Anti-TLR7 agents and compositions and methods of making and using the same

Abstract

Compositions and methods for making and using anti-TLR7 agents, such as monoclonal antibodies, TLR7 binding antibody fragments and derivatives, as well as kits, nucleic acids encoding such molecules, diagnostic reagents and kits comprising anti-TLR7 agents, and methods of making and using the same are described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 962,457, filed on January 17, 2020, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Field of the Invention

[0003] The present invention relates in part to agents that bind to Toll-like receptor 7, i.e., TLR7 and variants thereof, particularly monoclonal antibodies, antibody fragments, and antibody derivatives that specifically react with TLR7 under physiological and / or in vitro conditions. Such agents can be used for laboratory / research purposes (e.g., flow cytometry), and can be used to treat and / or prevent various diseases or conditions by delivering drugs or other compositions containing such agents. Background of the Invention

[0005] The following description includes information that may be helpful in understanding the present technology. No information provided herein, or any publication specifically or implicitly referenced herein, is admitted to be prior art or even particularly relevant to the presently claimed technology.

[0006] Toll-like receptors (TLRs) are present as transmembrane proteins on many cells of the immune system and in certain cells, and are usually involved in innate immune responses by recognizing pathogen-associated molecular patterns derived from various bacteria, fungi, parasites and viruses. The innate immune system responds rapidly to the invasion of microorganisms and acts as the first line of defense, in which innate immune cells such as macrophages or dendritic cells (DCs) play a central role in the production of proinflammatory cytokines or nitric oxide. TLRs also participate in the effective establishment of acquired immunity against pathogens by directly detecting molecules from microorganisms. The adaptive immune system produces highly specific responses to microorganisms by producing antibodies from B cells or by producing killer or helper T lymphocytes, thereby producing lifelong immune memory. This process may take weeks or even months to establish a sufficient level of immunity. Some TLR types are located on the cell surface (e.g., detecting and initiating responses to extracellular pathogens), while other TLR types are located inside the cell (e.g., detecting and initiating responses to intracellular pathogens).

[0007] Pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) specific to each pathogen are typically expressed on innate immune cells and distinguish between self or non-self structures. More than 10 members of the TLR family are present in mammals and function as PRRs, recognizing various PAMPs such as lipopolysaccharides, lipoproteins, nucleic acids, etc. In humans, 10 different TLR types have been identified, referred to as TLR1-10, while in mice, 13 TLR types have been identified. TLR1-TLR10 are conserved between humans and mice, although TLR10 is not functional in mice due to retroviral insertions. TLR11-TLR13 are not present in humans.

[0008] TLRs are type I transmembrane proteins that consist of three major domains: (1) a leucine-rich binding domain; (2) a transmembrane domain; and (3) a cytoplasmic TIR (i.e., Toll-IL-1R) domain. The TIR domain is a highly conserved intracellular domain found in IL-1R and various TLRs. For some TLRs (e.g., TLR1, TLR2, TLR4, TLR5, TLR6, TLR10), ligand recognition by TLRs is mediated by a binding domain containing leucine-rich repeats (LRRs), which consist of 19 to 25 tandem copies of the "xLxxLxLxx" motif.

[0009] TLRs can be divided into two types: extracellular and intracellular. TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11 recognize their ligands on the cell surface. In contrast, TLR3, TLR7, TLR8 and TLR9 are located intracellularly on the endoplasmic reticulum (ER) membrane of resting cells.

[0010] Ligand binding typically leads to dimerization of the cytoplasmic TIR domains, ultimately activating downstream intracellular signaling mediated by TLR domain-containing adaptor proteins. Similar to mammalian IL-1R signaling, TLR signaling also activates NF-κB as well as mitogen-activated protein kinases (MAPKs) to stimulate gene expression, including proinflammatory cytokines and co-stimulatory molecules.

[0011] Genomic nucleic acids from bacteria and viruses or their analogs stimulate the production of proinflammatory cytokines and type I interferons. Among them, immunostimulatory bacterial DNA was first discovered in Bacillus Calmette-Guerin bacilli, which can promote the antitumor activity of human peripheral blood leukocytes and induce type I interferons (IFN-α / β) and type II interferons (IFN-γ).

[0012] TLR7 is an intracellular receptor expressed on the endosomal membrane. TLR7 is closely related to TLR8, which also recognizes nucleosides and nucleotides from intracellular pathogens. TLR7 has two ligand binding sites. The first site, which binds small ligands, is conserved in both TLR7 and TLR8. The second site, which is different from that of TLR8, is used to bind single-stranded RNA (ssRNA) to enhance activation of the first site. Activation of TLR7 can induce type 1 interferon and inflammatory responses.

[0013] Described herein are specific monoclonal antibodies against TLR7 that provide superior target specificity, signal-to-noise ratio, etc. compared to other reported anti-TLR antibodies, as well as antigen-binding fragments of such antibodies that also bind to TLR7. SUMMARY OF THE INVENTION

[0015] In some aspects, provided herein are anti-TLR7 agents that bind to Toll-like receptor 7 (TLR7), including anti-TLR7 antibodies, TLR7-binding antibody fragments, derivatives and variants of such antibodies, and antibody fragments (including immunoconjugates, labeled antibodies and antigen-binding antibody fragments, etc.), diagnostic reagents containing such agents, containers and kits comprising the anti-TLR7 agents provided herein, and methods of making and using the same.

[0016] In certain aspects, provided herein are anti-TLR7 agents that bind to TLR7 under laboratory or physiological conditions, wherein the agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, wherein a) each immunoglobulin heavy chain variable domain of the anti-TLR7 agent comprises a first, a second, and a third heavy chain complementarity determining region (CDR), wherein the first heavy chain CDR (CDRH1) comprises a sequence identical to the amino acid sequence X1YX2X3X4 (SEQ ID NO: 30) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is D or N, X2 is W, Y or C, X3 is M, V or I, and X4 is S, A or H; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is D or N, X2 is W, Y or C, X3 is M, V or I, and X4 is S, A or H; 10 X 11 X 12 X 13 KX 14or (SEQ ID NO:31), wherein X1 is D, S, Y or F, X2 is K, T, S or N, X3 is Y, N or P, X4 is D, S, E or Y, X5 is G or S, X6 is S, T, R or G, X7 is F, T, S or Y, X8 is I or T, X9 is D, Y, H or N, X10 is D, S, Y or F, X11 is D, S, Y or F, X12 is K, T, S or N, X13 is Y, N or P, X14 is D, S, E or Y, X15 is G or S, X16 is S, T, R or G, X17 is F, T, S or Y, X18 is I or T, X19 is D, Y, H or N, X20 is K, T, S or N, X21 is Y, N or P, X22 is K, T, S or N, X23 is Y, N or P, X24 is D, S, E or Y, X25 is G or S, X26 is S, T, R or G, X27 is F, T, S or Y, X28 is I or T, X29 is D, Y, H or N, X30 is Y, N or P, X31 is D, S, Y or F, X32 is Y, N or P, X33 is Y, N or P, X34 is D, S, E or Y, X35 is Y, N or P, X36 is D, S, E or Y, X37 is Y, N or P, X38 is Y, N or P, X39 is Y, N or P, X40 is D, S, E or Y, X41 is D, S, E or Y, X42 is D 10 is A, R, G or N, X 11 is P, D or E, X 12 Is S or K, X 13 L, V or F, and X 14 is N, G, A, S or T; and the third heavy chain CDR (CDRH3) comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X 16 or (SEQ ID NO:32), wherein X1 is D, E, H or G, X2 is L, G or P, X3 is T, G or Y, X4 is no amino acid, T, D or G, X5 is no amino acid, L or G, X6 is no amino acid or Y, X7 is V, Y or S, X8 is V, S, P or G, X9 is D or N, X10 is D, E, H or G, X20 is L, G or P, X30 is T, G or Y, X40 is no amino acid, T, D or G, X50 is no amino acid, L or G, X60 is no amino acid or Y, X70 is V, Y or S, X80 is V, S, P or G, X90 is D or N, X110 is D, E, H or G, X120 is L, G or P, X130 is T, G or Y, X140 is no amino acid, T, D or G, X150 is no amino acid, L or G, X160 is no amino acid or Y, X170 is V, Y or S, X180 is V, S, P or G, X190 is D or N, X111 is D, E, H or G, X120 is L, G or P, X130 is T, G or Y, X140 is no amino acid, T, D or G, X140 is 10 is G, Y or W, X 11 Is no amino acid or N, X 12 No amino acid or Y, X 13 No amino acid or V, X 14 No amino acid, R or Y, X 15 A or D, and X 16 Y or F; and b) each immunoglobulin light chain variable domain of the anti-TLR7 agent comprises a first, a second and a third light chain CDR, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence X1X2SX3X4X5X6X7X8X9X 10(SEQ ID NO:33) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence wherein X1 is R or L, X2 is A or P, X3 is E or Q, X4 is D or S, X5 is I or L, X6 is Y or S, X7 is N or T, X8 is V, E, N or S, X9 is L or I, and ... 10 is A or H; the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein X1 is N or Y, X2 is N or S, X3 is R, N, S or Q, X4 is L or P, X5 is H, Q or I, and X6 is N, T, D or S; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein X1 is N or Y, X2 is N or S, X3 is R, N, S or Q, X4 is L or P, X5 is H, Q or I, and X6 is N, T, D or S. NO:35) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence wherein X1 is Q or L, X2 is Y, D or S, X3 is Y or S, X4 is D or S, X5 is Y or S, and X6 is N, H, W, F or Y.

[0017] Also provided in certain aspects is a first anti-TLR7 agent that binds to TLR7 under laboratory or physiological conditions, wherein the first agent competitively binds to a second anti-TLR7 agent, the second agent comprising at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, wherein a) each immunoglobulin heavy chain variable domain of the second agent comprises a first, a second and a third heavy chain complementarity determining region (CDR), wherein the first heavy chain CDR (CDRH1) comprises a residue identical to the amino acid sequence X1YX2X3X4 (SEQ ID NO: 30) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is D or N, X2 is W, Y or C, X3 is M, V or I, and X4 is S, A or H; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is D or N, X2 is W, Y or C, X3 is M, V or I, and X4 is S, A or H; 10 X 11 X 12 X 13 KX 14or (SEQ ID NO:31), wherein X1 is D, S, Y or F, X2 is K, T, S or N, X3 is Y, N or P, X4 is D, S, E or Y, X5 is G or S, X6 is S, T, R or G, X7 is F, T, S or Y, X8 is I or T, X9 is D, Y, H or N, X10 is D, S, Y or F, X11 is D, S, Y or F, X12 is K, T, S or N, X13 is Y, N or P, X14 is D, S, E or Y, X15 is G or S, X16 is S, T, R or G, X17 is F, T, S or Y, X18 is I or T, X19 is D, Y, H or N, X20 is K, T, S or N, X21 is Y, N or P, X22 is K, T, S or N, X23 is Y, N or P, X24 is D, S, E or Y, X25 is G or S, X26 is S, T, R or G, X27 is F, T, S or Y, X28 is I or T, X29 is D, Y, H or N, X30 is Y, N or P, X31 is D, S, Y or F, X32 is Y, N or P, X33 is Y, N or P, X34 is D, S, E or Y, X35 is Y, N or P, X36 is D, S, E or Y, X37 is Y, N or P, X38 is Y, N or P, X39 is Y, N or P, X40 is D, S, E or Y, X41 is D, S, E or Y, X42 is D 10 is A, R, G or N, X 11 is P, D or E, X 12 Is S or K, X 13 L, V or F, and X 14 is N, G, A, S or T; and the third heavy chain CDR (CDRH3) comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X 16 or (SEQ ID NO:32), wherein X1 is D, E, H or G, X2 is L, G or P, X3 is T, G or Y, X4 is no amino acid, T, D or G, X5 is no amino acid, L or G, X6 is no amino acid or Y, X7 is V, Y or S, X8 is V, S, P or G, X9 is D or N, X10 is D, E, H or G, X11 is D, E, H or G, X12 is L, G or P, X13 is T, G or Y, X14 is no amino acid, T, D or G, X15 is no amino acid, L or G, X16 is no amino acid or Y, X17 is V, Y or S, X18 is V, S, P or G, X19 is D or N, X20 is L, G or P, X21 is T, G or Y, X22 is L, G or P, X23 is T, G or Y, X24 is no amino acid, T, D or G, X25 is no amino acid, L or G, X26 is no amino acid or Y, X27 is V, Y or S, X28 is V, S, P or G, X29 is D or N, X30 is T, G or Y, X31 is T, G or Y, X32 is T, G or Y, X33 is T, G or Y, X34 is T, D or G, X35 is T, D or G, X36 is T, G or Y, X37 is T, D or G, X38 is T, G or P, X39 is T, G or Y, X40 is T, D or G, X41 is T, D or G, X42 is T, D or G, X43 is T, D or G, X44 10 is G, Y or W, X 11 Is no amino acid or N, X 12 No amino acid or Y, X 13 No amino acid or V, X 14 No amino acid, R or Y, X 15 A or D, and X 16 Y or F; and b) each immunoglobulin light chain variable domain of the anti-TLR7 agent comprises a first, a second and a third light chain CDR, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence X1X2SX3X4X5X6X7X8X9X 10(SEQ ID NO:33) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence wherein X1 is R or L, X2 is A or P, X3 is E or Q, X4 is D or S, X5 is I or L, X6 is Y or S, X7 is N or T, X8 is V, E, N or S, X9 is L or I, and ... 10 is A or H; the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein X1 is N or Y, X2 is N or S, X3 is R, N, S or Q, X4 is L or P, X5 is H, Q or I, and X6 is N, T, D or S; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein X1 is N or Y, X2 is N or S, X3 is R, N, S or Q, X4 is L or P, X5 is H, Q or I, and X6 is N, T, D or S. NO:35) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence wherein X1 is Q or L, X2 is Y, D or S, X3 is Y or S, X4 is D or S, X5 is Y or S, and X6 is N, H, W, F or Y.

[0018] Also provided in certain aspects is a first anti-TLR7 agent that binds to Toll-like receptor 7 (TLR7) under laboratory or physiological conditions, wherein the first agent binds to the same epitope as a second anti-TLR7 agent, the second agent comprising at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, wherein a) each immunoglobulin heavy chain variable domain of the second agent comprises a first, a second and a third heavy chain complementarity determining region (CDR), wherein the first heavy chain CDR (CDRH1) comprises a sequence identical to the amino acid sequence X1YX2X3X4 (SEQ ID NO: 30) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is D or N, X2 is W, Y or C, X3 is M, V or I, and X4 is S, A or H; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is D or N, X2 is W, Y or C, X3 is M, V or I, and X4 is S, A or H; 10 X 11 X 12 X 13 KX 14or (SEQ ID NO:31), wherein X1 is D, S, Y or F, X2 is K, T, S or N, X3 is Y, N or P, X4 is D, S, E or Y, X5 is G or S, X6 is S, T, R or G, X7 is F, T, S or Y, X8 is I or T, X9 is D, Y, H or N, X10 is D, S, Y or F, X11 is D, S, Y or F, X12 is K, T, S or N, X13 is Y, N or P, X14 is D, S, E or Y, X15 is G or S, X16 is S, T, R or G, X17 is F, T, S or Y, X18 is I or T, X19 is D, Y, H or N, X20 is K, T, S or N, X21 is Y, N or P, X22 is K, T, S or N, X23 is Y, N or P, X24 is D, S, E or Y, X25 is G or S, X26 is S, T, R or G, X27 is F, T, S or Y, X28 is I or T, X29 is D, Y, H or N, X30 is Y, N or P, X31 is D, S, Y or F, X32 is Y, N or P, X33 is Y, N or P, X34 is D, S, E or Y, X35 is Y, N or P, X36 is D, S, E or Y, X37 is Y, N or P, X38 is Y, N or P, X39 is Y, N or P, X40 is D, S, E or Y, X41 is D, S, E or Y, X42 is D 10 is A, R, G or N, X 11 is P, D or E, X 12 Is S or K, X 13 L, V or F, and X 14 is N, G, A, S or T; and the third heavy chain CDR (CDRH3) comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X 16 or (SEQ ID NO:32), wherein X1 is D, E, H or G, X2 is L, G or P, X3 is T, G or Y, X4 is no amino acid, T, D or G, X5 is no amino acid, L or G, X6 is no amino acid or Y, X7 is V, Y or S, X8 is V, S, P or G, X9 is D or N, X10 is D, E, H or G, X20 is L, G or P, X30 is T, G or Y, X40 is no amino acid, T, D or G, X50 is no amino acid, L or G, X60 is no amino acid or Y, X70 is V, Y or S, X80 is V, S, P or G, X90 is D or N, X110 is D, E, H or G, X120 is L, G or P, X130 is T, G or Y, X140 is no amino acid, T, D or G, X150 is no amino acid, L or G, X160 is no amino acid or Y, X170 is V, Y or S, X180 is V, S, P or G, X190 is D or N, X111 is D, E, H or G, X120 is L, G or P, X130 is T, G or Y, X140 is no amino acid, T, D or G, X140 is 10 is G, Y or W, X 11 Is no amino acid or N, X 12 No amino acid or Y, X 13 No amino acid or V, X 14 No amino acid, R or Y, X 15 A or D, and X 16 Y or F; and b) each immunoglobulin light chain variable domain of the anti-TLR7 agent comprises a first, a second and a third light chain CDR, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence X1X2SX3X4X5X6X7X8X9X 10(SEQ ID NO:33) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence wherein X1 is R or L, X2 is A or P, X3 is E or Q, X4 is D or S, X5 is I or L, X6 is Y or S, X7 is N or T, X8 is V, E, N or S, X9 is L or I, and ... 10 is A or H; the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein X1 is N or Y, X2 is N or S, X3 is R, N, S or Q, X4 is L or P, X5 is H, Q or I, and X6 is N, T, D or S; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein X1 is N or Y, X2 is N or S, X3 is R, N, S or Q, X4 is L or P, X5 is H, Q or I, and X6 is N, T, D or S. NO:35) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence wherein X1 is Q or L, X2 is Y, D or S, X3 is Y or S, X4 is D or S, X5 is Y or S, and X6 is N, H, W, F or Y.

[0019] Also provided in certain aspects are anti-TLR7 agents for detecting TLR7 in a heterogeneous immune cell population, wherein significant levels of TLR7 are detected in plasmacytoid dendritic cells (pDCs) and / or B cells in the cell population, and TLR7 is not significantly detected in other immune cells in the cell population.

[0020] Also provided in certain aspects are methods of detecting TLR7 in a heterogeneous population of immune cells, comprising contacting the population of cells with an anti-TLR7 agent provided herein, wherein TLR7 is not significantly detected in other immune cells in the population of cells.

[0021] On the one hand, the present invention provides an isolated, non-naturally occurring anti-TLR7 agent that binds to Toll-like receptor 7 (TLR7) under physiological conditions, particularly an antibody or an antigen-binding fragment or derivative thereof. In the case of an anti-TLR7 antibody or antigen-binding fragment, such a molecule typically comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. In such a molecule, each immunoglobulin heavy chain and light chain variable domain comprises the following arrangement of the first, second and third chain complementary determining regions (CDRs): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0022] In the heavy chain variable domain portion, the first heavy chain CDR (CDRH1) comprises an amino acid sequence having at least 65% sequence identity, optionally at least 80%, at least 90%, at least 95% and 100% sequence identity to the amino acid sequence DYWMS, NYYMA, DYYMA, DYCVH or DYYIH (as shown in SEQ ID NOs: 1-5, respectively), and the second heavy chain CDR (CDRH2) comprises an amino acid sequence having at least 65% sequence identity, optionally at least 80%, at least 90%, at least 95% and 100% sequence identity to the amino acid sequence DYWMS, NYYMA, DYYMA, DYCVH or DYYIH (as shown in SEQ ID NOs: 1-5, respectively). NO: 6-11) having at least 65% sequence identity, optionally having at least 80%, at least 90%, at least 95% and 100% sequence identity, and the third heavy chain CDR (CDRH3) comprises an amino acid sequence having at least 65% sequence identity, optionally having at least 80%, at least 90%, at least 95% and 100% sequence identity with the amino acid sequence DLTT--VVDG----FAY, DLTT--VVDG----FAY, EGGDLYYSNYNYVRFAY, HGG---YPNW---YFDF, GPYGGYSGDG----FDY or GPYGGYSGDG----FDY (as shown in SEQ ID NO: 12-15, respectively).

[0023] In the light chain variable domain portion, the first light chain CDR (CDRL1) comprises an amino acid sequence having at least 65% sequence identity, optionally at least 80%, at least 90%, at least 95% and 100% sequence identity to the amino acid sequence of RASEDIYNVLA, RASEDIYNELA, LPSEDIYNNLA or RASQSLSTSIH (as shown in SEQ ID NOs: 16-19, respectively), the second light chain CDR (CDRL2) comprises an amino acid sequence having at least 65% sequence identity, optionally 100% sequence identity to the amino acid sequence of NANRLHN, NANNLHT, NANSLHT, YASNLQD or YASQPIS (as shown in SEQ ID NOs: 20-24, respectively), and the third light chain CDR (CDRL3) comprises an amino acid sequence having at least 65% sequence identity, optionally 100% sequence identity to the amino acid sequence of QQYYDYPNT, QQYYDYPHT, QQYYDYPWT, LQDSDYPFT or QQSYSSPYT (as shown in SEQ ID NOs: 21-24, respectively). NO: 25-29) has at least 65% sequence identity, optionally at least 80%, at least 90%, at least 95% and 100% sequence identity.

