-17F, Figures 18D-18E). The overall effects on T cell activity are summarized in Figure 18F, which shows the results of an MLR assay with cells from four different donors. Molecules using C-terminal linkers, especially those with dimerization domains such as hinge or Fc, all succeeded in not producing substantial antagonistic effects while also blocking cleavage, making them ideal for immune stimulation in the context of excess sCD28 production.

[0422] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A single domain antibody (sdAb) comprising three CDRs and binding to membrane CD28, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1 (INSMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:2 (AINEKLLIYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3 (DLYGSDYWD); CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4 (INAMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:5 (AISGGGDTYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:6 (DMIEQQWWY); CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4 (INAMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:5 (AISGGGDTYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:7 (DTHRGVYWY); CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8 (IKTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9 (AINYIKEIYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10 (DVTKEDYWY); CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11 (INSMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 (AISNAREVYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13 (DVYFQEYWY); CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14 (INTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 (AINSISRTYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10 (DVTKEDYWY); CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8 (IKTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:16 (AIASDNRKYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10 (DVTKEDYWY); CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17 (IRTMA), CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18 (AISSGREVYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19 (DMYWQDYWW); or CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1 (INSMG), CDR2 comprises the amino acid sequence set forth in SEQ ID NO:20 (AISDRSEKYYADSVKG), and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:21 (DHHHSDWWT); Single domain antibodies (sdAbs).

2. 2. The sdAb of claim 1 which is a camelid antibody or a shark antibody.

3. The sdAb of claim 2 which is a VHH antibody.

4. The sequence from the N-terminus to CDR1 is X １ VQLVESGGGLVQX ２ GX ３ SLRLSCX ４ ASGSX ５ X ６ S (SEQ ID NO: X), wherein X １ is E or Q, and X ２ is A or P, and X ３ is E or G, and X ４ is A or K, and X ５ is I, L or T, and X ６ is A or F, and the sequence between CDR1 and CDR2 is WYRQAPGX ７ X ８ X ９ EX １０ VX １１ (SEQ ID NO: X), wherein X ７ is S or K, and X ８ is Q or G, and X ９ is R or L, and X １０ is L or R, and X １１ is one of A, S or T, and the sequence between CDR2 and CDR3 is １１ SRDNX １２ KX １３ TX １４ YLQMNX １５ LX １６ X １７ X １８ DX １９ X ２０ VYYCVV (SEQ ID NO: X), wherein X １１ is I or V, and X １２ is A or S, and X １３ is T or N, and X １４ is V, M or L, and X １５ is S or N, and X １６ is R, K or E, and X １７ is P or A, and X １８ is E or R, and X １９ is T or A, and X ２０ is A or G, and the sequence from the C-terminus to CDR3 is WGQGTX ２１ VTVSS (SEQ ID NO: X), wherein X ２１ is Q or L.

5. 5. The sdAb of claim 4, wherein the sequence from the N-terminus to CDR1 consists of EVQLVESGGGLVQAGESLRLSCAASGSIAS (SEQ ID NO: 22), the sequence between CDR1 and CDR2 consists of WYRQAPGSQRELVX (SEQ ID NO: 48), the sequence between CDR2 and CDR3 consists of RFTISRDNAKTTVYLQMNSLRPEDTAVYYCVV (SEQ ID NO: 24), and the sequence from the C-terminus to CDR3 consists of WGQGTQVTVSS (SEQ ID NO: 25), wherein X is A or T.