[0024] In some embodiments, the isolated, non-naturally occurring anti-TLR7 antibody or TLR7 binding fragment thereof comprises a first heavy chain CDR having the amino acid sequence DYYIH (SEQ ID NO: 5), a second heavy chain CDR having the amino acid sequence FINPDSGYTNYNEKFKT (SEQ ID NO: 11), a third heavy chain CDR having the amino acid sequence GPYGGYSGDG----FDY (SEQ ID NO: 15), a first light chain CDR having the amino acid sequence RASQSLSTSIH (SEQ ID NO: 19), a second light chain CDR having the amino acid sequence YASQPIS (SEQ ID NO: 24), and a third light chain CDR having the amino acid sequence QQSYSSPYT (SEQ ID NO: 29).

[0025] In some embodiments, the isolated anti-TLR7 agent comprises a non-naturally occurring anti-TLR7 antibody (mAb) comprising two immunoglobulin heavy chain variable domains comprising first, second, and third heavy chain complementarity determining regions (CDRH1-3, respectively), and two immunoglobulin light chain variable domains comprising first, second, and third light chain complementarity determining regions (CDRL1-3, respectively), wherein the antibody comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain having CDRH1-3 and CDRL1-3 selected from the group consisting of:

[0026]

[0027] In some embodiments, the isolated anti-TLR7 agent comprises a non-naturally occurring anti-TLR7 antibody (mAb) comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, wherein the immunoglobulin heavy chain variable domain has an amino acid sequence selected from SEQ ID NOs: 36-41 or an amino acid sequence having at least 65%-95% or more sequence identity with any such heavy chain variable domain sequence, and the immunoglobulin light chain variable domain is selected from SEQ ID NOs: 42-47 or an amino acid sequence having at least 65%-95% or more sequence identity with any such light chain variable domain sequence.

[0028] In some embodiments, when the anti-TLR7 agent is an antibody or an antigen-binding antibody fragment thereof, the antibody (or its fragment) is a monoclonal antibody and can be a camel, human, humanized, mouse, rabbit or other mammalian antibody or antigen-binding antibody fragment. In some embodiments, the antibody (antigen-binding antibody fragment) is IgG. In other embodiments, the IgG is IgG1, IgG2a or IgG2b or IgG3 or IgG4.

[0029] In certain embodiments where the anti-TLR7 antibodies and antigen-binding antibody fragments are other than fully human antibodies (i.e., antibodies produced or derived from a mammal capable of producing all or a portion of the human antibody repertoire), the molecules are chimeric or humanized anti-TLR7 antibodies and antigen-binding antibody fragments.

[0030] In some embodiments, the anti-TLR7 antibody, antigen-binding antibody fragment, or derivative or variant thereof comprises a detectable label.

[0031] In some embodiments, the anti-TLR7 agent, eg, an anti-TLR7 antibody, antigen-binding antibody fragment, or derivative or variant thereof, is part of an immunoconjugate, which further includes a cytotoxic agent, eg, a nucleic acid, a peptide, a polypeptide, a small molecule, or an aptamer.

[0032] A related aspect of the technology described herein relates to a composition comprising an anti-TLR7 agent, which is an isolated, non-naturally occurring anti-TLR7 antibody or antigen-binding antibody fragment according to the technology described herein. In addition to containing the anti-TLR7 antibody or antigen-binding antibody fragment described herein, such a composition generally also includes a carrier, such as a pharmaceutically acceptable carrier. Such a composition can be packaged in a container, and in some embodiments, these containers are further packaged into a kit that also includes instructions for use. In the context of a pharmaceutical composition, such a kit instruction is a package insert that contains not only instructions or uses, but also information about the active ingredient of the pharmaceutical (e.g., an anti-TLR7 antibody, an antigen-binding antibody fragment, or a derivative or variant thereof).

[0033] Another related aspect relates to a diagnostic kit configured to detect TLR7 in a biological sample, typically a biological sample taken from a subject. Such a kit includes a diagnostic reagent, which includes an anti-TLR7 agent as described herein, such as an anti-TLR7 antibody, antigen-binding antibody fragment, or a derivative or variant thereof conjugated to a detectable agent such as a fluorophore or an enzyme substrate and / or immobilized on a solid support.

[0034] Other aspects of the technology provided herein also relate to the production of anti-TLR7 agents described herein.In the case of anti-TLR7 antibodies (or antigen-binding antibody fragments or derivatives or variants thereof), one such aspect relates to isolated nucleic acid molecules encoding polypeptides provided herein. In some embodiments, such nucleic acids encode immunoglobulin heavy chain variable domains having: a first heavy chain CDR (CDRH1) comprising an amino acid sequence having at least 65%, optionally at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of DYWMS, NYYMA, DYYMA, DYCVH, or DYYIH (as shown in SEQ ID NOs: 1-5, respectively); and a second heavy chain CDR (CDRH2) comprising an amino acid sequence having at least 65%, optionally at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of DIKYDGSFIDYAPSLKN, DIKYDGTFIDYAPSLKN, SITNSGRTTYYRDSVKG, SISYEGSSTHYGDSVKA, YINPYSGYTNYNEKFKS, or FINPDSGYTNYNEKFKT (as shown in SEQ ID NOs: 1-5, respectively). NO:6-11) having at least 65%, optionally having at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% sequence identity, and a third heavy chain CDR (CDRH3) comprising an amino acid sequence having at least 65%, optionally having at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence DLTT--VVDG----FAY, EGGDLYYSNYNYVRFAY, HGG---YPNW---YFDF or GPYGGYSGDG----FDY (as shown in SEQ ID NO:12-15, respectively).Such nucleic acid may also encode an immunoglobulin light chain variable domain, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence having at least 65%, optionally at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence RASEDIYNVLA, RASEDIYNELA, LPSEDIYNNLA or RASQSLSTSIH (as shown in SEQ ID NOs: 16-19, respectively), and the second light chain CDR (CDRL2) comprises an amino acid sequence having at least 65%, optionally at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence NANRLHN, NANNLHT, NANSLHT, YASNLQD or YASQPIS (as shown in SEQ ID NOs: 16-19, respectively). NO: 20-24) having at least 65%, optionally having at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% sequence identity, and the third light chain CDR (CDRL3) comprises an amino acid sequence that has at least 65%, optionally having at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence QQYYDYPNT, QQYYDYPHT, QQYYDYPWT, LQDSDYPFT or QQSYSSPYT (as shown in SEQ ID NO: 25-29, respectively).

[0035] In certain embodiments, the nucleic acid molecules provided herein encode an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain, wherein the heavy chain variable domain has an amino acid sequence selected from SEQ ID NOs: 36-41, or an amino acid sequence that has at least 65%-95% or more sequence identity to any such heavy chain variable domain sequence, and the light chain variable domain has an amino acid sequence selected from SEQ ID NOs: 42-47, or an amino acid sequence that has at least 65%-95% or more sequence identity to any such light chain variable domain sequence.

[0036] Related aspects relate to plasmids, expression cassettes and vectors carrying the nucleic acids provided herein, and recombinant host cells transfected with such nucleic acid molecules.

[0037] Other aspects of the technology provided herein relate to methods for treating or preventing diseases or conditions associated with abnormal TLR7 levels (e.g., cancer, infection, tissue damage, autoimmune diseases, inflammation, etc.). This method includes administering to a subject in need of such treatment an amount of an anti-TLR7 agent provided herein (e.g., an anti-TLR7 antibody or its antigen-binding fragment, derivative, or variant) sufficient to achieve treatment, thereby treating or preventing the disease or condition. Such diseases and conditions that can be treated in this way include non-viral cancers (e.g., breast cancer, glioma, prostate cancer, non-small cell lung cancer, ovarian cancer, etc.), virus-related cancers such as cervical cancer associated with human papillomavirus (HPV) infection, cancer associated with HBV infection (e.g., hepatocellular carcinoma), cancer associated with Epstein-Barr virus (EBV) infection, cancer associated with polyomavirus infection, erythema nodosum leprosum (ENL), autoimmune diseases (e.g., systemic lupus erythematosus), autoimmune inflammation, Autoimmune thyroid disease associated with increased TLR7 expression, B cell lymphoma, T cell lymphoma, acute myeloid leukemia, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B cell leukemia, myelodysplastic syndrome, solid phase cancer, herpes virus infection and rejection of transplanted tissue or organ, etc. In some cases, the anti-TLR7 agents provided herein (e.g., anti-TLR7 antibodies or antigen-binding fragments, derivatives or variants thereof) can be used as adjuvants or in combination with adjuvants (e.g., for vaccines).

[0038] Other aspects of the technology provided herein relate to diagnostic methods, such as in vitro or in vivo diagnostic assays, using the anti-TLR7 agents provided herein to detect the presence of TLR7.

[0039] Certain embodiments are further described in the following description, examples, claims and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings illustrate certain embodiments of the present technology and are not limiting. For clarity and ease of illustration, the drawings are not drawn to scale, and in some cases, various aspects may be exaggerated or enlarged to aid in understanding specific embodiments.

[0042] Figure 1 The amino acid sequence of a representative full-length human TLR7 precursor is shown (SEQ ID NO: 48). In this sequence, the amino acid residues that make up the signal peptide (residues 1-26) are underlined. Residues 27-1,049 comprise the mature, processed form of the protein. See, e.g., NCBI Reference Sequence: NP_057646.1.

[0043] Figure 2The amino acid sequences of the immunoglobulin heavy chain (SEQ ID NO: 36-41) and light chain (SEQ ID NO: 42-47) variable domains of 6 different anti-TLR7 antibodies (AB 1-6) provided herein are shown. The CDR regions of each heavy chain and light chain are shown in bold and underlined. In each alignment, three characters ("*", ":", and ".") are used: "*" indicates a position with a single completely conserved residue; ":" indicates that one of the following "strong" residue groups is completely conserved: STA; NEQK; NHQK; NDEQ; QHRK; MILV; MILF; HY; and FYW; and "." indicates that one of the following "weaker" residue groups is completely conserved: CSA; ATV; SAG; STNK; STPA; SGND; SNDEQK; NDEQHK; NEQHRK; FVLIM; and HFY. These are all positive scoring residue groups that appear in the GonnetPam250 matrix. “Strong” and “weak” residue groups were defined as “strong” scores > 0.5 and “weak” scores, respectively. < 0.5.

[0044] Figure 3 Information on the anti-TLR7 antibodies used in the experiments described in the Examples below is listed.

[0045] 4A-4C are scatter plots showing the results of the gating strategy used for the flow cytometry experiments described in Example 3, below.

[0046] Figure 5 , Panels AD show the surface staining results of the experiments described in Example 3 below. Figure 5 , Figures AD show histograms of the experimental results described in Example 3 below.

[0047] Figure 6 , Panels AD show the intracellular staining results of the experiments described in Example 3 below. Figure 6 , Figures AD show histograms of the experimental results described in Example 3 below.

[0048] 7A-7F show Figure 6 The results presented in Figure 7A show mean fluorescence intensity (MFI) and signal-to-noise ratio (S:N; Figure 7B, 7D, and 7F) for pDCs, B cells, and monocytes.

[0049] Figures 8A and 8B show graphs of the results of the functional assays described in Example 4 below. Figures 8A and 8B show IL-8 secretion by 293XL-hTLR7 cells after treatment with R848 and hTLR7 antibodies provided herein. The dotted line shows cells treated with R848 alone. Figure 8A shows a representative graph showing IL-8 secretion. Figure 8B shows the fold change in IL-8 secretion with antibody treatment relative to R848 treated with isotype antibodies.

[0050] Fig. 9 The results of the blocking assay described in Example 5 are shown. The top two rows show the results of the isotype control (rat IgG1, κ; rat IgG2a, κ). The PE-conjugated anti-TLR7 antibody ( Fig. 9 , Figure AG) are listed on the y-axis. DETAILED DESCRIPTION OF THE INVENTION

[0052] Provided herein are agents that bind to TLR7. In some aspects, provided herein are antibodies and fragments thereof that bind to TLR7. For example, described herein are specific TLR7 monoclonal antibodies that provide superior target specificity, signal-to-noise ratio, etc. compared to other reported anti-TLR7 antibodies, as well as antigen-binding fragments of such antibodies that bind to TLR7. Also provided herein are methods for producing anti-TLR7 agents, particularly anti-TLR7 antibodies, having desired properties including affinity and / or specificity for TLR7 and / or its variants.

[0053] Antibody generation and characterization

[0054] The anti-TLR7 agents (e.g., anti-TLR7 antibodies) provided herein can have strong binding affinity and / or specificity to TLR7. In some embodiments, the anti-TLR7 agent can be a chimeric antibody. In some embodiments, the anti-TLR7 agent can be a humanized antibody. In some embodiments, the anti-TLR7 agent can be a variant antibody. For example, from a therapeutic perspective, the antibody can have a beneficial property. The assay for determining the activity of the anti-TLR7 antibodies provided herein includes, for example, cell-based ELISA (e.g., measuring the relative affinity of the antibody to the target on the cell), flow cytometry (e.g., measuring the cell specificity of the antibody) and cytotoxicity (e.g., measuring the potential of mediating direct or indirect killing of target cells expressing TLR7). In some cases, an immunogenic response cannot be induced after a therapeutically effective amount of humanized antibodies or variant antibodies are administered to a human patient. In some cases, if an immunogenic response is induced, the response can cause the antibody to still provide a therapeutic benefit for the patient treated with it.

[0055] In some embodiments, the anti-TLR7 agents herein (e.g., anti-TLR7 antibodies, humanized anti-TLR7 antibodies) bind to the same epitope. In order to screen for antibodies that bind to an epitope bound by an antibody of interest on TLR7 (e.g., those that block the binding of the antibody to TLR7), a cross-blocking assay is performed, as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). In some cases, epitope mapping can be performed to determine whether an antibody binds to an epitope of interest, as described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), Cunningham and Wells, Science 244: 1081–1085 (1989) or Davidson and Doranz, Immunology 143: 13–20 (2014).

[0056] The antibodies herein generally have a heavy chain variable domain comprising an amino acid sequence represented by the following formula: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, wherein "FRH1-4" represents 4 heavy chain framework regions, and "CDRH1-3" represents 3 hypervariable regions of the heavy chain variable domain of the anti-TLR7 antibody. FRH1-4 can be derived from a consensus sequence (e.g., the most common amino acids in a class, subclass, or subgroup of a heavy or light chain of a human immunoglobulin) or can be derived from a single human antibody framework region or from a combination of different framework region sequences. Many human antibody framework region sequences are compiled, for example, in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242. In one embodiment, the variable heavy chain FR is provided by the consensus sequence of the human immunoglobulin subgroup compiled by Kabat et al. as above.

[0057] The human variable heavy chain FR sequence may have substitutions therein, such as where a human FR residue is replaced by a corresponding non-human residue ("corresponding non-human residue" refers to a non-human residue with the same Kabat position number as the human residue of interest when aligning human and non-human sequences), but replacement with a non-human residue is not necessary. For example, replacement FR residues other than the corresponding non-human residue can be selected by phage display.

[0058] The antibody herein may have a light chain variable domain comprising an amino acid sequence represented by the formula: FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, wherein "FRL1-4" represents 4 framework regions, and "CDRL1-3" represents 3 hypervariable regions of the light chain variable domain of an anti-TLR7 antibody. FRL1-4 may be derived from a consensus sequence (e.g., the most common amino acids in a class, subclass, or subgroup of a heavy or light chain of a human immunoglobulin) or may be derived from a single human antibody framework region or from a combination of different framework region sequences. In one embodiment, the variable light chain FR is provided by a consensus sequence of a human immunoglobulin subgroup compiled by Kabat et al. as above.

[0059] The human variable light chain FR sequence may have a substitution therein, for example, wherein the human FR residue is replaced by the corresponding mouse residue, but the non-human residue substitution is not necessary. For example, the replacement residues other than the corresponding non-human residues can be selected by phage display. The method for producing the humanized anti-TLR7 antibody of interest herein is described in more detail below.

[0060] Anti-TLR7 agents

[0061] Provided herein are agents that bind to Toll-like receptor 7 (TLR7). Such agents may be referred to as anti-TLR7 agents and may include anti-TLR7 antibodies, anti-TLR7 antibody fragments (e.g., antigen binding fragments) and anti-TLR7 antibody derivatives. In some embodiments, the agent is isolated (e.g., isolated from components of its natural environment (e.g., animals, biological samples)). In some embodiments, the agent is non-naturally occurring (e.g., produced by human intervention). In some embodiments, the agent is a humanized antibody or an antigen binding fragment thereof. In some embodiments, the agent is a derivative of a humanized antibody that binds to TLR7. In some embodiments, the agent binds to TLR7 under laboratory conditions (e.g., in vitro binding to TLR7, in flow cytometry assays to TLR7, in ELISA to TLR7). In some embodiments, the agent binds to TLR7 under physiological conditions (e.g., binding to TLR7 in subject cells).

[0062] Generally, the anti-TLR7 agents provided herein include at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain. In some embodiments, the anti-TLR7 agents herein include two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. Generally, each immunoglobulin heavy chain variable domain of an anti-TLR7 agent includes the first, second and third heavy chain complementary determining regions (CDRs; CDRH1, CDRH2, CDRH3), and each immunoglobulin light chain variable domain of an anti-TLR7 agent includes the first, second and third light chain CDRs (CDRL1, CDRL2, CDRL3).

[0063] CDRH1

[0064] In some embodiments, the first heavy chain CDR (CDRH1) of the anti-TLR7 agent provided herein comprises an amino acid sequence at least 80% identical to the amino acid sequence X1YX2X3X4 (SEQ ID NO: 30), wherein X1 is D or N; X2 is W, Y or C; X3 is M, V or I; and X4 is S, A or H. In some embodiments, CDRH1 comprises an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO: 30. In some embodiments, CDRH1 comprises an amino acid sequence at least 95% identical to the amino acid sequence SEQ ID NO: 30. In some embodiments, CDRH1 comprises an amino acid sequence that is 100% identical to the amino acid sequence SEQ ID NO: 30.

[0065] The amino acid X1 of SEQ ID NO: 30 can be substituted by any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 30 is substituted by a conservative amino acid (e.g., conservatively substituted to D and / or N). In some embodiments, the amino acid X1 of SEQ ID NO: 30 is substituted by an acidic amino acid. In some embodiments, the amino acid is substituted by a basic amino acid.

[0066] The amino acid X2 of SEQ ID NO: 30 can be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 30 is substituted with a conservative amino acid (e.g., W, Y and / or C). In some embodiments, the amino acid X2 of SEQ ID NO: 30 is substituted with an aromatic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 30 is substituted with a neutral hydrophilic amino acid.

[0067] The amino acid X3 of SEQ ID NO: 30 can be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 30 is substituted with a conservative amino acid (e.g., conservatively substituted with M, V and / or I). In some embodiments, the amino acid X3 of SEQ ID NO: 30 is substituted with a hydrophobic amino acid.

[0068] The amino acid X4 of SEQ ID NO: 30 can be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 30 is substituted with a conservative amino acid (e.g., conservatively substituted with S, A and / or H). In some embodiments, the amino acid X4 of SEQ ID NO: 30 is substituted with a neutral hydrophilic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 30 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 30 is substituted with a basic amino acid.

[0069] In some embodiments, the CDRH1 of the anti-TLR7 agents provided herein comprises an amino acid sequence selected from DYWMS (SEQ ID NO: 1), NYYMA (SEQ ID NO: 2), DYYMA (SEQ ID NO: 3), DYCVH (SEQ ID NO: 4), and DYYIH (SEQ ID NO: 5).

[0070] CDRH2

[0071] In some embodiments, the second heavy chain CDR (CDRH2) of the anti-TLR7 agent provided herein comprises an amino acid sequence X1IX2X3X4X5X6X7X8X9YX 10 X 11 X 12 X 13 KX 14 (SEQ ID NO:31) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence of (SEQ ID NO:32), wherein X1 is D, S, Y or F; X2 is K, T, S or N; X3 is Y, N or P; X4 is D, S, E or Y; X5 is G or S; X6 is S, T, R or G; X7 is F, T, S or Y; X8 is I or T; X9 is D, Y, H or N; ... 10 is A, R, G or N; X 11 is P, D or E; X 12 is S or K; X 13 is L, V or F; and X 14is N, G, A, S or T. In some embodiments, the CDRH2 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CDRH2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CDRH2 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0072] The amino acid X1 of SEQ ID NO: 31 can be substituted by any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 31 is substituted by a conservative amino acid (e.g., conservative substitutions are D, S, Y and / or F). In some embodiments, the amino acid X1 of SEQ ID NO: 31 is substituted by an acidic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 31 is substituted by a neutral hydrophilic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 31 is substituted by an aromatic amino acid.

[0073] The amino acid X2 of SEQ ID NO: 31 can be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., conservatively substituted with K, T, S and / or N). In some embodiments, the amino acid X2 of SEQ ID NO: 31 is substituted with a basic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 31 is substituted with a neutral hydrophilic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 31 is substituted with a basic amino acid.

[0074] The amino acid X3 of SEQ ID NO: 31 can be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., a conservative substitution with Y, N and / or P). In some embodiments, the amino acid X3 of SEQ ID NO: 31 is substituted with an aromatic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 31 is substituted with a basic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 31 is substituted with an amino acid residue that affects the chain direction.

[0075] The amino acid X4 of SEQ ID NO: 31 can be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., conservative substitutions are D, S, E and / or Y). In some embodiments, the amino acid X4 of SEQ ID NO: 31 is substituted with an acidic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 31 is substituted with a neutral hydrophilic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 31 is substituted with an aromatic amino acid.

[0076] The amino acid X5 of SEQ ID NO: 31 can be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., conservatively substituted with G and / or S). In some embodiments, the amino acid X5 of SEQ ID NO: 31 is substituted with an amino acid residue that affects the chain direction. In some embodiments, the amino acid X5 of SEQ ID NO: 31 is substituted with a neutral hydrophilic amino acid.

[0077] The amino acid X6 of SEQ ID NO: 31 can be substituted by any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 31 is substituted by a conservative amino acid (e.g., conservative substitutions are S, T, R and / or G). In some embodiments, the amino acid X6 of SEQ ID NO: 31 is substituted by a neutral hydrophilic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 31 is substituted by a basic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 31 is substituted by an amino acid that affects the chain direction.

[0078] The amino acid X7 of SEQ ID NO: 31 can be substituted by any amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 31 is substituted by a conservative amino acid (e.g., conservative substitutions are F, T, S and / or Y). In some embodiments, the amino acid X7 of SEQ ID NO: 31 is substituted by an aromatic amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 31 is substituted by a neutral hydrophilic amino acid.

[0079] The amino acid X8 of SEQ ID NO: 31 can be substituted by any amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 31 is substituted by a conservative amino acid (e.g., conservatively substituted to I and / or T). In some embodiments, the amino acid X8 of SEQ ID NO: 31 is substituted by a hydrophobic amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 31 is substituted by a neutral hydrophilic amino acid.

[0080] The amino acid X9 of SEQ ID NO: 31 can be substituted by any amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 31 is substituted by a conservative amino acid (e.g., conservative substitutions are D, Y, H and / or N). In some embodiments, the amino acid X9 of SEQ ID NO: 31 is substituted by an acidic amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 31 is substituted by an aromatic amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 31 is substituted by a basic amino acid.

[0081] Amino acid X of SEQ ID NO: 31 10 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 31 10 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., conservatively substituted with A, R, G and / or N). 10 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a hydrophobic amino acid. 10 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a basic amino acid. 10 Substitutions by amino acid residues that affect chain direction.

[0082] Amino acid X of SEQ ID NO: 31 11 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 31 11 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., a conservative substitution with P, D and / or E). 11 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with an amino acid residue that affects the chain direction. 11 Replaced by acidic amino acids.

[0083] Amino acid X of SEQ ID NO: 31 12 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 31 12 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., a conservative substitution with S and / or K). 12 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a neutral hydrophilic amino acid. 12 Replaced by basic amino acids.

[0084] Amino acid X of SEQ ID NO: 31 13Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 31 13 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., a conservative substitution with L, V and / or F). 13 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a hydrophobic amino acid. 13 Replaced by aromatic amino acids.

[0085] Amino acid X of SEQ ID NO: 31 14 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 31 14 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a conservative amino acid (e.g., a conservative substitution with N, G, A, S and / or T). 14 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a basic amino acid. 14 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with an amino acid that affects the residues in the chain. 14 In some embodiments, amino acid X of SEQ ID NO: 31 is substituted with a hydrophobic amino acid. 14 Replaced by neutral hydrophilic amino acids.

[0086] In some embodiments, the CDRH2 of the anti-LR7 agent provided herein comprises an amino acid sequence selected from DIKYDGSFIDYAPSLKN (SEQ ID NO: 6), DIKYDGTFIDYAPSLKN (SEQ ID NO: 7), SITNSGRTTYYRDSVKG (SEQ ID NO: 8), SISYEGSSTHYGDSVKA (SEQ ID NO: 9), YINPYSGYTNYNEKFKS (SEQ ID NO: 10), and FINPDSGYTNYNEKFKT (SEQ ID NO: 11).

[0087] CDRH3

[0088] In some embodiments, the third heavy chain CDR (CDRH3) of the anti-TLR7 agent provided herein comprises an amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X 16(SEQ ID NO:32) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is D, E, H or G; X2 is L, G or P; X3 is T, G or Y; X4 is no amino acid, T, D or G; X5 is no amino acid, L or G; X6 is no amino acid or Y; X7 is V, Y or S; X8 is V, S, P or G; X9 is D or N; ... 10 is G, Y or W; X 11 Is no amino acid or N; X 12 Is no amino acid or Y; X 13 No amino acid or V; X 14 is no amino acid, R or Y; X 15 is A or D; and X 16 is Y or F. In some embodiments, the third heavy chain CDR (CDRH3) of the anti-TLR7 agent provided herein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CDRH3 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CDRH3 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0089] The amino acid X1 of SEQ ID NO: 32 can be substituted by any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 32 is substituted by a conservative amino acid (e.g., conservative substitutions are D, E, H and / or G). In some embodiments, the amino acid X1 of SEQ ID NO: 32 is substituted by an acidic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 32 is substituted by a basic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 32 is substituted by an amino acid residue that affects the chain direction.

[0090] The amino acid X2 of SEQ ID NO: 32 can be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., conservative substitutions to L, G and / or P). In some embodiments, the amino acid X2 of SEQ ID NO: 32 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 32 is substituted with an amino acid residue that affects the chain direction.

[0091] The amino acid X3 of SEQ ID NO: 32 can be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., a conservative substitution of T, G and / or Y). In some embodiments, the amino acid X3 of SEQ ID NO: 32 is substituted with a neutral hydrophilic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 32 is substituted with an amino acid that affects the chain direction. In some embodiments, the amino acid X3 of SEQ ID NO: 32 is substituted with an aromatic amino acid.

[0092] The amino acid X4 of SEQ ID NO: 32 can be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., a conservative substitution of T, D and / or G). In some embodiments, the amino acid X4 of SEQ ID NO: 32 is substituted with a neutral hydrophilic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 32 is substituted with an acidic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 32 is substituted with an amino acid residue that affects the chain direction.

[0093] The amino acid X5 of SEQ ID NO: 32 can be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., conservatively substituted with L and / or G). In some embodiments, the amino acid X5 of SEQ ID NO: 32 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 32 is substituted with an amino acid residue that affects the chain direction.

[0094] The amino acid X6 of SEQ ID NO: 32 can be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., conservatively substituted with Y). In some embodiments, the amino acid X6 of SEQ ID NO: 32 is substituted with an aromatic amino acid.

[0095] The amino acid X7 of SEQ ID NO: 32 can be substituted by any amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 32 is substituted by a conservative amino acid (e.g., conservative substitutions are V, Y and / or S). In some embodiments, the amino acid X7 of SEQ ID NO: 32 is substituted by a hydrophobic amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 32 is substituted by an aromatic amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 32 is substituted by a neutral hydrophilic amino acid.

[0096] The amino acid X8 of SEQ ID NO: 32 can be substituted by any amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 32 is substituted by a conservative amino acid (e.g., conservative substitutions are V, S, P and / or G). In some embodiments, the amino acid X8 of SEQ ID NO: 32 is substituted by a hydrophobic amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 32 is substituted by a neutral hydrophilic amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 32 is substituted by an amino acid residue that affects the chain direction.

[0097] The amino acid X9 of SEQ ID NO: 32 can be substituted by any amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 32 is substituted by a conservative amino acid (e.g., conservatively substituted to D and / or N). In some embodiments, the amino acid X9 of SEQ ID NO: 32 is substituted by an acidic amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 32 is substituted by a basic amino acid.

[0098] Amino acid X of SEQ ID NO:32 10 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 32 10 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., conservatively substituted with G, Y and / or W). 10 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with an amino acid residue that affects the chain direction. 10 Replaced by aromatic amino acids.

[0099] Amino acid X of SEQ ID NO:32 11 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 32 11 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a conservative amino acid (eg, a conservative substitution with N). 11 Replaced by basic amino acids.

[0100] Amino acid X of SEQ ID NO:32 12 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 32 12 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a conservative amino acid (eg, a conservative substitution with Y). 12 Replaced by aromatic amino acids.

[0101] Amino acid X of SEQ ID NO:3213 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 32 13 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., a conservative substitution with V). 13 Replaced by hydrophobic amino acids.

[0102] Amino acid X of SEQ ID NO:32 14 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 32 14 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a conservative amino acid (eg, a conservative substitution with R and / or Y). 14 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a basic amino acid. 14 Replaced by aromatic amino acids.

[0103] Amino acid X of SEQ ID NO:32 15 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 32 15 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., conservatively substituted with A and / or D). 15 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a hydrophobic amino acid. 15 Replaced by acidic amino acids.

[0104] Amino acid X of SEQ ID NO:32 16 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 32 16 In some embodiments, amino acid X of SEQ ID NO: 32 is substituted with a conservative amino acid (e.g., a conservative substitution with Y and / or F). 16 Replaced by aromatic amino acids.

[0105] In some embodiments, the CDRH3 of the anti-TLR7 agent provided herein comprises an amino acid sequence selected from DLTT--VVDG----FAY (SEQ ID NO: 12), EGGDLYYSNYNYVRFAY (SEQ ID NO: 13), HGG---YPNW---YFDF (SEQ ID NO: 14), and GPYGGYSGDG----FDY (SEQ ID NO: 15).

[0106] CDRL1

[0107] In some embodiments, the first light chain CDR (CDRL1) of the anti-TLR7 agent provided herein comprises an amino acid sequence X1X2SX3X4X5X6X7X8X9X 10 (SEQ ID NO: 33) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein X1 is R or L, X2 is A or P; X3 is E or Q; X4 is D or S; X5 is I or L; X6 is Y or S; X7 is N or T; X8 is V, E, N or S; X9 is L or I; and ... 10 is A or H. In some embodiments, CDRL1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, CDRL1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, CDRL1 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0108] The amino acid X1 of SEQ ID NO: 33 can be substituted by any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 33 is substituted by a conservative amino acid (e.g., conservatively substituted to R and / or L). In some embodiments, the amino acid X1 of SEQ ID NO: 33 is substituted by a basic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 33 is substituted by a hydrophobic amino acid.

[0109] The amino acid X2 of SEQ ID NO: 33 can be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 33 can be substituted with a conservative amino acid (e.g., conservative substitutions to A and / or P). In some embodiments, the amino acid X2 of SEQ ID NO: 33 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 33 is substituted with an amino acid that affects chain direction.

[0110] The amino acid X3 of SEQ ID NO: 33 can be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 33 can be substituted with a conservative amino acid (e.g., conservative substitutions to E and / or Q). In some embodiments, the amino acid X3 of SEQ ID NO: 33 is substituted with an acidic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 33 is substituted with a basic amino acid.

[0111] The amino acid X4 of SEQ ID NO: 33 can be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 33 can be substituted with a conservative amino acid (e.g., conservative substitutions to D and / or S). In some embodiments, the amino acid X4 of SEQ ID NO: 33 is substituted with an acidic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 33 is substituted with a neutral hydrophilic amino acid.

[0112] The amino acid X5 of SEQ ID NO: 33 may be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 33 may be substituted with a conservative amino acid (e.g., conservatively substituted with I and / or L). In some embodiments, the amino acid X5 of SEQ ID NO: 33 is substituted with a hydrophobic amino acid.

[0113] The amino acid X6 of SEQ ID NO: 33 can be substituted by any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 33 can be substituted by a conservative amino acid (e.g., conservatively substituted to Y and / or S). In some embodiments, the amino acid X6 of SEQ ID NO: 33 is substituted by an aromatic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 33 is substituted by a neutral hydrophilic amino acid.

[0114] The amino acid X7 of SEQ ID NO: 33 can be substituted by any amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 33 can be substituted by a conservative amino acid (e.g., conservatively substituted to N and / or T). In some embodiments, the amino acid X7 of SEQ ID NO: 33 is substituted by a basic amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 33 is substituted by a neutral hydrophilic amino acid.

[0115] The amino acid X8 of SEQ ID NO: 33 may be substituted by any amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 33 may be substituted by a conservative amino acid (e.g., conservative substitutions are V, E, N and / or S). In some embodiments, the amino acid X8 of SEQ ID NO: 33 is substituted by a hydrophobic amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 33 is substituted by an acidic amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 33 is substituted by a basic amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 33 is substituted by a neutral hydrophilic amino acid.

[0116] The amino acid X9 of SEQ ID NO: 33 may be substituted with any amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 33 may be substituted with a conservative amino acid (e.g., conservative substitution to L and / or I). In some embodiments, the amino acid X9 of SEQ ID NO: 33 is substituted with a hydrophobic amino acid.

[0117] Amino acid X of SEQ ID NO: 33 10 Can be substituted by any amino acid. In some embodiments, amino acid X of SEQ ID NO: 33 10 Can be substituted with conservative amino acids (e.g., conservative substitutions to A and / or H). In some embodiments, amino acid X of SEQ ID NO: 33 10 In some embodiments, amino acid X of SEQ ID NO: 33 is substituted with a hydrophobic amino acid. 10 Replaced by basic amino acids.

[0118] In some embodiments, the CDRL1 of the anti-TLR7 agents provided herein comprises an amino acid sequence selected from RASEDIYNVLA (SEQ ID NO: 16), RASEDIYNELA (SEQ ID NO: 17), LPSEDIYNNLA (SEQ ID NO: 18), and RASQSLSTSIH (SEQ ID NO: 19).

[0119] CDRL2

[0120] In some embodiments, the second light chain CDR (CDRL2) of the anti-TLR7 agent provided herein comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein X1 is N or Y, X2 is N or S, X3 is R, N, S or Q, X4 is L or P, X5 is H, Q or I, and X6 is N, T, D or S. In some embodiments, CDRL2 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 34. In some embodiments, CDRL2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO: 34. In some embodiments, CDRL2 comprises an amino acid sequence that is 100% identical to the amino acid sequence SEQ ID NO: 34.

[0121] The amino acid X1 of SEQ ID NO: 34 can be substituted with any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 34 is substituted with a conservative amino acid (e.g., conservatively substituted with N and / or Y). In some embodiments, the amino acid X1 of SEQ ID NO: 34 is substituted with a basic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 34 is substituted with an aromatic amino acid.

[0122] The amino acid X2 of SEQ ID NO: 34 can be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 34 is substituted with a conservative amino acid (e.g., conservatively substituted with N and / or S). In some embodiments, the amino acid X2 of SEQ ID NO: 34 is substituted with a basic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 34 is substituted with a neutral hydrophilic amino acid.

[0123] The amino acid X3 of SEQ ID NO: 34 can be substituted by any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 34 is substituted by a conservative amino acid (e.g., conservatively substituted by R, N, S and / or Q). In some embodiments, the amino acid X3 of SEQ ID NO: 34 is substituted by a basic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 34 is substituted by a neutral hydrophilic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 34 is substituted by a basic amino acid.

[0124] The amino acid X4 of SEQ ID NO: 34 can be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 34 is substituted with a conservative amino acid (e.g., conservatively substituted with L and / or P). In some embodiments, the amino acid X4 of SEQ ID NO: 34 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 34 is substituted with an amino acid that affects chain direction.

[0125] The amino acid X5 of SEQ ID NO: 34 can be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 34 is substituted with a conservative amino acid (e.g., conservatively substituted with H, Q and / or I). In some embodiments, the amino acid X5 of SEQ ID NO: 34 is substituted with a basic amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 34 is substituted with a hydrophobic amino acid.

[0126] The amino acid X6 of SEQ ID NO: 34 can be substituted by any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 34 is substituted by a conservative amino acid (e.g., a conservative substitution of N, T, D and / or S). In some embodiments, the amino acid X6 of SEQ ID NO: 34 is substituted by a basic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 34 is substituted by a neutral hydrophilic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 34 is substituted by an acidic amino acid.

[0127] In some embodiments, the CDRL2 of the anti-TLR7 agent provided herein comprises an amino acid sequence selected from NANRLHN (SEQ ID NO: 20), NANNLHT (SEQ ID NO: 21), NANSLHT (SEQ ID NO: 22), YASNLQD (SEQ ID NO: 23), and YASQPIS (SEQ ID NO: 24).

[0128] CDRL3

[0129] In some embodiments, the third light chain CDR (CDRL3) of the anti-TLR7 agent provided herein comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1QXXXPX6T (SEQ ID NO: 35), wherein X1 is Q or L; X2 is Y, D or S; X3 is Y or S; X4 is D or S; X5 is Y or S; and X6 is N, H, W, F or Y. In some embodiments, CDRL3 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 35. In some embodiments, CDRL3 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO: 35. In some embodiments, CDRL3 comprises an amino acid sequence that is 100% identical to the amino acid sequence SEQ ID NO: 35.

[0130] The amino acid X1 of SEQ ID NO: 35 can be substituted with any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 35 is substituted with a conservative amino acid (e.g., conservatively substituted with Q and / or L). In some embodiments, the amino acid X1 of SEQ ID NO: 35 is substituted with a basic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 35 is substituted with a hydrophobic amino acid.

[0131] The amino acid X2 of SEQ ID NO: 35 can be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 35 is substituted with a conservative amino acid (e.g., a conservative substitution of Y, D and / or S). In some embodiments, the amino acid X2 of SEQ ID NO: 35 is substituted with an aromatic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 35 is substituted with an acidic amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 35 is substituted with a neutral hydrophilic amino acid.

[0132] The amino acid X3 of SEQ ID NO: 35 can be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 35 is substituted with a conservative amino acid (e.g., conservatively substituted with Y and / or S). In some embodiments, the amino acid X3 of SEQ ID NO: 35 is substituted with an aromatic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 35 is substituted with a neutral hydrophilic amino acid.

[0133] The amino acid X4 of SEQ ID NO: 35 can be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 35 is substituted with a conservative amino acid (e.g., conservatively substituted with D and / or S). In some embodiments, the amino acid X4 of SEQ ID NO: 35 is substituted with an acidic amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 35 is substituted with a neutral hydrophilic amino acid.

[0134] The amino acid X5 of SEQ ID NO: 35 can be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 35 is substituted with a conservative amino acid (e.g., conservatively substituted with Y and / or S). In some embodiments, the amino acid X5 of SEQ ID NO: 35 is substituted with an aromatic amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 35 is substituted with a neutral hydrophilic amino acid.

[0135] The amino acid X6 of SEQ ID NO: 35 can be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 35 is substituted with a conservative amino acid (e.g., a conservative substitution of N, H, W, F and / or Y). In some embodiments, the amino acid X6 of SEQ ID NO: 35 is substituted with a basic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 35 is substituted with an aromatic amino acid.

[0136] In some embodiments, the CDRL3 of the anti-TLR7 agent provided herein comprises an amino acid sequence selected from QQYYDYPNT (SEQ ID NO: 25), QQYYDYPHT (SEQ ID NO: 26), QQYYDYPWT (SEQ ID NO: 27), LQDSDYPFT (SEQ ID NO: 28), and QQSYSSPYT (SEQ ID NO: 29).

[0137] CDR Group

[0138] In some embodiments, the anti-TLR7 agent comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain, the heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3), and the light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, the anti-TLR7 agent herein comprises two immunoglobulin heavy chain variable domains, each of which comprises a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains, each of which comprises a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). The CDR group may comprise any combination of the CDR amino acid sequences provided herein (i.e., CDRH1, CDRH2, CDRH3; and CDRL1, CDRL2, CDRL3). In some embodiments, the immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2 and CDRH3 amino acid sequences, and the immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2 and CDRL3 amino acid sequences, and the amino acid sequences are selected from Groups 1 to 6 provided in the following table. For an anti-TLR7 agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences, and each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences, and the amino acid sequences are selected from Groups 1 to 6 provided in the following table.

[0139]

[0140] In some embodiments, all CDRs are from the same group. For example, for an anti-TLR7 agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain can comprise a set of CDRH1, CDRH2, and CDRH3 amino acid sequences from Group 1, and each immunoglobulin light chain variable domain can comprise a set of CDRL1, CDRL2, and CDRL3 amino acid sequences from Group 1.

[0141] In some embodiments, the CDRs are from different groups. For example, for an anti-TLR7 agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain can comprise a set of CDRH1, CDRH2, and CDRH3 amino acid sequences from Group 1, and each immunoglobulin light chain variable domain can comprise a set of CDRL1, CDRL2, and CDRL3 amino acid sequences from Group 2. In another example, for an anti-TLR7 agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, one immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from Group 1, and the other immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from Group 2; and one immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from Group 1, and the other immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from Group 2.