6. 2. The sdAb of claim 1, comprising a sequence selected from the group consisting of: EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAINEKLLIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDLYGSDYWDWGQGTQVTVSS (SEQ ID NO: 26); b. EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMIEQQWWYWGQGTQVTVSS (SEQ ID NO: 27); c. EVQLVESGGGLVQAGESLRLSCAASGSIASINAMGWYRQAPGSQRELVAAISGGGDTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDTHRGVYWYWGQGTQVTVSS (SEQ ID NO: 28); d. EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVAAINYIKEIYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO: 29); e. EVQLVESGGGLVQAGESLRLSCAASGSIASINSMAWYRQAPGSQRELVAAISNAREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVYFQEYWYWGQGTQVTVSS (SEQ ID NO: 30); f. EVQLVESGGGLVQAGESLRLSCAASGSIASINTMAWYRQAPGSQRELVAAINSISRTYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO: 31); g. EVQLVESGGGLVQAGESLRLSCAASGSIASIKTMAWYRQAPGSQRELVTAIASDNRKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDVTKEDYWYWGQGTQVTVSS (SEQ ID NO: 32); h. EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKQRELVTAIASDNRKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 70); i. EVQLVESGGGLVQPGGSLRLSCKASGSIASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 71); j. EVQLVESGGGLVQPGGSLRLSCAASGSTASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 72); k. EVQLVESGGGLVQPGGSLRLSCKASGSTASIKTMAWYRQAPGKGLELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 73); EVQLVESGGGLVQPGGSLRLSCAASGSIASIKTMAWYRQAPGKGRELVTAIASDNRKYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDVTKEDYWYWGQGTLVTVSS (SEQ ID NO: 74); m. EVQLVESGGGLVQAGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDMYWQDYWWWGQGTQVTVSS (SEQ ID NO: 33); n. EVQLVESGGGLVQPGESLRLSCAASGSIASIRTMAWYRQAPGSQRELVAAISSGREVYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTQVTVSS (SEQ ID NO: 75); EVQLVESGGGLVQPGGSLRLSCKASGSIASIRTMAWYRQAPGKGLELVAAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 76); p.EVQLVESGGGLVQPGGSLRLSCKASGSTASIRTMAWYRQAPGKGLELVSAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 77); EVQLVESGGGLVQPGGSLRLSCAASGSIASIRTMAWYRQAPGKGLELVSAISSGREVYYADSVKGRFTISRDNSKTTVYLQMNSLRAEDTAVYYCVVDMYWQDYWWWGQGTLVTVSS (SEQ ID NO: 78); or r.EVQLVESGGGLVQAGESLRLSCAASGSIASINSMGWYRQAPGSQRELVAAISDRSEKYYADSVKGRFTISRDNAKTTVYLQMNSLRPEDTAVYYCVVDHHHSDWWTWGQGTQVTVSS (SEQ ID NO: 34).

7. is not a CD28 agonist; and / or is not a CD28 antagonist; and / or does not degrade mCD28 and does not inhibit mCD28-mediated immune cell activation; 2. The sdAb of claim 1.

8. The sdAb of claim 1, wherein the agent binds within the stalk region of CD28.

9. 9. The sdAb of claim 8, wherein the stalk region comprises the amino acid sequence GKHLCPSPLFPGPSKP (SEQ ID NO: 35) or KGKHLCPSPLFPGPS (SEQ ID NO: 36), or consists of the amino acid sequence HVKGKHLCPSPLFPGPSKP (SEQ ID NO: 37).

10. A dimeric agent comprising at least two membrane CD28 (mCD28)-binding single domain antibodies (sdAbs), wherein a first mCD28-binding sdAb is linked to a second mCD28-binding sdAb by a linker.

11. 11. The dimeric agent of claim 10 comprising the sdAb of claim 1.

12. 11. The dimeric agent of claim 10, wherein the first sdAb and the second sdAb comprise the same sequence.

13. 11. The dimeric agent of claim 10, wherein the first sdAb and the second sdAb comprise different sequences.

14. The dimeric agent of claim 10, which inhibits proteolytic cleavage of mCD28.

15. 11. The dimeric agent of claim 10, wherein the first sdAb, the second sdAb or both when not part of a dimeric agent are CD28 antagonists, and the dimeric agent is not a CD28 antagonist.

16. 11. The dimeric agent of claim 10, comprising a first polypeptide comprising the first sdAb and a second polypeptide comprising the second sdAb, wherein the linker links the first polypeptide and the second polypeptide.

17. 17. The dimeric agent of claim 16, wherein the first polypeptide comprises a first free cysteine ​​amino acid on the outside of the first sdAb, the second polypeptide comprises a second free cysteine ​​amino acid on the outside of the second sdAb, and the linker comprises a bond between the first and second free cysteine ​​amino acids.

18. 18. The dimeric agent of claim 17, wherein the first free cysteine, the second free cysteine, or both are C-terminal amino acids.

19. 17. The dimerization agent of claim 16, wherein the first polypeptide comprises the first sdAb and a first dimerization domain, the second polypeptide comprises the second sdAb and a second dimerization domain, and the linker comprises the dimerization domains, a bond between the dimerization domains, or both.

20. 20. The dimerization agent of claim 19, wherein the first dimerization domain comprises a first immunoglobulin (Ig) hinge domain, the second dimerization domain comprises a second Ig hinge domain, and the linker comprises a disulfide bond between the first Ig hinge domain and the second Ig hinge domain.

21. 20. The dimerization agent of claim 19, wherein the first sdAb is N-terminal to the first dimerization domain, the second sdAb is N-terminal to the second dimerization domain, or both.

22. 21. The dimeric agent of claim 20, wherein the Ig hinge domain is a human Ig hinge domain comprising the amino acid sequence DKTHTCPPCPAPE (SEQ ID NO: 83) or ESKYGPPCPPCPAPEFEGG (SEQ ID NO: 85).