[0142] VH

[0143] The anti-TLR7 agents provided herein may comprise a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence ELQLVESGGGLVKPGASLKLSCVASGFTFSDYWMSWVRQTPGKTMEWIGDIKYDGSFIDYAPSLKNRFTISRDNAKNTLYLQMSNVRSEDTATYYCARDLTTVVDGFAYWGQGTLVTVSS (SEQ ID NO: 36), for example, to the amino acid sequence SEQ IDNO: 36 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VH comprises a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 36.

[0144] The anti-TLR7 agents provided herein may comprise a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence ELQLVESGGGLVKPGASLKLSCVASGFTFSDYWMSWVRQTPGKTMEWIGDIKYDGT FIDYAPSLKNRFTISRDNAKNTLYLQMSNVRSEDTATYYCARDLTTVVDGFAYWGHG T LVTVSS (SEQ ID NO: 37), for example, to the amino acid sequence SEQ ID NO:37 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VH comprises a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO:37.

[0145] The anti-TLR7 agents provided herein may comprise a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence EVQLVESGGGLLQPGRSLKLSCAASGFTFTNYYMAWVRQAPTKGLEWVASITNSGRTTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCTREGGDLYYSNYNYVRFA YWGQGTLVTVSS (SEQ ID NO: 38), for example, to the amino acid sequence SEQ ID NO:38 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VH comprises a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO:38.

[0146] The anti-TLR7 agents provided herein may comprise a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence EVQLVESGGGLVQPGRSLKLSCAASGFTFRDYYMAWVRQAPKKGLEWVASISYEGSS THYGDSVKARFTVSRDDAKSTLYLQMNSLRSEDTATYYCGRHGGYPNWYFDFWGP GT MVTVSS (SEQ ID NO: 39), for example, to the amino acid sequence SEQ ID NO: 39 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VH comprises a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VH comprises a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VH comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 39.

[0147] The anti-TLR7 agents provided herein may comprise a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence QVNLLQSGAALVKPGASVKLSCKASGYTFTDYCVHWVKQSHGKSLEWIGYINPYSG YTNYNEKFKSKATLTVDTSTNTAYMELSRLTSDDSATCYCTRGPYGGYSGDG----FDYWGQGV MVTVSS (SEQ ID NO: 40), for example, to the amino acid sequence SEQ ID NO:40 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VH comprises a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH comprises a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO:40.

[0148] The anti-TLR7 agents provided herein may comprise a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence QVNLLQSGAALVKPGASVKLSCKASGYTFTDYYIHWVKQSHVKSLEWFGFINPDSG YTNYNEKFKTKATLTVDKSTNTAYMELSRLTSEGSATYYCTRGPYGGYSGDGFDYWGQGVMVTVSS (SEQ ID NO: 41), for example, to the amino acid sequence SEQ ID NO: 42. NO:41 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VH comprises a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO:41.

[0149] In some embodiments, the VH of the anti-TLR7 agent provided herein comprises a polypeptide selected from SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

[0150] V L

[0151] The anti-TLR7 agents provided herein may comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence NIVLTQSPATLSVTPGESVSLSCRASQSLSTSIHWYQQKPNESPRLLIRYASQPISGIPSRF SGSGSGTDFTLSINRVESEDFSIYYCQQSYSSPYTFGAGTKLELK (SEQ ID NO: 42), for example, to the amino acid sequence SEQ ID NO: 42 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VL comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VL comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 42.

[0152] The anti-TLR7 agents provided herein may comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence NIVLTQSPATLSVTPGESVSLSCRASQSLSTSIHWYQQMPNESPRLLIRYASQPISGIPSR FSGSGSGTDFTLSINRVESEDFSIYYCQQSYSSPYTFGAGTRLELK (SEQ ID NO: 43), for example, to the amino acid sequence SEQ ID NO: 43 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VL comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the VL comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 43.

[0153] The anti-TLR7 agents provided herein may comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence DIQMTQSPASLSASLGETVSIECLPSEDIYNNLAWYQQKPGKSPQLLIHYASNLQDGVPSRFSGSGSGTQYSLKIKSLESEDAATYFCLQDSDYPFTFGSGTKLEIK (SEQ ID NO: 44), for example, to the amino acid sequence SEQ ID NO: 44 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the VL comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the VL comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0154] The anti-TLR7 agents provided herein may comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence DIQMTQSPASLSASLGETVTIECRASEDIYNELAWYQQKPGKSPQLLIYNANSLHTGVP SRFSGSGSGTQYSLKINSLQSEDVASYFCQQYYDYPWTFGGGTKLELK (SEQ ID NO: 45), for example, to the amino acid sequence SEQ ID NO: 45 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the VL comprises a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the VL comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0155] The anti-TLR7 agents provided herein may comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence DIQMTQSPASLSASLGETVTIECRASEDIYNVLAWYQQKPGKSPQLLISNANRLHNGV PSRFSGSGSGTQYSLKINSLQSEDVASYFCQQYYDYPNTFGAGTKLELK (SEQ ID NO: 46), for example, to the amino acid sequence SEQ ID NO: 46 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the VL comprises a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the VL comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 46.

[0156] The anti-TLR7 agents provided herein may comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of the anti-TLR7 agents provided herein comprises a polypeptide that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence DIQMTQSPASLSASLGETVTIECRASEDIYNVLAWYQQKPGKSPQLLISNANNLHTGV PSRFSGSESGTQYSLKINSLQSEDVASYFCQQYYDYPHTFGAGTKLELK (SEQ ID NO: 47), for example, to the amino acid sequence SEQ ID NO: 47 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VL comprises a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VL comprises a polypeptide that is 100% identical to the amino acid sequence of SEQ ID NO: 47.

[0157] In some embodiments, the VL of the anti-TLR7 agent provided herein comprises a polypeptide selected from the group consisting of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47.

[0158] Fc

[0159] The anti-TLR7 agents provided herein may include a fragment crystallizable region (Fc region). The Fc region generally forms the tail of an antibody and may interact with certain cell surface receptors and certain components of the complement system. The Fc region may include, for example, two polypeptides, each of which is derived from the second (CH2) and third (CH3) constant domains of an antibody heavy chain.

[0160] The amino acid sequence of the wild-type CH2-CH3 portion of the Fc region is provided below (as located by EU index in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242) (SEQ ID NO: 51).

[0161]

[0162] In some embodiments, the Fc region includes one or more modifications (e.g., one or more amino acid substitutions, insertions or deletions relative to a comparable wild-type Fc region). Relative to an agent comprising a wild-type Fc region, an agent comprising a modified Fc region (variant agent) generally has an altered phenotype. The variant agent phenotype can be expressed as an altered serum half-life, an altered stability, an altered susceptibility to cellular enzymes, or an altered effector function (e.g., as determined in an NK-dependent or macrophage-dependent assay). Fc region modifications that alter effector function may include modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and reduce binding to inhibitory receptors (e.g., FcγRIIB (CD32B)) (see, e.g., Stavenhagen, JB et al. (2007) Cancer Res. 57 (18): 8882-8890)). Examples of human IgG1 Fc region variants with reduced binding to CD32B and / or increased binding to CD16A contain F243L, R292P, Y300L, V305I and / or P396L substitutions. The amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.

[0163] In some embodiments, the Fc region includes one or more modifications that reduce or eliminate Fc binding to Fc receptors. Such modifications may include amino acid substitutions at positions 234, 235, 265, and 297 (see, e.g., U.S. Patent No. 5,624,821, which is incorporated herein by reference). Exemplary substitutions include one or more of L234A, L235A, D265A, and N297Q. The amino acid positions correspond to the amino acid numbering of the CH2-CH3 domains provided above.

[0164] In some embodiments, the Fc region comprises one or more modifications that alter (relative to a wild-type Fc region) the ratio of affinity of the modified Fc region for an activating FcγR (e.g., FcγRIIA or FcγRIIIA) versus an inhibitory FcγR (e.g., FcγRIIB):

[0165]

[0166] When the modified Fc region has an affinity ratio greater than 1, the anti-TLR7 agents herein may have specific uses in providing therapeutic or prophylactic treatment for a disease, disorder or infection, or in improving symptoms thereof, wherein enhanced efficacy of effector cell function (e.g., ADCC) mediated by FcγR is desired, such as cancer or infectious diseases. In the case where the modified Fc region has an affinity ratio less than 1, the anti-TLR7 agents herein may have specific uses in providing therapeutic or prophylactic treatment for a disease or disorder, or in improving symptoms thereof, wherein enhanced efficacy of effector cell function mediated by FcγR is desired, such as autoimmune or inflammatory disorders. Table 3 lists exemplary single, double, triple, quadruple and quintuple amino acid substitutions with affinity ratios greater than 1 or less than 1 (see, e.g., PCT Publication Nos. WO 04 / 063351; WO 06 / 088494; WO 07 / 024249; WO 06 / 113665; WO 07 / 021841; WO 07 / 106707; WO 2008 / 140603, all of which are incorporated herein by reference). The amino acid positions correspond to the amino acid numbers of the CH2-CH3 domains provided above.

[0167]

[0168] In certain embodiments, an antibody of the present disclosure may comprise: (1) a heavy chain variable region having CDRH1, CDRH2 and CDRH3 as set forth in SEQ ID NOs: 4, 10 and 15, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequence of SEQ ID NO: 40, and (2) a light chain variable region having CDRL1, CDRL2 and CDRL3 as set forth in SEQ ID NOs: 19, 24 and 29, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequence of SEQ ID NO: 46. Such an antibody may be IgG1, IgG2, IgG3 or IgG4.

[0169] In certain embodiments, the antibody comprises a heavy chain having a heavy chain variable region having the sequence of SEQ ID NO:40 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55:

[0170] QVNLLQSGAALVKPGASVKLSCKASGYTFTDYCVHWVKQSHGKSLEWIGYINPYSG

[0171] YTNYNEKFKSKATLTVDTSTNTAYMELSRLTSDDSATCYCTRGPYGGYSGDGFDYWG

[0172] QGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[0173] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH

[0174] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

[0175] EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA

[0176] KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having a light chain variable region having the sequence of SEQ ID NO:46 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52:

[0177] DIQMTQSPASSLSASLGETVTIECRASEDIYNVLAWYQQKPGKSPQLLISNANRLHNGV

[0178] PSRFSGGSGTQYSLKINSLQSEDVASYFCQQYYDYPNTFGAGTKLELKRTVAAPSVFI

[0179] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0180] In certain embodiments, an antibody of the present disclosure may comprise: (1) a heavy chain variable region having CDRH1, CDRH2 and CDRH3 as set forth in SEQ ID NOs: 5, 11 and 15, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequence of SEQ ID NO: 41, and (2) a light chain variable region having CDRL1, CDRL2 and CDRL3 as set forth in SEQ ID NOs: 19, 24 and 29, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequence of SEQ ID NO: 47. Such an antibody may be IgG1, IgG2, IgG3 or IgG4.

[0181] In certain embodiments, the antibody comprises a heavy chain having a heavy chain variable region having the sequence of SEQ ID NO:41 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53:

[0182] QVNLLQSGAALVKPGASVKLSCKASGYTFTDYYIHWVKQSHVKSLEWFGFINPDSG

[0183] YTNYNEKFKTKATLTVDKSTNTAYMELSRLTSEGSATYYCTRGPYGGYSGDGFDYW

[0184] GQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

[0185] GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT

[0186] HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

[0187] VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0188] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having a light chain variable region having the sequence of SEQ ID NO:47 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:54:

[0189] DIQMTQSPASSLSASLGETVTIECRASEDIYNVLAWYQQKPGKSPQLLISNANNLHTGV

[0190] PSRFSGESGTQYSLKINSLQSEDVASYFCQQYYDYPHTFGAGTKLELKRTVAAPSVFI

[0191] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0192] Agents that competitively bind to anti-TLR7 agents

[0193] Provided herein is an anti-TLR7 agent that is competitively bound or capable of competitively binding to one or more anti-TLR7 agents described herein. In particular, provided herein is an anti-TLR7 agent that competes with or can compete for binding to TLR7 with one or more anti-TLR7 agents described herein. Such an agent that competes or can compete with an anti-TLR7 agent described herein can be referred to as a competitor agent. In some cases, when a competitor binds to the same TLR7 common region (i.e., an extracellular region or a leucine-rich binding domain) as an anti-TLR7 agent described herein, it can be considered that the agent (i.e., a competitor agent) competes for binding to TLR7. In some cases, when a competitor binds to the same TLR7 region as an anti-TLR7 agent described herein (e.g., the same peptide (linear epitope) or the same surface amino acid (conformational epitope)), it can be considered that the agent (i.e., a competitor agent) competes for binding to TLR7. In some cases, when the competitor binds to the same TLR7 common region as the anti-TLR7 agents described herein under suitable assay conditions (i.e., the extracellular region or the leucine-rich binding domain), it can be considered that the agent (i.e., the competitor agent) is able to compete for binding to TLR7. In some cases, when the competitor binds to the exact same TLR7 region as the anti-TLR7 agents described herein under suitable assay conditions (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)), it can be considered that the agent (i.e., the competitor agent) is able to compete for binding to TLR7.

[0194] In some cases, when a competitor blocks the binding of one or more anti-TLR7 agents described herein to TLR7, the agent (i.e., competitor agent) can be considered to compete for binding to TLR7. In some cases, when a competitor blocks the binding of one or more anti-TLR7 agents described herein to TLR7 under suitable assay conditions, the agent (i.e., competitor agent) can be considered to be able to compete for binding to TLR7. Whether a competitor blocks the binding of one or more anti-TLR7 agents described herein to TLR7 can be determined using a suitable competition assay or blocking assay, such as the blocking assay described in Example 6 herein. In a competition or blocking assay, a competitor agent can block the binding of one or more anti-TLR7 agents described herein to TLR7 by 50% or more, whereas, in a competition or blocking assay, one or more anti-TLR7 agents described herein can block the binding of a competitor agent to TLR7 by about 50% or more. For example, the agent (i.e., competitor agent) can block the binding of one or more anti-TLR7 agents described herein to TLR7 by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in a competition or blocking assay, whereas one or more anti-TLR7 agents described herein block the binding of the competitor agent to TLR7 by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in a competition or blocking assay.

[0195] In some cases, when a competitor binds to TLR7 with an affinity similar to one or more anti-TLR7 agents described herein, the agent (i.e., competitor agent) can be considered to compete for binding to TLR7. In some cases, when a competitor binds to TLR7 with an affinity similar to one or more anti-TLR7 agents described herein under suitable assay conditions, the agent (i.e., competitor agent) can be considered to be able to compete for binding to TLR7. In some embodiments, when a competitor binds to TLR7 with an affinity of at least about 50% of the affinity of one or more anti-TLR7 agents described herein, the agent (i.e., competitor agent) is considered to compete for binding to TLR7. For example, when a competitor binds to TLR7 with an affinity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the affinity of one or more anti-TLR7 agents described herein, the agent (i.e., competitor agent) can be considered to compete for binding to TLR7. Competitor agents may comprise any of the features described herein for anti-TLR7 agents.

[0196] Also provided herein is an anti-TLR7 agent that binds to or can bind to the same epitope as one or more anti-TLR7 agents described herein. In particular, provided herein is an anti-TLR7 agent that competes with one or more anti-TLR7 agents described herein for binding to the same epitope on TLR7. This agent that binds to the same epitope may be referred to as an epitope competitor. In some cases, an epitope competitor may bind to exactly the same TLR7 region (e.g., exactly the same peptide (linear epitope) or exactly the same surface amino acid (conformational epitope)) as an anti-TLR7 agent described herein. In some cases, an epitope competitor blocks the binding of one or more anti-TLR7 agents described herein to TLR7. In a competitive assay, an epitope competitor may block the binding of one or more anti-TLR7 agents described herein to TLR7 by about 50% or more, whereas, in a competitive assay, one or more anti-TLR7 agents described herein may block the binding of an epitope competitor to TLR7 by 50% or more. In some cases, an epitope competitor binds to TLR7 with an affinity similar to that of one or more anti-TLR7 agents described herein. In some embodiments, the epitope competitor binds to TLR7 with an affinity of at least about 50% of the affinity of one or more anti-TLR7 agents described herein. For example, the epitope competitor can bind to TLR7 with an affinity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the affinity of one or more anti-TLR7 agents described herein. The epitope competitor can comprise any of the features of the anti-TLR7 agents described herein.

[0197] Antibody preparation

[0198] In some embodiments, the anti-TLR7 agent is an antibody. Methods for producing anti-TLR7 antibodies and anti-TLR7 antibody variants are described in the following examples. In some embodiments, the anti-TLR7 agent is a humanized antibody or an antigen-binding fragment thereof. In some embodiments, the anti-TLR7 agent is a humanized antibody or a derivative thereof that binds to TLR7. Humanized anti-TLR7 antibodies can be prepared based on non-human anti-TLR7 antibodies. Fully human antibodies can also be prepared, for example, in genetically engineered (i.e., transgenic) mice (e.g., from Medarex), and when present with an immunogen, mice can produce human antibodies that do not necessarily require CDR transplantation. These antibodies are fully human (100% human protein sequence) antibodies from animals such as mice, in which non-human antibody genes are suppressed and replaced by human antibody gene expression. When TLR7 is presented to these genetically engineered mice or other animals that can produce the human framework of the relevant CDR, antibodies against TLR7 can be produced.

[0199] In the case of generating variants, a parent antibody is prepared. The following section describes exemplary techniques for generating such non-human antibodies and parent antibodies.

[0200] Antigen preparation

[0201] The antigen used to generate the antibody can be, for example, a complete TLR7, particularly a portion of TLR7 expressed in a cell (e.g., an N-terminal domain, a C-terminal domain, a cytoplasmic domain, an intracellular domain, a transmembrane domain, an extracellular domain, an extracellular domain, a TIR domain, a leucine-rich domain, or a TLR7 fragment containing a desired epitope). Other antigenic forms that can be used to generate antibodies are obvious to those skilled in the art.

[0202] Polyclonal antibodies

[0203] Polyclonal antibodies can be produced in animals (vertebrates or invertebrates, including mammals, birds and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It can be used to conjugate the relevant antigen to a protein or other carrier that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, using bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR, where R and R 1 Non-protein carriers (such as colloidal gold) can also be used for antibody production.

[0204] Animals are immunized against antigens, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted by subcutaneous injection of one-fifth to one-tenth of the original amount of peptide or conjugate in Freund's complete adjuvant at multiple sites. After 7 to 14 days, the animals are bled and the antibody titer of the serum is determined. The animals are boosted until the titer plateaus. Typically, animals are boosted with the same antigen but conjugates conjugated to different proteins and / or conjugates conjugated by different cross-linking agents. Conjugates can also be made into protein fusions in recombinant cell cultures. In addition, agglutinating agents such as alum are also suitable for enhancing immune responses.

[0205] Monoclonal antibodies

[0206] Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975) or can be prepared by other methods, such as recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, mice or other suitable host animals such as hamsters or macaques are immunized as described above to stimulate lymphocytes to produce or be able to produce antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0207] The hybridoma cells thus prepared are inoculated and grown in a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0208] Preferred myeloma cells are those that effectively fuse, support the selected antibody-producing cells to stably produce antibodies at high levels, and are sensitive to culture media such as HAT culture media. Among them, preferred myeloma cell lines are mouse myeloma cell lines, such as SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA, in Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0209] The production of monoclonal antibodies against the antigen in the culture medium in which the hybridoma cells are grown is determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation, in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), or by flow cytometry analysis of cells expressing membrane antigens. For example, the binding affinity of the monoclonal antibodies can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).

[0210] After hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting dilution methods and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown as ascites tumors in animals. Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites, or serum by conventional immunoglobulin purification methods such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0211] The DNA encoding the monoclonal antibody is easily separated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the heavy chain and light chain of the monoclonal antibody encoding the monoclonal antibody). Alternatively, cDNA can be prepared from mRNA, and then the cDNA is subjected to DNA sequencing. Hybridoma cells are used for cDNA preparation as a preferred source of such genomic DNA or RNA. Once separated, the DNA can be placed in expression vectors well known in the art, and then transfected into host cells such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not produce immunoglobulins in addition, to obtain the synthesis of monoclonal antibodies in recombinant host cells. The recombinant production of antibodies will be described in more detail below.

[0212] Humanization and amino acid sequence variants

[0213] General methods for antibody humanization are described in, for example, U.S. Patent Nos. 5861155, 6479284, 6407213, 6639055, 6500931, 5530101, 5585089, 5693761, 5693762, 6180370, 5714350, 6350861, 5777085, 5834597, 5882644, 5932448, 6013256, 6129914, 6210671, 6329511, 5225539, 6548640 and 5624821, all of which are incorporated herein by reference. In certain embodiments, it may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly where these variants improve the binding affinity or other biological properties of the antibody.

[0214] The amino acid sequence variants of anti-TLR7 antibodies are prepared by introducing appropriate nucleotide changes into the anti-TLR7 antibody DNA or by peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the anti-TLR7 antibodies of the embodiments herein. As long as the final construct has the desired characteristics, any combination of deletions, insertions and substitutions can be performed to obtain the final construct. Amino acid changes can also change the post-translational processes of humanized or variant anti-TLR antibodies, such as changing the number or position of glycosylation sites.

[0215] As described in Cunningham and Wells Science, 244: 1081-1085 (1989), a method for identifying certain residues or regions of anti-TLR7 antibodies as preferred positions for mutagenesis is called "alanine scanning mutagenesis". Here, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced by neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of amino acids with the TLR7 antigen. The amino acid positions that show functional sensitivity to substitution are then refined by introducing further or other variants at or for the substitution site. Therefore, although the site for introducing amino acid sequence changes is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the mutation performance of a given site, alanine scanning or random mutagenesis is performed at the target codon or region and the expressed anti-TLR7 antibody variants are screened for the desired activity. Amino acid sequence insertion includes amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertion variants include the fusion of an enzyme or polypeptide that increases the serum half-life of the antibody to the N- or C-terminus of the antibody.

[0216] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted at this position. The sites of greatest interest for substitution mutagenesis include hypervariable regions, but FR changes are also contemplated. Conservative substitutions are preferred, but more substantial changes can be introduced and the products can be screened. Examples of substitutions are shown below:

[0217] Examples of amino acid residue substitutions

[0218] Ala(A)val;leu;ile;val

[0219] Arg(R)lys;gln;asn;lys

[0220] Asn(N)gln;his;asp,lys;gln;arg

[0221] Asp(D)glu;asn

[0222] Cys(C)ser;ala

[0223] Gln(Q)asn;glu

[0224] Glu(E)asp;gln

[0225] Gly(G)ala

[0226] His(H)asn;gln;lys;arg

[0227] Ile(I)leu;val;met;ala;leu;phe;norleucine

[0228] Leu(L)norleucine;ile;val;ile;met;ala;phe

[0229] Lys(K)arg;gln;asn

[0230] Met(M)leu;phe;ile

[0231] Phe(F)leu;val;ile;ala;tyr

[0232] Pro(P)ala

[0233] Ser(S)thr

[0234] Thr(T)ser

[0235] Trp(W)tyr;phe

[0236] Tyr(Y)trp;phe;thr;ser

[0237] Val(V)ile;leu;met;phe;ala;norleucine

[0238] Substantial modification in the biological properties of antibodies is achieved by selecting substitutions whose effects vary significantly in maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Based on common side chain properties, naturally occurring residues are divided into the following categories:

[0239] (1) Hydrophobicity: norleucine, met, ala, val, leu, ile;

[0240] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0241] (3) Acidic: asp, glu;

[0242] (4) Basic: asn, gln, his, lys, arg;

[0243] (5) Residues that affect chain direction: gly, pro; and

[0244] (6) Aromatic: trp, tyr, phe.

[0245] Non-conservative substitutions replace a member of one of these classes with another class.

[0246] Any cysteine ​​residues not involved in maintaining the correct conformation of the antibody may also be substituted to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine ​​bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0247] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variant selected for further development has improved biological properties relative to the parent antibody that produces it. A convenient way to produce this substitution variant is to use phage-displayed affinity maturation. In short, several hypervariable region sites (e.g., 6 to 7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are displayed in a monovalent manner from filamentous phage particles as a fusion with the gene III product of the M13 packaged in each particle. The variants displayed by phage are then screened for their biological activity (e.g., binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or in addition, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the antigen. This contact residue and adjacent residue are candidate residues that are substituted according to the technology set forth herein. Once such variants are generated, the panel of variants is screened as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0248] Another type of amino acid variant of an antibody changes the original glycosylation pattern of the antibody. The change refers to the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is usually N-linked and / or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the most common recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0249] Addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be accomplished by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0250] Nucleic acid molecules encoding amino acid sequence variants of anti-TLR7 antibodies herein are prepared by various methods known in the art. These methods include, but are not limited to, separation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variants or non-variant forms of previously prepared anti-TLR7 antibodies.

[0251] Human Antibodies

[0252] As an alternative to humanization, human antibodies can be generated. For example, transgenic animals (e.g., mice) can be generated that, upon immunization, are capable of producing a complete repertoire of human antibodies without producing endogenous immunoglobulins. For example, chimeric and germline mutant mice can produce a complete repertoire of antibody heavy chain joining regions (J-J) H Homozygous deletion of the ) gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice can result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807). Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); and U.S. Pat. Nos. 5,565,332 and 5,573,905). Human antibodies can also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0253] Antigen-binding antibody fragment

[0254] In certain embodiments, the anti-TLR7 agent is an antibody fragment that retains at least one desired activity (including antigen binding). A variety of techniques have been developed to produce antibody fragments. In some cases, these fragments are derived from proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In some cases, these fragments are produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). In some cases, the leucine zipper GCN4 is used to promote the assembly of F(ab')2 molecules to form F(ab')2. According to another method, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0255] Multispecific antibodies and other agents

[0256] In some embodiments, the anti-TLR7 agent comprises a first binding moiety and a second binding moiety, wherein the first binding moiety specifically reacts with a first molecule of TLR7 and the second binding moiety specifically reacts with a second molecule of a molecular species different from the first molecule. Such an agent may comprise a plurality of first binding moieties, a plurality of second binding moieties, or a plurality of first binding moieties and a plurality of second binding moieties. Preferably, the ratio of the first binding moiety to the second binding moiety is about 1:1, although it may be in the range of about 1000:1 to about 1:1000, wherein the ratio may be measured according to valence.

[0257] In those embodiments where the first part is an antibody, the second binding moiety may also be an antibody. In some embodiments, the first and second parts are connected by a linker moiety, which may have two to hundreds or even thousands of valencies to connect the first and second binding moieties by one or different chemical structures. Examples of bispecific antibodies include those antibodies reactive to two different epitopes; in some cases, one epitope is a TLR7 epitope, and the second epitope is on an unrelated soluble molecule. In some embodiments, bispecific antibodies are reactive to epitopes on TLR7 and to epitopes on different molecules found on the same cell surface. In some embodiments, bispecific antibodies are reactive to epitopes on TLR7 and epitopes on different molecules found on different cell surfaces.

[0258] The composition herein may also include a first agent and a second agent, wherein the first agent includes a first binding moiety that specifically reacts with a first molecule (e.g., TLR7) and the second agent includes a second binding moiety that specifically reacts with a second molecule, the second molecule being a molecular species different from the first molecule. The first and / or second agent may be an antibody. The ratio of the first agent to the second agent may be in the range of about 1,000:1 to 1:1,000, although the preferred ratio is about 1:1. In some embodiments, it may be necessary to produce a multispecific (e.g., bispecific) anti-TLR7 antibody that has binding specificity to at least two different epitopes. Certain bispecific antibodies can bind to two different epitopes of TLR7. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0259] According to one approach for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises the C H At least a portion of the 3 domains. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule. This provides a mechanism for increasing heterodimer production to exceed other unwanted end products such as homodimers (see, for example, WO96 / 27011 published on September 6, 1996).

[0260] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, and some cross-linking techniques are also well known.

[0261] Any suitable technology can be used to produce bispecific antibodies from antibody fragments. For example, chemical bonds can be used to prepare bispecific antibodies. In some methods, intact antibodies are proteolytically cleaved to produce F (ab ') 2 fragments (see, for example, Brennan et al., Science 229: 81 (1985), which is incorporated herein by reference). These fragments are reduced in the presence of dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab ' fragments produced are then converted into thionitrobenzoate (TNB) derivatives. Then one of the Fab '-TNB derivatives is reconverted to Fab '-thiol by reduction with mercaptoethylamine and mixed with another Fab '-TNB derivative of equimolar amount to form bispecific antibodies. In another embodiment, the Fab '-SH fragments directly recovered from Escherichia coli can be chemically coupled in vitro to form bispecific antibodies (see, for example, Shalaby et al., J. Exp. Med. 175: 217-225 (1992), which is incorporated herein by reference).

[0262] Any suitable technique for preparing and isolating bispecific antibody fragments directly from recombinant cell culture can be used. For example, bispecific antibodies have been produced using leucine zippers (see, e.g., Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992), which is incorporated herein by reference). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced to form monomers at the hinge region and then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides another mechanism for preparing bispecific antibody fragments. The fragments contain a heavy chain variable domain (V H ), the heavy chain variable domain (V H ) is connected to the light chain variable domain (V L ), the linker is too short to allow pairing between the two domains on the same chain. H and V L The domain is forced to align with the complementary V L and V HThe domains are paired to form two antigen binding sites. Another strategy is to prepare bispecific antibody fragments by using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994), which is incorporated herein by reference). In some cases, bispecific antibodies can be "linear antibodies" produced as described in Zapata et al. Protein Eng. 8 (10): 1057-1062 (1995), which is incorporated herein by reference.

[0263] Antibodies with two or more valencies are contemplated herein. Antibodies (or polymers or polypeptides) in which each arm or fragment thereof comprises one or more binding sites will be referred to herein as "multivalent" antibodies. For example, a "bivalent" antibody herein is one in which each Fab or fragment thereof comprises two binding sites, while a "trivalent" polypeptide herein is one in which each Fab or fragment thereof comprises three binding sites. In the multivalent polymers herein, the two or more binding sites of each Fab may bind to the same or different antigens. For example, the two or more binding sites in the multivalent polypeptides herein may be directed against the same antigen, such as against the same portion or epitope of the antigen or against two or more identical or different portions or epitopes of the antigen; and / or may be directed against different antigens; or a combination thereof. Thus, the bivalent polypeptides herein may, for example, comprise two identical binding sites, may comprise a first binding site for a first portion or epitope of an antigen and a second binding site for the same portion or epitope of the antigen or against another portion or epitope of the antigen; or may comprise a first binding site for a first portion or epitope of an antigen and a second binding site for a different antigen. However, as will be clear from the above description, the technology herein is not limited thereto, as the multivalent polypeptides herein may comprise any number of binding sites for the same or different antigens. In one embodiment, the multivalent polypeptide comprises at least two ligand binding elements, one of which contains one or more CDR peptide sequences set forth herein. In another embodiment, the multivalent polypeptide comprises three ligand binding sites, each independently selected from the CDR sequences disclosed herein.

[0264] In certain embodiments, at least one ligand binding element binds TLR7. In one embodiment, at least one ligand binding element binds another target. In one embodiment, there are up to 10,000 binding elements in the multivalent binding molecule, and the ligand binding elements can be attached to a scaffold.

[0265] Antibodies (or polymers or polypeptides) containing at least two binding sites per Fab or fragment thereof herein may also be referred to as "multispecific", wherein at least one binding site is for a first antigen and a second binding site is for a second antigen different from the first antigen. Thus, a "bispecific" polymer comprises at least one site for a first antigen and at least one second site for a second antigen, while a "trispecific" is a polymer comprising at least one binding site for a first antigen, at least one further binding site for a second antigen, and at least one further binding site for a third antigen; and so on. Thus, in its simplest form, a bispecific polypeptide herein is a bivalent polypeptide (per Fab) of the technology provided herein. However, as will be clear from the above description, the technology herein is not limited thereto, as the multispecific polypeptides herein may comprise any number of binding sites for two or more different antigens.

[0266] Other modifications

[0267] Other modifications of anti-TLR7 agents are contemplated. For example, the technology herein also relates to immunoconjugates comprising antibodies described herein (e.g., anti-TLR7 antibodies) that are conjugated to cytotoxic agents such as toxins (e.g., enzyme-active toxins of bacterial, fungal, plant or animal origin or fragments thereof) or radioactive isotopes (e.g., radioactive conjugates) or cytotoxic drugs. Such conjugates are sometimes referred to as "agent-drug conjugates" or "ADCs". Conjugates are made using a variety of bifunctional protein coupling agents such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0268] Anti-TLR7 agents disclosed herein (e.g., anti-TLR7 antibodies) can be formulated into immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556. For example, liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of limited pore size to produce liposomes with a desired diameter. Fab' fragments of the antibodies provided herein can be conjugated to liposomes by a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257:286-288 (1982).Another active ingredient is optionally contained within the liposome.

[0269] Enzymes or other polypeptides can be covalently bound to anti-TLR7 agents (e.g., anti-TLR7 antibodies) by techniques well known in the art, such as using heterobifunctional cross-linking reagents discussed above. In some embodiments, fusion proteins comprising at least the antigen binding region of an antibody provided herein linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312: 604-608 (1984)).

[0270] In certain embodiments, for example, it may be desirable to use antibody fragments rather than complete antibodies to increase penetration of target tissues and cells. In this case, it may be desirable to modify the antibody fragment to increase its serum half-life. This can be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag and then fusing it to the antibody fragment at either end or in the middle, for example, by DNA or peptide synthesis methods; see, for example, WO96 / 32478, published on October 17, 1996).

[0271] Covalent modification of anti-TLR7 agents (e.g., anti-TLR7 antibodies) is also included in the scope of the present technology. For example, modification can be performed by chemical synthesis or by enzymatic or chemical cleavage of anti-TLR7 antibodies. By reacting the targeted amino acid residues of the antibody with an organic derivatizing agent that can react with the selected side chain or N- or C-terminal residue, other types of covalent modifications of the antibody are introduced into the molecule. Exemplary covalent modifications of polypeptides are described in U.S. Patent No. 5,534,615, which is specifically incorporated herein by reference. Preferred covalent modification types of antibodies include connecting the antibody to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0272] Nucleic acid, vector, host cell and recombinant method

[0273] The technology described herein also provides isolated nucleic acids encoding anti-TLR7 agents (eg, anti-TLR7 antibodies), vectors and host cells comprising the nucleic acids, and recombinant techniques for producing the agents or antibodies. The nucleic acids herein may include one or more subsequences, each of which is referred to as a polynucleotide.

[0274] Provided herein are nucleic acids (e.g., isolated nucleic acids) comprising nucleotide sequences encoding anti-TLR7 agents or antibodies or fragments thereof. In some embodiments, the nucleic acids encode immunoglobulin heavy chain variable domains of anti-TLR7 agents provided herein. In some embodiments, the nucleic acids encode immunoglobulin light chain variable domains of anti-TLR7 agents provided herein. In some embodiments, the nucleic acids encode immunoglobulin heavy chain variable domains and immunoglobulin light chain variable domains of anti-TLR7 agents provided herein. In some embodiments, the nucleic acids comprise nucleotide sequences encoding the amino acid sequences of any of SEQ ID NOs: 1-29 and 36-47. For example, the nucleic acids may comprise nucleotide sequences encoding the amino acid sequences of any of the CDRs of SEQ ID NOs: 1-29 and 36-47. The nucleic acids may comprise nucleotide sequences encoding the amino acid sequences of any of the immunoglobulin heavy chain variable domains of SEQ ID NOs: 36-41. The nucleic acids may comprise nucleotide sequences encoding the amino acid sequences of any of the immunoglobulin light chain variable domains of SEQ ID NOs: 42-47.

[0275] For the recombinant production of anti-TLR7 agents or antibodies, nucleic acids encoding anti-TLR7 agents or antibodies can be isolated and inserted into replicable vectors for further cloning (DNA amplification) or for expression. In some cases, anti-TLR7 agents or antibodies can be produced by homologous recombination, such as described in U.S. Patent No. 5,204,244, which is specifically incorporated herein by reference. DNA encoding anti-TLR7 agents or antibodies can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequences, replication origins, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, such as described in U.S. Patent No. 5,534,615, issued on July 9, 1996, which is specifically incorporated herein by reference.

[0276] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, e.g., Gram-negative or Gram-positive organisms, for example, the Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans and Shigella, and Bacilli, e.g., B. subtilis and B. licheniformis (e.g., B. licheniformis 41P, DD published April 12, 1989). 266,710), Pseudomonas such as Pseudomonas aeruginosa and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and not restrictive.

[0277] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-TLR7 agent / antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms. Many other genera, species and strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0278] Suitable host cells for expressing anti-TLR7 agents / antibodies (e.g., glycosylated anti-TLR7 agents / antibodies) are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding licensed insect host cells from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori (silk moths). Many virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, which can be used as viruses herein according to the present technology, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0279] Suitable host cells for expressing anti-TLR7 agents / antibodies may also include vertebrate cells (eg, mammalian cells). Vertebrate cells may be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse supporting cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).

[0280] Host cells can be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0281] Host cells for producing the agents / antibodies herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58: 44 (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Re. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM ), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art at appropriate concentrations may also be included. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

[0282] When using recombinant technology, the agent / antibody can be produced in the cell, periplasmic space or directly secreted into the culture medium. If the antibody is produced in the cell, as a first step, the particle debris, i.e., the fragment of the host cell or cracking, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for separating antibodies secreted into the periplasmic space of Escherichia coli. In short, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. In the case where the antibody is secreted into the culture medium, the supernatant of this expression system is usually concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis and antibiotics can be included to prevent the growth of foreign contaminants.

[0283] Pharmaceutical / antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is typically agarose, but other matrices may also be used. Compared to agarose, mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter processing times. In antibodies containing C H3 In the case of a domain, Bakerbond ABX.TM resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques, such as ion exchange column fractionation, ethanol precipitation, reversed phase HPLC, silica gel chromatography, heparin SEPHAROSE, can also be used depending on the antibody to be recovered. TM Chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

[0284] Following any preliminary purification steps, the mixture comprising the agent or antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and can be performed at low salt concentrations (e.g., about 0-0.25 M salt).

[0285] Drug formulations, dosages and routes of administration

[0286] The present technology provides anti-TLR7 agents and antibodies and related compositions, which can be used, for example, to eliminate TLR7-expressing cells from the body, and to identify and quantify the number of TLR7-expressing cells in a tissue sample.

[0287] The therapeutic methods and compositions of the present technology may be referred to as "TLR7-based" to indicate that these therapies can alter the relative or absolute amount of unwanted or toxic TLR7-expressing cells, such as lymphoma or autoimmune B lymphocytes.

[0288] For example, one way to control the amount of unwanted TLR7 expressing cells in a patient is to provide a composition comprising one or more anti-TLR7 antibodies to induce cytotoxic activity against TLR7 expressing cells.

[0289] Anti-TLR7 agents / antibodies can be formulated in pharmaceutical compositions for a variety of purposes, including the treatment of diseases, disorders, or physical trauma. Pharmaceutical compositions comprising one or more anti-TLR7 agents / antibodies herein can be incorporated into kits and medical devices for such treatment. Medical devices can be used to administer the pharmaceutical compositions herein to patients in need, and according to one embodiment of the present technology, kits comprising such devices are provided. Such devices and kits can be designed for routine administration of the pharmaceutical compositions herein, including self-administration.

[0290] Therapeutic formulations of the agent or antibody can be prepared by mixing the agent / antibody with the desired purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) and stored in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the doses and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residual yl) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0291] The formulations herein may also contain more than one active compound as required to treat a particular indication, preferably those having complementary activities and which do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0292] Active ingredients can also be embedded in microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. This technology is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0293] Formulations for in vivo administration are generally sterile. This can be achieved, for example, by filtration through sterile filtration membranes.

[0294] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the agent / antibody, which are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot.TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for shorter periods of time. When encapsulated agents / antibodies remain in the body for a long time, they may be exposed to 37 ℃ Moisture may cause denaturation or aggregation, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be designed to achieve stabilization based on the mechanisms involved. For example, if the aggregation mechanism is found to be the formation of intermolecular S--S bonds through sulfo-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0295] For therapeutic applications, the anti-TLR7 agents provided herein, such as antibodies, are administered to mammals, such as humans, in a pharmaceutically acceptable dosage form as described above, including intravenous administration to humans as a bolus or by continuous infusion over a period of time or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes.

[0296] For the prevention or treatment of disease, the appropriate dosage of the agent / antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0297] Depending on the type and severity of the disease, an antibody of about 1 μg / kg to about 50 mg / kg (e.g., 0.1-20 mg / kg) can be an initial candidate dose for administration to a patient, whether, for example, administered alone or by continuous infusion once or more. Depending on the above factors, a typical daily or weekly dosage range may be from about 1 μg / kg to about 20 mg / kg or more. For repeated administration over several days or longer, depending on the condition, repeated treatment occurs until desired disease symptom suppression occurs. However, other dosage regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiography. Antibodies can be used to determine detection methods for TLR7 levels in body fluids or tissues to optimize patient exposure to therapeutic antibodies.

[0298] In some embodiments, the composition comprising the anti-TLR7 agent herein (e.g., mAb interfering with TLR7 activity) is administered as a monotherapy, and in some embodiments, the composition comprising the anti-TLR7 agent is administered as part of a combination therapy. In some cases, the effectiveness of the agent / antibody in preventing or treating a disease can be increased by continuous administration or in combination with another agent effective for these purposes, such as a chemotherapeutic drug for treating cancer or microbial infection. In other cases, the anti-TLR7 agent can be used to enhance or sensitize cells to chemotherapy, thereby exerting efficacy at lower doses and lower toxicity. In addition to administering a composition comprising an agent that reduces the number of TLR7 expressing cells, some combination therapies also include delivering a second treatment regimen selected from the following: administration of chemotherapeutic agents, radiotherapy, surgery, and any combination of the above.