23. 21. The dimeric agent of claim 20, wherein the first sdAb is separated from the first Ig hinge domain by an amino acid linker, the second sdAb is separated from the second Ig hinge domain by an amino acid linker, or both.

24. 24. The dimeric agent of claim 23, wherein the amino acid linker is a flexible linker comprising a sequence selected from (GGGGS)n, (GS)n, (GGS)n, (GSGGS)n, (EGGGS)n, (EGGS)n and combinations thereof, where n is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8.

25. 20. The dimerization agent of claim 19, wherein the first dimerization domain, the second dimerization domain, or both, further comprise an Ig heavy chain CH2 domain, an Ig heavy chain CH3 domain, and wherein the hinge domain is N-terminal to the CH2 domain and the CH2 domain is N-terminal to the CH3 domain.

26. 20. The dimeric agent of claim 19, wherein the dimerization domain contains at least one mutation that does not induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) or that reduces ADCC or CDC.

27. the dimerizing agent comprises a first polypeptide comprising the first sdAb and a first dimerization domain, and a second polypeptide comprising the second sdAb and a second dimerization domain; The dimerization domain is selected from the group consisting of DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39) and ESKYGPPCPPPCPAP 11. The dimeric agent of claim 10, comprising EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 92), or a sequence having at least 95% identity thereto.

28. 28. The dimerization agent of claim 27, comprising an sdAb consisting of SEQ ID NO: 74, a dimerization domain consisting of SEQ ID NO: 92 or a sequence with at least 95% identity thereto, and a linker connecting the sdAb to the dimerization domain, wherein the linker comprises five repeats of SEQ ID NO:

118.

29. 29. The dimeric agent of claim 28, consisting of, from N-terminus to C-terminus, SEQ ID NO: 74, five repeats of SEQ ID NO: 118, and SEQ ID NO:

92.

30. 11. The dimeric agent of claim 10, comprising a single polypeptide, wherein the single polypeptide comprises the first sdAb N-terminal to the second sdAb; (a) the first sdAb and the second sdAb are separated by an amino acid linker of less than 13 amino acids, and the dimeric agent is not a CD28 antagonist, inhibits ligand binding to CD28 by less than 50%, or both; or (b) the first sdAb and the second sdAb are separated by an amino acid linker of 10 or more amino acids, and the dimeric agent is a CD28 antagonist; Dimerizing agents.

31. 31. A pharmaceutical composition comprising the sdAb of any one of claims 1 to 9 or the dimeric agent of any one of claims 10 to 30, and a pharmaceutically acceptable carrier, excipient or adjuvant.

32. The pharmaceutical composition of claim 31 for use in the treatment and / or prevention of cancer in a subject in need thereof, for improving PD-1 and / or PD-L1 based immunotherapy for the treatment of cancer in a subject in need thereof, or both, wherein the cancer may be selected from melanoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, and colorectal cancer.

33. At least one sdAb according to any one of claims 1 to 9 or a dimerising agent according to any one of claims 10 to 30; at least one of: (a) an anti-PD-1 and / or PD-L1 immunotherapy; and (b) a label stating that the agent of the invention is for use in conjunction with a PD-1 and / or PD-L1-based immunotherapy; Includes a kit.

34. 1. A method for producing a dimeric agent that inhibits proteolytic cleavage of mCD28 on the surface of a cell, comprising: a. i. Obtaining an agent that binds to cell surface mCD28 and blocks cleavage of said mCD28 by proteases; ii. linking a first portion of the drug to a second portion of the drug via a linker to form a dimeric drug; iii. testing the ability of the dimeric agent to block cleavage of mCD28 on the cell surface by a protease; and iv. selecting a dimeric agent that blocks cleavage of mCD28 on the cell surface; and b. Culturing a host cell comprising one or more vectors comprising one or more nucleic acid sequences encoding a dimeric agent, wherein said one or more nucleic acid sequences are: i. obtaining an agent that binds to mCD28 on the cell surface and blocks cleavage of said mCD28 by proteases (the obtained agent may be an sdAb); ii. linking a first portion of the drug to a second portion of the drug via a linker to form a dimeric drug; iii. Testing the ability of the dimeric agent to block cleavage of mCD28 on the cell surface by proteases; and iv. Selecting agents that block cleavage of mCD28 on the cell surface wherein the dimerization agent is a dimerization agent selected by and assaying mCD28 downstream signaling in the presence of the resulting dimeric agents, and selecting at least one dimeric agent that (a) neither substantially agonizes nor substantially antagonizes mCD28 signaling; or (b) substantially antagonizes mCD28 signaling; thereby generating an agent that inhibits the proteolytic cleavage of mCD28 on the surface of cells, method.