[0299] Such other agents may be present in the composition administered or may be administered separately.In addition, the anti-TLR agent may be appropriately administered consecutively or in combination with other agents or treatment modalities, such as chemotherapeutic drugs or radiation or immunosuppressive drugs used to treat cancer, infection, etc.

[0300] The research and diagnostic applications of the anti-TLR7 agents herein include clinical diagnostic applications

[0301] Provided herein are diagnostic reagents comprising anti-TLR7 agents described herein. For example, anti-TLR7 agents such as antibodies provided herein can be used to detect and / or purify TLR7, such as expressed from body fluids or on cells in body fluids or tissues. Also provided herein are methods for detecting TLR7. For example, a method may include contacting a sample (e.g., a biological sample known or suspected to contain TLR7) with an anti-TLR7 agent provided herein, and if the sample contains TLR7, then detecting TLR7: anti-TLR7 complexes. Also provided herein are reagents comprising anti-TLR7 agents described herein and methods for detecting TLR7 for research purposes.

[0302] For example, anti-TLR7 antibodies can be used in diagnostic assays of TLR7, such as detecting its presence in specific cells, tissues or body fluids. Such diagnostic methods can be used to diagnose, for example, hyperproliferative diseases or disorders. Therefore, clinical diagnostic applications as well as research applications are included herein.

[0303] In some embodiments, the anti-TLR7 agent / antibody comprises a detectable marker or label. In some embodiments, the anti-TLR7 agent / antibody is conjugated to a detectable marker or label. For example, for research and diagnostic applications, the anti-TLR7 agent / antibody can be labeled with a detectable moiety. There are many labels available, which are generally divided into the following categories:

[0304] (a) Radioisotopes, e.g. 35 S. 14 C. 125 I. 3 H and 131 I. Antibodies can be labeled with radioactive isotopes using, for example, the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. (1991), and the radioactivity can be measured using scintillation counting.

[0305] (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, Texas Red and Brilliant Violet TM Fluorescent labels can be conjugated to antibodies using, for example, techniques disclosed in Current Protocols in Immunology above. Fluorescence can be quantified using a flow cytometer, an imaging microscope, or a fluorimeter.

[0306] (c) A variety of enzyme-substrate labels are available, some of which are reviewed in U.S. Patent No. 4,275,149. The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be measured using a variety of techniques. For example, the enzyme can catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme can alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying changes in fluorescence are described above. Chemiluminescent substrates are electronically excited by a chemical reaction and can then emit measurable light (e.g., using a chemiluminometer) or provide energy to a fluorescent receptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. The technique of conjugating enzymes to antibodies is described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

[0307] Examples of enzyme-substrate combinations include, for example:

[0308] (i) horseradish peroxidase (HRP) with catalase as substrate, where catalase oxidizes a dye precursor (e.g. o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));

[0309] (ii) alkaline phosphatase (AP) using p-nitrophenyl phosphate as a chromogenic substrate; and

[0310] (iii) β-D-galactosidase (.beta.-D-Gal) and a chromogenic substrate (e.g. p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0311] Many other enzyme-substrate combinations can be used (eg, as described in US Pat. Nos. 4,275,149 and 4,318,980, each of which is incorporated herein by reference).

[0312] In some cases, the label is conjugated to the agent / antibody indirectly. Those skilled in the art will be aware of various techniques for achieving this. For example, an antibody can be conjugated to biotin, any of the three major categories of labels described above can be conjugated to avidin, and vice versa. Biotin selectively binds to avidin, so the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the antibody, the antibody is conjugated to a small hapten (e.g., digoxin) and one of the different types of labels described above is conjugated to an anti-hapten antibody (e.g., an anti-digoxin antibody). Thus, indirect conjugation of the label to the antibody can be achieved.

[0313] In some embodiments, the anti-TLR7 agent / antibody need not be labeled, and its presence can be detected, for example, using a labeled antibody that binds to the anti-TLR7 antibody.

[0314] In some embodiments, the anti-TLR7 agent / antibody herein is fixed on a solid support or substrate. In some embodiments, the anti-TLR7 agent / antibody herein is non-diffusively fixed on a solid support (e.g., the anti-TLR7 agent / antibody will not detach from the solid support). The solid support or substrate can be any physically separable solid, and the anti-TLR7 agent / antibody can be directly or indirectly attached thereto, including but not limited to surfaces provided by microarrays and holes, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles and nanoparticles. Solid supports can also include, for example, chips, columns, optical fibers, tissues, filters (e.g., planar filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylic resins, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethane, TEFLON TM , polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF), etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon, silica gel and modified silicon, Carbon, metals (e.g., steel, gold, silver, aluminum, silicon, and copper), inorganic glasses, conductive polymers (including polymers such as polypyrrole and polyindole); micro- or nanostructured surfaces, such as nucleic acid mosaic arrays (tiling arrays), nanotubes, nanowires, or nanoparticles decorated surfaces; or porous surfaces or gels, such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibrous or chain polymers. In some embodiments, the solid support or substrate can be coated with any number of materials using passive or chemically derived coatings, including polymers such as dextran, acrylamide, gelatin, or agarose. The beads and / or particles can be free or interconnected (e.g., hot pressed). In some embodiments, the solid support or substrate can be a collection of particles. In some embodiments, the particles can comprise silica, and the silica can comprise silicon dioxide. In some embodiments, the silica can be porous, and in certain embodiments, the silica can be non-porous. In some embodiments, the particles further comprise an agent that imparts paramagnetism to the particles. In certain embodiments, the agent comprises a metal, and in certain embodiments, the agent is a metal oxide (e.g., iron or an iron oxide, wherein the iron oxide comprises Fe 2+ and Fe 3+ The anti-TLR7 agent / antibody may be linked to the solid support by covalent bonds or by non-covalent interactions and may be linked to the solid support directly or indirectly (e.g., via a spacer molecule or an intermediate such as biotin).

[0315] The agents and antibodies provided herein can be used in any known assay method, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instrument), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0316] Flow cytometry and mass spectrometry cytometry assays typically involve the use of a single primary antibody to specifically identify the presence of a target molecule expressed on the surface of individual cells in a dispersed suspension. Dispersed cells are typically obtained from biological fluid samples such as blood, but can also be obtained from single cell dispersions prepared from solid tissue samples such as spleen or tumor biopsy tissue. A primary antibody can be directly conjugated to a detectable portion, such as a fluorophore such as phycoerythrin for flow cytometry or a heavy metal chelate for mass spectrometry cytometry. Alternatively, a primary antibody can be unlabeled or labeled with an undetectable label such as biotin, and then the primary antibody is detected by a detectably labeled secondary antibody that specifically recognizes the primary antibody itself or a label on the primary antibody. The labeled cells are then analyzed in an instrument such as a flow cytometer, a mass spectrometer, a fluorescence microscope, or a bright field optical microscope that can perform single cell detection to identify those individual cells that express the target recognized by the primary antibody in a dispersed cell population or tissue sample. A detailed description of the technical basis and practical application of the flow cytometry principle can be found, for example, in Shapiro, Practical Flow Cytometry, 4 th Edition,Wiley,2003.

[0317] Sandwich assays involve the use of two antibodies, each of which can bind to different immunogenic parts or epitopes of the detected protein. In sandwich assays, the test sample analyte is bound by a first antibody fixed on a solid support, and then a second antibody is bound to the analyte, thereby forming an insoluble three-part complex. See, for example, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable portion (direct sandwich assay) or can be measured using an anti-immunoglobulin antibody labeled with a detectable portion (indirect sandwich assay). For example, a type of sandwich assay is an ELISA assay, in which case the detectable portion is an enzyme. In cell ELISA, a target cell population can be attached to a solid support using antibodies that are first attached to a support and recognize different cell surface proteins. These first antibodies capture cells on a support. TLR7 on the cell surface can then be detected by adding anti-TLR7 antibodies to the captured cells and detecting the amount of TLR7 antibodies attached to the cells. In some cases, fixed and permeabilized cells can be used, in which case surface TLR7 and intracellular TLR7 can be detected.

[0318] For immunohistochemistry, blood or tissue samples may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin.

[0319] The agents / antibodies herein can also be used in in vivo diagnostic assays. Typically, the antibodies are treated with radionuclides (e.g. 111 In, 99 Tc, 14C. 131 I. 125 I. 3 H. 32 P or 35 S) so that bound target molecules can be localized using immunoscintigraphy.

[0320] Detection of TLR7 in immune cells

[0321] Provided herein are agents and methods for detecting TLR7 in immune cells. Detection of TLR7 in immune cells may refer to detection on the surface of immune cells (e.g., by surface staining) and / or inside immune cells (e.g., by intracellular staining). In some embodiments, agents and methods for detecting TLR7 in heterogeneous immune cell groups are provided. Heterogeneous immune cell groups may include two or more types of immune cells. For example, heterogeneous immune cell groups may include two or more B cells, plasmacytoid dendritic cells (pDC), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, bone marrow dendritic cells (mDC), innate lymphocytes, mast cells, eosinophils, basophils, natural killer cells, etc. In some embodiments, heterogeneous immune cell groups include peripheral blood mononuclear cells (PBMC), which may include, for example, T cells, B cells, natural killer cells, and monocytes.

[0322] Typically, cells are contacted with an anti-TLR7 agent as described herein (e.g., in a flow cytometry assay as described in Example 3; or any suitable protein or cell detection assay). In some embodiments, an anti-TLR7 agent as described herein detects significant levels of TLR7 in certain immune cells. TLR7 can be detected at significant levels in certain immune cells by an anti-TLR7 agent as described herein, but not significantly detected in other immune cells. The level of TLR7 detection in certain immune cells may vary according to certain factors, such as the type of detection assay, the type of detection reagent (e.g., the type of dye), antibody concentration, donor cell variability, etc.

[0323] Detecting TLR7 at a significant level can refer to a particular signal-to-noise (S:N) ratio (eg, a threshold or range) measured in a flow cytometry assay.

[0324] In some embodiments, significant levels of TLR7 are detected in plasmacytoid dendritic cells (pDCs). In some cases, plasmacytoid dendritic cells (pDCs) can be identified by the expression of CD304. Thus, in some embodiments, in the presence of CD304 +Significant levels of TLR7 have been detected in plasmacytoid dendritic cells (pDCs). CD304, also known as neuropilin-1, BDCA-4, and VEGF165R, is a 140 kD type I transmembrane protein. Its extracellular region contains two CUB, two FV / FVIII, and one MAM domain; soluble isoforms are generated by alternative mRNA splicing. CD304 is involved in angiogenesis, neural development, and tumor metastasis, and can be expressed by plasmacytoid dendritic cells, thymocytes, neurons, endothelial cells, and a subset of TFH cells. CD304 may also be expressed in several carcinomas, and high expression of this molecule in prostate cancer may be associated with poor prognosis.

[0325] In some embodiments, significant levels of TLR7 are detected in B cells. In some cases, B cells can be identified by CD19 expression and lack of CD3 expression. Thus, in some embodiments, in CD19 + CD3 - Significant levels of TLR7 have been detected in B cells. CD19 is a 95kD type I transmembrane glycoprotein, also known as B4. It is a member of the immunoglobulin superfamily expressed on B cells (from pre-B cells to embryonic B cells, not present on plasma cells) and follicular dendritic cells. CD19 is generally involved in B cell development, activation and differentiation. CD19 forms a complex with CD21 (CR2) and CD81 (TAPA-1) and acts as a BCR co-receptor. CD3, also known as T3, is a member of the Ig superfamily and is mainly expressed on T cells, NK-T cells, and is expressed on thymocytes at varying levels during T cell differentiation. CD3 consists of CD3ε, δ, γ and ζ chains, and forms a TCR complex by binding to the TCRα / β or γ / δ chains. CD3 participates in TCR signal transduction, T cell activation and antigen recognition by binding to peptide / MHC antigen complexes.

[0326] In some embodiments, the anti-TLR7 agents described herein do not significantly detect TLR7 in certain immune cells. For example, in certain lymphocytes (e.g., CD3 - CD19 - lymphocytes) and / or T cells (e.g. CD3 + CD19 - TLR7 may not be significantly detected in some lymphocytes (e.g., CD3 - CD19 - lymphocytes) and / or T cells (e.g. CD3 + CD19 -T cells) can refer to a specific signal-to-noise (S:N) ratio (e.g., a threshold or range) measured in flow cytometry. In some embodiments, in certain lymphocytes (e.g., CD3 - CD19 - lymphocytes) and / or T cells (e.g. CD3 + CD19 - TLR7 is not significantly detected in certain lymphocytes (e.g., CD3 T cells) means that the signal-to-noise ratio (S:N) is about 1.5 or less. - CD19 - lymphocytes) and / or T cells (e.g. CD3 + CD19 - TLR7 was not significantly detected in T cells (T cells) means that the signal-to-noise ratio (S:N) was about 1 or less.

[0327] Provided herein are reagents and methods for detecting TLR7 inside immune cells (e.g., by intracellular staining). In some embodiments, in plasmacytoid dendritic cells (pDCs) (e.g., CD304 + TLR7 is detected inside plasmacytoid dendritic cells (pDCs). In some embodiments, in a flow cytometry assay, TLR7 is detected inside plasmacytoid dendritic cells (pDCs) (e.g., CD304 + TLR7 is detected in plasmacytoid dendritic cells (pDCs). In some embodiments, TLR7 is detected in plasmacytoid dendritic cells (pDCs) (e.g., CD304 + In some embodiments, in a flow cytometry assay, TLR7 is detected within plasmacytoid dendritic cells (pDCs) (e.g., CD304 + In some embodiments, in a flow cytometry assay, TLR7 is detected within plasmacytoid dendritic cells (pDCs) (e.g., CD304 + The signal-to-noise ratio (S:N) for detecting TLR7 inside plasmacytoid dendritic cells (pDC) is about 14 or greater. The above-mentioned example of the signal-to-noise ratio (S:N) for detecting TLR7 inside plasmacytoid dendritic cells (pDC) may vary depending on certain factors, such as the type of detection assay, the type of detection reagent (e.g., dye type), antibody concentration, donor cell variability, etc.

[0328] In some embodiments, in B cells (e.g., CD19 + CD3 -In some embodiments, TLR7 is detected inside B cells (e.g., CD19 + CD3 - In some embodiments, TLR7 is detected inside B cells (e.g., CD19 + CD3 - B cells) with a signal-to-noise ratio (S:N) of about 2 or greater. In some embodiments, in a flow cytometry assay, TLR7 is detected inside B cells (e.g., CD19 + CD3 - B cells) with a signal-to-noise ratio (S:N) of about 3 or greater. In some embodiments, in a flow cytometry assay, TLR7 is detected inside B cells (e.g., CD19 + CD3 - B cells) with a signal-to-noise ratio (S:N) of about 4 or greater. In some embodiments, in a flow cytometry assay, TLR7 is detected inside B cells (e.g., CD19 + CD3 - B cells) internal detection of TLR7, the signal to noise ratio (S: N) is about 4.02 or greater. The above-mentioned signal to noise ratio (S: N) of detecting TLR7 inside B cells may vary according to certain factors, such as the type of detection assay, the type of detection reagent (e.g., the type of dye), the antibody concentration, the variability of donor cells, etc.

[0329] In some embodiments, in monocytes (e.g., CD14 + In some embodiments, TLR7 is detected in monocytes (e.g., CD14 + In some embodiments, TLR7 is detected in monocytes (e.g., CD14 + In some embodiments, TLR7 is detected in monocytes (e.g., CD14 TLR7) with a signal-to-noise ratio (S:N) of about 2 or greater in a flow cytometry assay. + TLR7 is detected inside monocytes (e.g., CD14 TLR7) with a signal-to-noise ratio (S:N) of about 3 or greater. In some embodiments, in a flow cytometry assay, TLR7 is detected inside monocytes (e.g., CD14 TLR7) with a signal-to-noise ratio (S:N) of about 3 or greater. + The above-mentioned example of the signal-to-noise ratio (S:N) for detecting TLR7 inside monocytes may vary depending on certain factors, such as the type of detection assay, the type of detection reagent (e.g., the type of dye), the antibody concentration, the variability of donor cells, etc.

[0330] Kit containing the anti-TLR7 agent of the present invention

[0331] The anti-TLR7 agent (e.g., anti-TLR7 antibody) herein can be provided in a kit, such as a packaged combination of a predetermined amount of reagent and instructions for use (e.g., instructions for performing a diagnostic assay; instructions for performing a laboratory assay). In some embodiments, the kit is a diagnostic kit for detecting TLR7 in a sample (e.g., a biological sample). In the case of labeling an anti-TLR7 agent with a fluorophore, the kit may include an isotype negative control unrelated antibody of the same type to control the non-specific binding of the anti-TLR7 agent. In the case of labeling an anti-TLR7 agent with an enzyme, the kit may include substrates and cofactors required for the enzyme (e.g., providing a substrate precursor that can detect a chromophore or fluorophore). Other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents can vary widely to provide a concentration of the reagent in the solution that substantially optimizes the sensitivity of the assay. In some cases, the reagent may be provided as a dry powder (e.g., a lyophilized powder), including an excipient that will provide a reagent solution with an appropriate concentration when dissolved.

[0332] Production products

[0333] In another aspect of the present technology, an article of manufacture containing materials that can be used to treat or diagnose the above-mentioned diseases is provided. The article of manufacture may include a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be made of a variety of materials, such as glass or plastic. The container may contain a composition effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The active anti-TLR7 agent in the composition may be an anti-TLR7 antibody. The label on or associated with the container indicates that the composition is used to treat or diagnose the selected condition. The article of manufacture may also include a second container containing a pharmaceutically acceptable buffer such as phosphate buffer, Ringer's solution, and dextrose solution; and may also include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0334] definition

[0335] As defined herein, "acceptor human framework" generally refers to a framework comprising an amino acid sequence of a heavy chain variable domain (VH) framework or a light chain variable domain (VL) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof or it may contain amino acid sequence changes. In some embodiments, the number of framework amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the VH and / or VL acceptor human framework is identical to the VH and / or VL human immunoglobulin framework amino acid sequence or a human consensus framework amino acid sequence.

[0336] "Framework" or "FR" generally refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1; FR2; FR3; and FR4. Thus, HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0337] "Human consensus framework" generally refers to a framework that represents the most common amino acid residues in a selected human immunoglobulin VL or VH framework sequence. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the sequence subgroup is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols.1-3. In some embodiments, for VL, the subgroup is subgroup kappa I as described above by Kabat et al. In some embodiments, for VH, the subgroup is subgroup III as described above by Kabat et al.

[0338] The term "hypervariable region" or "HVR" generally refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody contains six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally contain amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs), the latter having the highest sequence variability and / or being involved in antigen recognition.

[0339] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using a number of well-known protocols, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745," ("Contact" numbering scheme); Martin et al., Proc. Natl. Acad. Sci., 86:9268-9272 (1989) ("AbM" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan; 27(1): 55-77 ("IMGT" numbering scheme); and those described in Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8; 309(3): 657-70 ("Aho" numbering scheme).

[0340] The boundaries of a given CDR or FR vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions indicated by inserted letters (e.g., "30a"), while deletions occur in some antibodies. Both schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0341] Table 4 below lists example position boundaries for CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and CDRL3 identified by the Kabat, Chothia and Contact schemes, respectively. For CDRH1, the residue numbers are listed using the Kabat and Chothia numbering schemes. FRs are located between CDRs, e.g., FRH1 is located between CDRH1 and CDRH2, and so on. Note that since the Kabat numbering scheme shown places insertions at H35A and H35B, the ends of the Chothia CDRH1 loop vary between H32 and H34 when numbered using the Kabat numbering scheme shown, depending on the length of the loop.

[0342]

[0343] 1-Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5 th Ed.Public Health Service,National Institutes of Health,Bethesda,MD

[0344] 2-Al-Lazikani et al., (1997) JMB 273,927-948

[0345] CDRs also contain "specificity determining residues" or "SDRs," which are residues that make contact with specific antigens. Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein as described in Kabat et al., supra.

[0346] The term "variable region" or "variable domain" generally refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to separate antibodies that bind to the antigen to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0347] "Affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described elsewhere herein. In some cases, the antibodies herein bind to a target (e.g., TLR7) with high affinity, e.g., no more than about 1×10 -7 M, preferably not more than about 1×10 -8 M and preferably not more than about 5×10 -9 Kd value of M.

[0348] An "affinity matured" antibody generally refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations. Preferably, such alterations result in an improvement in the affinity of the antibody for its target antigen.

[0349] The term "anti-TLR7 agent" generally refers to a molecule that is or comprises one or more anti-TLR7 antibodies, TLR7 binding antibody fragments, or TLR7 binding antibody derivatives.

[0350] The terms "anti-TLR7 antibody" and "antibody that binds to TLR7" generally refer to an antibody that can bind to TLR7 with sufficient affinity and / or specificity so that the antibody can be used as a research tool, diagnostic agent and / or therapeutic agent in targeting TLR7. In some embodiments, the degree of binding of the anti-TLR7 antibody (or its antigen-binding fragment) to an unrelated non-TLR7 protein is less than about 10% of the binding of the antibody to TLR7, for example, as measured by radioimmunoassay (RIA) or by Scatchard analysis or by surface plasmon resonance (e.g., Biacore). In certain embodiments, the dissociation constant (Kd) of the antibody that binds to TLR7 is 0.1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM (e.g., 10 -7 M or less, for example, from 10 -7 M to 10 - 13 M). In certain embodiments, an anti-TLR7 antibody binds to an epitope of TLR7 that is conserved among TLR7 from different species.

[0351] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0352] "Antibody derivatives" generally refer to molecules other than intact antibodies, which contain a portion derived from an intact antibody (or its antigen-binding fragment) and bind to the antigen bound by the intact antibody (or its antigen-binding fragment). Examples of antibody derivatives include, but are not limited to, single-chain variable fragments (scFv), diabodies, tribodies, etc., aptamers, single-chain variable fragments, diabodies, tribodies, etc., containing multiple antigen-binding antibody fragments.

[0353] "Antibody fragments" or "antigen-binding antibody fragments" generally refer to molecules other than intact antibodies that contain a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, and multispecific antibodies formed from antibody fragments.

[0354] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0355] The term "Fc region" generally refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also referred to as the EU index numbering, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0356] With respect to a reference antibody (e.g., an antibody that binds TLR7), "an antibody that binds to the same epitope" generally means that the antibody blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay.

[0357] The term "chimeric" antibody generally refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0358] "Human antibody" generally refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes "humanized" antibodies comprising non-human antigen-binding residues.

[0359] "Humanized" antibodies generally refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all FRs correspond to those of human antibodies. Humanized antibodies may optionally include at least a portion of an antibody constant region derived from a human antibody. In some embodiments, a humanized antibody (or its antigen-binding fragment or derivative), when compared with an antibody from which a receptor framework region is derived, is derived, including one or more amino acid substitutions (or deletions or insertions) at a desired position. In some such embodiments, the amino acid residues substituted (or inserted or deleted) at a specific position of a human (or other) or other FR correspond to the amino acid residues at the corresponding position in the parent antibody (i.e., the non-human antibody from which CDRs or HVRs are derived). A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0360] The term "antibody drug conjugate" (ADC) or "immunoconjugate" generally refers to a specific class of agent-drug conjugates. Here, an "agent-drug conjugate" is an anti-TLR7 agent (e.g., an anti-TLR7 antibody or a TLR7 binding fragment or derivative thereof) conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0361] The term "cytotoxic agent" generally refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0362] A "diagnostic agent" generally refers to a compound used to perform a diagnostic assay, such as a target-specific antibody (or antigen-binding fragment thereof).

[0363] "Effector functions" generally refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0364] An "effective amount" of an agent (eg, a pharmaceutical formulation) generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0365] The term "epitope" generally refers to a specific site on an antigen molecule to which an antibody binds.

[0366] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and generally refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the progeny derived therefrom, without regard to the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.

[0367] A "rabbit antibody" generally refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a rabbit or rabbit cells or derived from a non-rabbit source utilizing a rabbit antibody repertoire or other rabbit antibody encoding sequences.

[0368] "Immunoconjugate" generally refers to an antibody (or antigen-binding fragment or derivative thereof) conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent. Immunoconjugate is equivalent to the term "antibody drug conjugate" (ADC).

[0369] An "individual" or "patient" or "subject" is typically a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0370] "Isolated" molecules (e.g., nucleic acids, antibodies) generally refer to molecules that have been separated from components of their original environment (e.g., the natural environment if the molecule is naturally occurring or the host cell if the molecule is exogenously expressed), and thus have been altered from their original environment by human intervention (e.g., "by human hands"). In some embodiments, for example, antibodies determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC) are purified to greater than 95% or 99% purity. An isolated nucleic acid may refer to a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or in a chromosomal location different from its natural chromosomal location. In some embodiments, an isolated nucleic acid may be provided that has a non-nucleic acid component (e.g., protein, lipid) less than the amount of the component present in the source sample. A composition comprising an isolated nucleic acid may be about 50% to greater than 99% free of non-nucleic acid components. A composition comprising an isolated nucleic acid may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of non-nucleic acid components.

[0371] "Isolated nucleic acid encoding an anti-TLR7 antibody" or "isolated polynucleotide encoding an anti-TLR7 antibody" generally refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a recombinant host cell.

[0372] The term "TLR7" generally refers to any natural, mature TLR7 that is produced by processing of a TLR7 precursor protein in a cell. Unless otherwise indicated, the term includes TLR7 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring TLR7 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary full-length human TLR7 precursor protein is shown in Figure 1 As shown in (SEQ ID NO: 48; of the 1,049 amino acid residues, the first 26 are a signal peptide, and residues 27 to 1,049 constitute the mature processed protein).

[0373] The term "TLR7-positive cell" generally refers to any cell that expresses TLR7 on its surface or on an intracellular membrane or organelle (e.g., endosome, ER, Golgi, lysosome, etc.). Some cells, including those infected by microorganisms or associated with certain cancer types and tumors, show upregulation of TLR7 expression.

[0374] The term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous antibody group, i.e., the individual antibodies that make up the group are identical (e.g., assessed at the level of Ig heavy chain and / or light chain amino acid sequence) and / or bind to the same epitope, except for possible variant antibodies, such as mutations that contain naturally occurring mutations or mutations that are produced during the production of monoclonal antibody preparations, which variants are generally present in small amounts. Compared to polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents that an antibody is derived from a substantially homogeneous antibody group, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibody used according to the present technology can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and this method and other exemplary methods for preparing monoclonal antibodies are described herein.

[0375] The term "package insert" generally refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0376] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence generally refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0377] The term "pharmaceutical composition" generally refers to a preparation in such a form that the biological activity of the active ingredient contained therein is effective, and that contains no other ingredients that would be unacceptably toxic to a subject to which the formulation would be administered.

[0378] "Pharmaceutically acceptable carrier" generally refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0379] As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treatment") generally refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be used for prevention or performed during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies herein are used to delay the development of the disease or slow the progression of the disease.

[0380] The term "vector" generally refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors." Example

[0381] The examples set forth below illustrate certain embodiments and do not limit the present technology.

[0382] Example 1: Generation and identification of anti-TLR7 hybridomas

[0383] Hybridomas that secrete monoclonal antibodies reactive with TLR7 expressed in vivo can be prepared as described in this Example. The resulting anti-TLR7 antibodies can be used for a variety of purposes, including diagnostic assays, examples of which are provided in Examples 3 and 4 below.

[0384] The common strains of laboratory mice such as BALB / c or C57 / Bl6 or rats such as Sprague Dawley are suitable hosts for immunization with TLR7 immunogens. After successful immunization of mice, hybridomas are formed using standard protocols to fuse myeloma cells with spleen and draining lymph node cells collected from animals. After selecting successful fusions in HAT culture medium and cloning into about 1 cell / well in microtiter plates, culture supernatants can be detected by flow cytometry for cell transfectants (such as HEK 293 or RBL) expressing TLR7. The wells with successful staining patterns are then subcultured in larger containers until there are enough cells to carry out subcloning. The cells transfected with TLR7 can be used again by flow cytometry to carry out the further identification of hybridoma subclone candidate. Then, for example, by flow cytometry, the clone selected as the best candidate is further screened for the human blood cells that are divided into different subgroups (such as lymphocytes, monocytes, etc.) and for one or more cell lines produced from sick and / or infected human cells. The percentage of positive cells in each blood cell subset can be quantified compared to the isotype control.

[0385] An example of an ideal candidate clone has strong reactivity to immune cells (such as plasmacytoid dendritic cells (pDC), B cells, monocytes) expressing TLR7, but has no obvious reactivity to other blood cell populations (such as some lymphocytes, T cells). In some cases, the ideal candidate clone can detect the TLR7 on the cell surface of the plasmacytoid dendritic cells (pDC). In some cases, the ideal candidate clone can detect intracellular TLR7. In some cases, the ideal candidate clone can detect the intracellular TLR7 in plasmacytoid dendritic cells (pDC). In some cases, the ideal candidate clone can detect the intracellular TLR7 in B cells. In some cases, the ideal candidate clone can detect the intracellular TLR7 in monocytes. In some cases, the ideal candidate clone can detect the intracellular TLR7 in plasmacytoid dendritic cells (pDC), B cells and monocytes.

[0386] Example 2: Sequencing of anti-TLR7 antibody variable regions

[0387] Cells from well-performing anti-TLR7 hybridoma cell lines (as described in Example 1 above) were grown in standard mammalian tissue culture medium. Total RNA was isolated from hybridoma cells expressing various clones of anti-TLR monoclonal antibodies using a procedure based on the RNeasy Mini kit (Qiagen). The RNA was used to generate the first strand cDNA. Both light and heavy chain variable domain cDNAs were amplified by 5'-RACE technology. Positive clones were prepared by PCR, and then DNA sequencing was performed on multiple clones.

[0388] Figure 2 Shown are the amino acid sequences of the heavy and light chain variable domains (CDR and framework regions) of 6 different antibodies (clones) referred to as AB 1-6 (also referred to herein as antibody 1-6 and clone 1-6), including CDR1, CDR2 and CDR3 regions. Various heavy and light chain CDR sequences are shown in Table 5 below.

[0389]

[0390]

[0391] Example 3: Detection of cells expressing TLR7

[0392] This example describes flow cytometry-based detection of human peripheral blood mononuclear cells (PBMCs) using certain anti-TLR7 monoclonal antibodies provided herein.

[0393] Materials and methods

[0394] PBMC isolation

[0395] Human peripheral blood mononuclear cells (PBMCs) were isolated under sterile conditions by Ficoll-Paque PLUS (GE Healthcare) gradient according to the manufacturer's instructions.

[0396] Surface and intracellular staining

[0397] PBMCs were counted and adjusted to a cell density of 0.7-1×10 in cell staining buffer (BioLegend, cat.#420201). 7 cells / mL. 100 μl of cell suspension (0.7-1×10 6 Total cells) were stained with CD3, CD19, and CD304 for 15 min in the dark. To detect surface CD289 / TLR7, isotype (rat IgG2a, kappa or mouse IgG1, kappa) or monoclonal TLR7 antibodies (see Figure 3 ; AB1, AB2, AB3, AB4, AB5, AB6 or commercial ABI) are included in the surface mixture and stained in the dark for 15 minutes. The cells are washed twice with 2mL cell staining buffer. In order to detect surface CD289 / TLR7, the cells are resuspended in 300μL of 1% paraformaldehyde and acquired on a cytometer. In order to detect intracellular CD289 / TLR7, the cells are resuspended in 300-500μL fixed buffer (BioLegend, cat.#420801) for 20 minutes in the dark. The cells are washed twice with 2mL of 1× intracellular staining permeabilization wash buffer (BioLegend, cat.#421002), and then stained in the dark for 30 minutes with isotype (rat IgG2a, k or mouse IgG1, k) or monoclonal TLR7 antibody (AB1, AB2, AB3, AB4, AB5, AB6 or commercial ABI). The cells were washed with 2 mL of 1× intracellular staining permeabilization wash buffer and then washed with 2 mL of cell staining buffer. The cells were resuspended in 300 μL of cell staining buffer and acquired on a cytometer. Samples were acquired on a BD CantoII and analyzed using FlowJo software.

[0398] Unless otherwise stated, all reagents used were from BioLegend.

[0399] Flow Cytometry / Gating Hierarchy

[0400] Lymphocytes and monocytes were gated together based on their forward (FSC) and side scatter (SSC) patterns. lo SSC lo , monocytes are FSC intSSC int (Figure 4A). From the lymphocyte and monocyte gates, B cells are CD19 + CD3 - , T cells are CD3 + CD19 - (Figure 4B). Some lymphocytes (FSC lo SSC lo ) are not T cells or B cells (CD19 - CD3 - ). Monocytes are CD14 + CD304 + Plasmacytoid dendritic cells (pDCs) are CD14 - CD304 + cells (Figure 4C).

[0401] result

[0402] PBMCs were intracellularly stained with each TLR7 antibody as well as an isotype control antibody (control) Figure 6 ). Compared with the control, no staining was observed on T cells. B cells, monocytes and pDC showed positive staining. The mean fluorescence intensity (MFI) and signal-to-noise ratio (S:N) results of pDC, B cells and monocytes are shown in Figures 7A-7F. In some cases, the signal-to-noise ratio (S:N) is higher than that of commercially available anti-TLT7 antibodies. For example, in pDC cell populations, the S:N of AB6 is 12.0 (0.5 μg / T), and the S:N of commercially available anti-TLT7 antibodies is 8.4 (optimal 1 μg / T); S:N ratio is calculated as MFI hTLR7 staining / MFI isotype staining.

[0403] PBMCs were surface stained with each TLR7 antibody as well as an isotype control antibody ( Figure 5 ). No staining was observed on T cells, B cells or monocytes. Dark staining was observed on pDCs compared to the isotype.

[0404] Example 4: Functional assay

[0405] This example describes a functional assay based on the detection of TLR7 in cells overexpressing the target.

[0406] experiment

[0407] HEK293 cells overexpressing human TLR7 (293XL-hTLR7, Invivogen) were cultured and stimulated according to the manufacturer's instructions. TLR7 recognizes small synthetic molecules, such as the imidazoquinoline compound R848 (Sigma Aldrich). This cell line expresses human interleukin 8 through TLR7 stimulation in the presence of R848. In brief, 800,000 293XL-hTLR7 cells were seeded into wells of a 96-well plate, cultured overnight, and then incubated with different concentrations of TLR7 antibodies at 37°C for 15 minutes. 0.01 μg of R848 was added to each well and the plate was incubated at 37°C for 20 hours. The supernatant was collected and the secretion of IL-8 was evaluated (BioLegend cat. #431504).

[0408] result

[0409] The amount of R848-induced human IL-8 antibody secreted by the cells decreased inversely with the concentration of TLR7 antibody present (Figure 8, panel A). The fold change in IL-8 secretion was calculated as the ratio of hIL-8 secreted in the presence of the indicated concentration of TLR7 antibody to hIL-8 secreted in the presence of 10 μg / mL isotype control antibody.

[0410] Example 5: Blocking assay

[0411] This example describes a blocking assay using the anti-TLR7 antibodies described herein.

[0412] PBMC isolation

[0413] Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque PLUS (GE Healthcare) gradient according to the manufacturer's instructions.

[0414] Blocking assay

[0415] For blocking studies, PBMCs were stained with CD3, CD19, and CD304, fixed, permeabilized, and then incubated in the dark for 30 minutes with 5 μg of purified antibodies (rat IgG2a, rat IgG1, AB1, AB2, AB3, AB4, AB5, or AB6). Rat IgG2a, rat IgG1, AB1, AB2, AB3, AB4, AB5, and AB6 were used as blocking agents. In some cases, no blocking agent was used. The cells were then incubated with PE-conjugated antibodies or isotype controls for 30 minutes. The cells were washed with 1× permeabilization buffer (intracellular staining permeabilization wash buffer (BioLegend, cat.#421002), acquired on BD CantoII, and analyzed using FlowJo software.

[0416] Calculation of blocking %

[0417] The raw MFI percentage is calculated by dividing the MFI of samples blocked with isotype control, AB1, AB2, AB3, AB4, AB5, or AB6 by the unblocked MFI. This value is subtracted from 100 to obtain the blocking percentage. The formula is as follows:

[0418]

[0419] result

[0420]

[0421] Example 6: Sequence Example

[0422] Examples of amino acid sequences relevant to the technology described herein are provided below.

[0423]

[0424] CDRH1

[0425] X1YX2X3X4 (SEQ ID NO: 30)

[0426] X1=D or N

[0427] X2=W,Y or C

[0428] X3=M,V or I

[0429] X4=S,A or H

[0430] CDRH2

[0431] X1IX2X3X4X5X6X7X8X9YX 10 X 11 X 12 X 13 KX 14 (SEQ ID NO: 31) X1 = D, S, Y or F

[0432] X2=K,T,S or N

[0433] X3=Y,N or P

[0434] X4=D,S,E or Y

[0435] X5=G or S

[0436] X6=S,T,R or G

[0437] X7=F,T,S or Y

[0438] X8=I or T

[0439] X9=D,Y,H or N

[0440] X 10 =A, R, G or N

[0441] X 11 =P, D or E

[0442] X 12 =S or K

[0443] X 13 =L, V or F

[0444] X 14 =N, G, A, S or T

[0445] CDRH3

[0446] X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X 16 (SEQ ID NO: 32) X1 = D, E, H or G

[0447] X2=L,G or P

[0448] X3=T,G or Y

[0449] X4 = no amino acid, T, D or G

[0450] X5 = No amino acid, L or G

[0451] X6=no amino acid or Y

[0452] X7=V,Y or S

[0453] X8=V,S,P or G

[0454] X9=D or N

[0455] X 10 =G, Y or W

[0456] X 11 = No amino acid or N

[0457] X 12 = No amino acid or Y

[0458] X 13 = No amino acid or V

[0459] X 14 = No amino acid, R or Y

[0460] X 15 =A or D

[0461] X 16 =Y or F

[0462] CDRL1

[0463] X1X2XX3X4X5X6X7X8X9X 10 (SEQ ID NO: 33) X1 = R or L

[0464] X2=A or P

[0465] X3=E or Q

[0466] X4=D or S

[0467] X5=I or L

[0468] X6=Y or S

[0469] X7=N or T

[0470] X8=V,E,N or S

[0471] X9=L or I

[0472] X 10 =A or H

[0473] CDRL2

[0474] X1AX2X3X4X5X6 (SEQ ID NO: 34)

[0475] X1=N or Y

[0476] X2=N or S

[0477] X3=R,N,S or Q

[0478] X4=L or P

[0479] X5=H,Q or I

[0480] X6=N,T,D or S

[0481] CDRL3

[0482] X1QX2X3X4X5PX6T (SEQ ID NO: 35) X1=Q or L

[0483] X2=Y,D or S

[0484] X3=Y or S

[0485] X4=D or S

[0486] X5=Y or S

[0487] X6=N,H,W,F or Y

[0488]

[0489]

[0490] Precursor human TLR7 protein sequence (UniProtKB-Q9NYK1 (TLR7_HUMAN) / NCBI reference sequence: NP_057646.1) (SEQ ID NO: 48)

[0491]

[0492] Human TLR7 signal sequence (SEQ ID NO: 49)

[0493] mvfpmwtlkr qililfniil iskllg

[0494] Mature human TLR7 protein sequence (SEQ ID NO: 50)

[0495]

[0496] Example 7: Example of implementation scheme

[0497] The examples set forth below illustrate certain embodiments and do not limit the technology.

[0498] A1. An anti-TLR7 agent that binds to Toll-like receptor 7 (TLR7) under laboratory or physiological conditions, wherein the agent comprises at least one immunoglobulin heavy chain variable domain and / or at least one immunoglobulin light chain variable domain, wherein:

[0499] a) Each immunoglobulin heavy chain variable domain of the anti-TLR7 agent comprises a first, a second and a third heavy chain complementarity determining region (CDR), wherein

[0500] The first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1YX2X3X4 (SEQ ID NO: 30), wherein

[0501] X1 is D or N,

[0502] X2 is W, Y or C,

[0503] X3 is M, V or I, and

[0504] X4 is S, A or H;

[0505] The second heavy chain CDR (CDRH2) comprises the amino acid sequence X1IX2X3X4X5X6X7X8X9YX 10 X 11 X 12 X 13 KX 14 (SEQ ID NO: 31) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein

[0506] X1 is D, S, Y or F,

[0507] X2 is K, T, S or N,

[0508] X3 is Y, N or P,

[0509] X4 is D, S, E or Y,

[0510] X5 is G or S,

[0511] X6 is S, T, R or G,

[0512] X7 is F, T, S or Y,

[0513] X8 is I or T,

[0514] X9 is D, Y, H or N,

[0515] X 10 is A, R, G or N,

[0516] X 11 is P, D or E,

[0517] X 12 is S or K,

[0518] X 13 is L, V or F, and

[0519] X 14 is N, G, A, S, or T; and

[0520] The third heavy chain CDR (CDRH3) comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X 16(SEQ ID NO: 32) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein

[0521] X1 is D, E, H or G,

[0522] X2 is L, G or P,

[0523] X3 is T, G or Y,

[0524] X4 is no amino acid, T, D or G,

[0525] X5 is no amino acid, L or G,

[0526] X6 is no amino acid or Y,

[0527] X7 is V, Y or S,

[0528] X8 is V, S, P or G,

[0529] X9 is D or N,

[0530] X 10 is G, Y or W,

[0531] X 11 is no amino acid or N,

[0532] X 12 is no amino acid or Y,

[0533] X 13 Is no amino acid or V,

[0534] X 14 is no amino acid, R or Y,

[0535] X 15 is A or D, and

[0536] X 16 is Y or F; and / or

[0537] b) each immunoglobulin light chain variable domain of the anti-TLR7 agent comprises a first, a second and a third light chain CDR, wherein

[0538] The first light chain CDR (CDRL1) comprises the amino acid sequence X1X2SX3X4X5X6X7X8X9X 10 (SEQ ID NO:33) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein

[0539] X1 is R or L,

[0540] X2 is A or P,

[0541] X3 is E or Q,

[0542] X4 is D or S,

[0543] X5 is I or L,

[0544] X6 is Y or S,

[0545] X7 is N or T,

[0546] X8 is V, E, N or S,

[0547] X9 is L or I, and

[0548] X 10 is A or H;

[0549] The second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein

[0550] X1 is N or Y,

[0551] X2 is N or S,

[0552] X3 is R, N, S or Q,

[0553] X4 is L or P,

[0554] X5 is H, Q or I, and

[0555] X6 is N, T, D, or S; and

[0556] The third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1QX2X3X4X5PX6T (SEQ ID NO:35), wherein

[0557] X1 is Q or L,

[0558] X2 is Y, D or S,

[0559] X3 is Y or S,

[0560] X4 is D or S,

[0561] X5 is Y or S, that is

[0562] X6 is N, H, W, F, or Y.

[0563] A2. The anti-TLR7 agent of embodiment A1, wherein CDRH1 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:30.

[0564] A3. The anti-TLR7 agent of embodiment A1, wherein CDRH1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:30.

[0565] A4. The anti-TLR7 agent of embodiment A1, wherein CDRH1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:30.

[0566] A5. The anti-TLR7 agent of embodiment A1, wherein CDRH1 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:30.

[0567] A6. The anti-TLR7 agent of any one of embodiments A1 to A5, wherein CDRH2 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:31.

[0568] A7. The anti-TLR7 agent of any one of embodiments A1 to A5, wherein CDRH2 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:31.

[0569] A8. The anti-TLR7 agent of any one of embodiments A1 to A5, wherein CDRH2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:31.

[0570] A9. The anti-TLR7 agent of any one of embodiments A1 to A5, wherein CDRH2 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0571] A10. The anti-TLR7 agent of any one of embodiments A1 to A9, wherein CDRH3 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:32.

[0572] A11. The anti-TLR7 agent of any one of embodiments A1 to A9, wherein CDRH3 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:32.

[0573] A12. The anti-TLR7 agent of any one of embodiments A1 to A9, wherein CDRH3 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:32.

[0574] A13. The anti-TLR7 agent of any one of embodiments A1 to A9, wherein CDRH3 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0575] A14 The anti-TLR7 agent of any one of embodiments A1 to A13, wherein CDRL1 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:33.

[0576] A15. The anti-TLR7 agent of any one of embodiments A1 to A13, wherein CDRL1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:33.

[0577] A16. The anti-TLR7 agent of any one of embodiments A1 to A13, wherein CDRL1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:33.

[0578] A17. The anti-TLR7 agent of any one of embodiments A1 to A13, wherein the CDRL1 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0579] A18. The anti-TLR7 agent of any one of embodiments A1 to A17, wherein CDRL2 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:34.

[0580] A19. The anti-TLR7 agent of any one of embodiments A1 to A17, wherein CDRL2 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:34.

[0581] A20. The anti-TLR7 agent of any one of embodiments A1 to A17, wherein CDRL2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:34.

[0582] A21. The anti-TLR7 agent of any one of embodiments A1 to A17, wherein CDRL2 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 34.

[0583] A22. The anti-TLR7 agent of any one of embodiments A1 to A21, wherein CDRL3 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:35.

[0584] A23. The anti-TLR7 agent of any one of embodiments A1 to A21, wherein CDRL3 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:35.

[0585] A24. The anti-TLR7 agent of any one of embodiments A1 to A21, wherein CDRL3 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:35.

[0586] A25. The anti-TLR7 agent of any one of embodiments A1 to A21, wherein CDRL3 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 35.

[0587] A26. The anti-TLR7 agent of any one of embodiments A1 to A25, wherein CDRH1 comprises an amino acid sequence selected from the group consisting of DYWMS (SEQ ID NO: 1), NYYMA (SEQ ID NO: 2), DYYMA (SEQ ID NO: 3), DYCVH (SEQ ID NO: 4) and DYYIH (SEQ ID NO: 5).

[0588] A27. The anti-TLR7 agent of any one of embodiments A1 to A26, wherein CDRH2 comprises an amino acid sequence selected from the group consisting of DIKYDGSFIDYAPSLKN (SEQ ID NO: 6), DIKYDGTFIDYAPSLKN (SEQ ID NO: 7), SITNSGRTTYYRDSVKG (SEQ ID NO: 8), SISYEGSSTHYGDSVKA (SEQ ID NO: 9), YINPYSGYTNYNEKFKS (SEQ ID NO: 10), and FINPDSGYTNYNEKFKT (SEQ ID NO: 11).

[0589] A28. The anti-TLR7 agent of any one of embodiments A1 to A27, wherein CDRH3 comprises an amino acid sequence selected from the group consisting of DLTT--VVDG----FAY (SEQ ID NO: 12), EGGDLYYSNYNYVRFAY (SEQ ID NO: 13), HGG---YPNW---YFDF (SEQ ID NO: 14), and GPYGGYSGDG----FDY (SEQ ID NO: 15).

[0590] A29. The anti-TLR7 agent of any one of embodiments A1 to A28, wherein CDRL1 comprises an amino acid sequence selected from the group consisting of: RASEDIYNVLA (SEQ ID NO: 16), RASEDIYNELA (SEQ ID NO: 17), LPSEDIYNNLA (SEQ ID NO: 18) and RASQSLSTSIH (SEQ ID NO: 19).

[0591] A30. The anti-TLR7 agent of any one of embodiments A1 to A29, wherein CDRL2 comprises an amino acid sequence selected from the group consisting of: NANRLHN (SEQ ID NO: 20), NANNLHT (SEQ ID NO: 21), NANSLHT (SEQ ID NO: 22), YASNLQD (SEQ ID NO: 23) and YASQPIS (SEQ ID NO: 24).

[0592] A31. The anti-TLR7 agent of any one of embodiments A1 to A30, wherein CDRL3 comprises an amino acid sequence selected from the group consisting of: QQYYDYPNT (SEQ ID NO: 25), QQYYDYPHT (SEQ ID NO: 26), QQYYDYPWT (SEQ ID NO: 27), LQDSDYPFT (SEQ ID NO: 28), and QQSYSSPYT (SEQ ID NO: 29).

[0593] A32. The anti-TLR7 agent of any one of embodiments A1 to A31, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.

[0594] A33. The anti-TLR7 agent of embodiment A32, wherein the two immunoglobulin heavy chain variable domains each comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences.

[0595] A34. The anti-TLR7 agent of embodiment A32 or A33, wherein the two immunoglobulin light chain variable domains each comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences.

[0596] A35. The anti-TLR7 agent of any one of embodiments A1 to A34, wherein each immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2 and CDRH3 amino acid sequences, and each immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2 and CDRL3 amino acid sequences, and the amino acid sequences are selected from the following groups 1 to 6:

[0597]

[0598] A36. The anti-TLR7 agent of embodiment A35, wherein all CDR sequences are from the same group.

[0599] A37. The anti-TLR7 agent of any one of embodiments A1 to A36, wherein the agent is isolated.

[0600] A38. The anti-TLR7 agent of any one of embodiments A1 to A37, wherein the agent is non-natural.

[0601] A39. The anti-TLR7 agent of any one of embodiments A1 to A38, wherein the agent is an antibody or an antigen-binding fragment thereof.

[0602] A40. The anti-TLR7 agent of any one of embodiments A1 to A38, wherein the agent is an antibody or a derivative thereof.

[0603] A41 The anti-TLR7 agent of any one of Embodiments A1 to A40, wherein the agent is a humanized antibody or an antigen-binding fragment thereof.

[0604] A42. The anti-TLR7 agent of any one of embodiments A1 to A40, wherein the agent is a derivative of a humanized antibody that binds TLR7.

[0605] A43. The anti-TLR7 agent of any one of embodiments A1 to A42, wherein the agent comprises a detectable marker or label.

[0606] A44. The anti-TLR7 agent of any one of embodiments A1 to A43, wherein the agent is conjugated to a detectable marker or label.

[0607] A45. The anti-TLR7 agent of any one of embodiments A1 to A44, wherein the agent is non-diffusively immobilized on a solid support.

[0608] A46. A diagnostic reagent comprising the anti-TLR7 agent of any one of embodiments A1 to A45.

[0609] A47. A kit comprising the anti-TLR7 agent of any one of embodiments A1 to A45 or the diagnostic agent of embodiment A46.

[0610] A48. A diagnostic kit configured to detect Toll-like receptor 7 (TLR7) in a biological sample, wherein the kit comprises the anti-TLR7 agent of any one of embodiments A1 to A45 or the diagnostic reagent of embodiment A46.

[0611] A49. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the immunoglobulin heavy chain variable domain of the anti-TLR7 agent of any one of embodiments A1 to A45.

[0612] A50. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the immunoglobulin light chain variable domain of the anti-TLR7 agent of any one of embodiments A1 to A45.

[0613] A51. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule, the nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an anti-TLR7 agent of any one of embodiments A1 to A45, and the second expression cassette comprises a promoter and a nucleic acid molecule, the nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of an anti-TLR7 agent of any one of embodiments A1 to A45.

[0614] A52. A recombinant host cell transfected with the recombinant expression vector of embodiment A51.

[0615] A53 A method for detecting TLR7, comprising contacting a sample known or suspected to contain TLR7 with the anti-TLR7 agent of any one of embodiments A1 to A45, and if the sample contains TLR7, detecting a TLR7:anti-TLR7 complex.

[0616] B1. A first anti-TLR7 agent that binds to Toll-like receptor 7 (TLR7) under laboratory or physiological conditions, wherein the first agent competitively binds or is capable of competitively binding with a second anti-TLR7 agent, the second agent being the anti-TLR7 agent of any one of embodiments A1 to A36.

[0617] B2. A first anti-TLR7 agent that binds to Toll-like receptor 7 (TLR7) under laboratory or physiological conditions, wherein the first agent binds or is capable of binding to the same epitope as a second anti-TLR7 agent, the second agent being the anti-TLR7 agent of any one of embodiments A1 to A36.

[0618] B3. The first anti-TLR7 agent of embodiment B1 or B2, wherein the first agent and / or the second agent are isolated.

[0619] B4. The first anti-TLR7 agent of any one of embodiments B1 to B3, wherein the first agent and / or the second agent is non-natural.

[0620] B5. The first anti-TLR7 agent of any one of embodiments B1 to B4, wherein the first agent and / or the second agent is an antibody or an antigen-binding fragment thereof.

[0621] B6. The first anti-TLR7 agent of any one of embodiments B1 to B4, wherein the first agent and / or the second agent is an antibody or a derivative thereof.

[0622] B7. The first anti-TLR7 agent of any one of embodiments B1 to B6, wherein the first agent and / or the second agent is a humanized antibody or an antigen-binding fragment thereof.

[0623] B8. The first anti-TLR7 agent of any one of embodiments B1 to B6, wherein the first agent and / or the second agent is a derivative of a humanized antibody that binds TLR7.

[0624] B9. The first anti-TLR7 agent of any one of embodiments B1 to B8, comprising a detectable marker or label.

[0625] B10. The first anti-TLR7 agent of any one of embodiments B1 to B9, wherein the first agent is conjugated to a detectable marker or label.

[0626] B11. The first anti-TLR7 agent of any one of embodiments B1 to B10, wherein the first agent is non-diffusively immobilized on a solid support.

[0627] B12. A diagnostic reagent comprising the first anti-TLR7 agent of any one of embodiments B1 to B11.

[0628] B13. A kit comprising the first anti-TLR7 agent of any one of embodiments B1 to B11 or the diagnostic agent of embodiment B12.

[0629] B14. A diagnostic kit configured to detect Toll-like receptor 7 (TLR7) in a biological sample, wherein the kit comprises the first anti-TLR7 agent of any one of embodiments B1 to B11 or the diagnostic reagent of embodiment B12.

[0630] B15. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the immunoglobulin heavy chain variable domain of the first anti-TLR7 agent of any one of embodiments B1 to B11.

[0631] B16. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the immunoglobulin light chain variable domain of the first anti-TLR7 agent of any one of embodiments B1 to B11.

[0632] B17. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of a first anti-TLR7 agent of any one of embodiments B1 to B11, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of a first anti-TLR7 agent of any one of embodiments B1 to B11.

[0633] B18. A recombinant host cell transfected with the recombinant expression vector of embodiment B17.

[0634] B19. A method of detecting TLR7, comprising contacting a sample known or suspected to contain TLR7 with a first anti-TLR7 agent of any one of embodiments B1 to B11, and if the sample contains TLR7, detecting a TLR7:anti-TLR7 complex.

[0635] C1. An anti-TLR7 agent for detecting TLR7 in a heterogeneous immune cell population, wherein a significant level of TLR7 is detected in plasmacytoid dendritic cells, monocytes and / or B cells of the cell population, while TLR7 is not significantly detected in other immune cells of the cell population.

[0636] C2. The anti-TLR7 agent of embodiment C1, wherein the anti-TLR7 agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, wherein:

[0637] i) each immunoglobulin heavy chain variable domain of the anti-TLR7 agent comprises a first, a second and a third heavy chain complementarity determining region (CDR); and

[0638] ii) Each immunoglobulin light chain variable domain of the anti-TLR7 agent comprises a first, a second and a third light chain CDR.

[0639] C3. The anti-TLR7 agent of embodiment C2, wherein the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1YX2XXX4 (SEQ ID NO: 30), wherein

[0640] X1 is D or N,

[0641] X2 is W, Y or C,

[0642] X3 is M, V or I, and

[0643] X4 is S, A or H.

[0644] C4. The anti-TLR7 agent of embodiment C2 or C3, wherein the second heavy chain CDR (CDRH2) comprises an amino acid sequence X1IX2X3X4X5X6X7X8X9YX 10 X 11 X 12 X 13 KX 14 (SEQ ID NO:31) is at least 80% identical (e.g., 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical) to an amino acid sequence, wherein

[0645] X1 is D, S, Y or F,

[0646] X2 is K, T, S or N,

[0647] X3 is Y, N or P,

[0648] X4 is D, S, E or Y,

[0649] X5 is G or S,

[0650] X6 is S, T, R or G,

[0651] X7 is F, T, S or Y,

[0652] X8 is I or T,

[0653] X9 is D, Y, H or N,

[0654] X 10 is A, R, G or N,

[0655] X 11 is P, D or E,

[0656] X 12 is S or K,

[0657] X 13 is L, V or F, and

[0658] X 14 Is N, G, A, S or T.

[0659] C5. The anti-TLR7 agent of embodiment C2, C3 or C4, wherein the third heavy chain CDR (CDRH3) comprises an amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 FX 15 X16 (SEQ ID NO:32) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein

[0660] X1 is D, E, H or G,

[0661] X2 is L, G or P,

[0662] X3 is T, G or Y,

[0663] X4 is no amino acid, T, D or G,

[0664] X5 is no amino acid, L or G,

[0665] X6 is no amino acid or Y,

[0666] X7 is V, Y or S,

[0667] X8 is V, S, P or G,

[0668] X9 is D or N,

[0669] X 10 is G, Y or W,

[0670] X 11 is no amino acid or N,

[0671] X 12 is no amino acid or Y,

[0672] X 13 Is no amino acid or V,

[0673] X 14 is no amino acid, R or Y,

[0674] X 15 is A or D, and

[0675] X 16 It is Y or F.

[0676] C6. The anti-TLR7 agent of any one of embodiments C2 to C5, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence X1X2SX3X4X5X6X7X8X9X 10 (SEQ ID NO:33) is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an amino acid sequence, wherein

[0677] X1 is R or L,

[0678] X2 is A or P,

[0679] X3 is E or Q,

[0680] X4 is D or S,

[0681] X5 is I or L,

[0682] X6 is Y or S,

[0683] X7 is N or T,

[0684] X8 is V, E, N or S,

[0685] X9 is L or I, and

[0686] X 10 It is A or H.

[0687] C7. The anti-TLR7 agent of any one of embodiments C2 to C6, wherein the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1AX2XXXX6 (SEQ ID NO: 34), wherein

[0688] X1 is N or Y,

[0689] X2 is N or S,

[0690] X3 is R, N, S or Q,

[0691] X4 is L or P,

[0692] X5 is H, Q or I, and

[0693] X6 is N, T, D or S.

[0694] C8. The anti-TLR7 agent of any one of embodiments C2 to C7, wherein the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence X1QX2X3X4X5PX6T (SEQ ID NO: 35), wherein

[0695] X1 is Q or L,

[0696] X2 is Y, D or S,

[0697] X3 is Y or S,

[0698] X4 is D or S,

[0699] X5 is Y or S, and

[0700] X6 is N, H, W, F, or Y.

[0701] C9. The anti-TLR7 agent of any one of embodiments C2 to C8, wherein CDRH1 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 30.

[0702] C10. The anti-TLR7 agent of any one of embodiments C2 to C8, wherein CDRH1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:30.

[0703] C11. The anti-TLR7 agent of any one of embodiments C2 to C8, wherein CDRH1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:30.

[0704] C12. The anti-TLR7 agent of any one of embodiments C2 to C8, wherein CDRH1 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0705] C13. The anti-TLR7 agent of any one of embodiments C2 to C12, wherein CDRH2 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:31.

[0706] C14. The anti-TLR7 agent of any one of embodiments C2 to C12, wherein CDRH2 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:31.

[0707] C15. The anti-TLR7 agent of any one of embodiments C2 to C12, wherein CDRH2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:31.

[0708] C16. The anti-TLR7 agent of any one of embodiments C2 to C12, wherein CDRH2 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0709] C17. The anti-TLR7 agent of any one of embodiments C2 to C16, wherein CDRH3 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 32.

[0710] C18. The anti-TLR7 agent of any one of embodiments C2 to C16, wherein CDRH3 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:32.

[0711] C19. The anti-TLR7 agent of any one of embodiments C2 to C16, wherein CDRH3 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 32.

[0712] C20. The anti-TLR7 agent of any one of embodiments C2 to C16, wherein CDRH3 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0713] C21. The anti-TLR7 agent of any one of embodiments C2 to C20, wherein CDRL1 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:33.

[0714] C22. The anti-TLR7 agent of any one of embodiments C2 to C20, wherein CDRL1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:33.

[0715] C23. The anti-TLR7 agent of any one of embodiments C2 to C20, wherein CDRL1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:33.

[0716] C24. The anti-TLR7 agent of any one of embodiments C2 to C20, wherein CDRL1 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0717] C25. The anti-TLR7 agent of any one of embodiments C2 to C24, wherein CDRL2 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:34.

[0718] C26. The anti-TLR7 agent of any one of embodiments C2 to C24, wherein CDRL2 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:34.

[0719] C27. The anti-TLR7 agent of any one of embodiments C2 to C24, wherein CDRL2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:34.

[0720] C28. The anti-TLR7 agent of any one of embodiments C2 to C24, wherein CDRL2 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 34.

[0721] C29. The anti-TLR7 agent of any one of embodiments C2 to C28, wherein CDRL3 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 35.

[0722] C30. The anti-TLR7 agent of any one of embodiments C2 to C28, wherein CDRL3 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:35.

[0723] C31. The anti-TLR7 agent of any one of embodiments C2 to C28, wherein CDRL3 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:35.

[0724] C32. The anti-TLR7 agent of any one of embodiments C2 to C28, wherein CDRL3 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 35.

[0725] C33. The anti-TLR7 agent of any one of embodiments C2 to C32, wherein CDRH1 comprises an amino acid sequence selected from DYWMS (SEQ ID NO: 1), NYYMA (SEQ ID NO: 2), DYYMA (SEQ ID NO: 3), DYCVH (SEQ ID NO: 4) and DYYIH (SEQ ID NO: 5).

[0726] C34. An anti-TLR7 agent according to any one of embodiments C2 to C33, wherein CDRH2 comprises an amino acid sequence selected from DIKYDGSFIDYAPSLKN (SEQ ID NO: 6), DIKYDGTFIDYAPSLKN (SEQ ID NO: 7), SITNSGRTTYYRDSVKG (SEQ ID NO: 8), SISYEGSSTHYGDSVKA (SEQ ID NO: 9), YINPYSGYTNYNEKFKS (SEQ ID NO: 10) and FINPDSGYTNYNEKFKT (SEQ ID NO: 11).

[0727] C35. The anti-TLR7 agent of any one of embodiments C2 to C34, wherein CDRH3 comprises an amino acid sequence selected from DLTT--VVDG----FAY (SEQ ID NO: 12), EGGDLYYSNYNYVRFAY (SEQ ID NO: 13), HGG---YPNW---YFDF (SEQ ID NO: 14) and GPYGGYSGDG----FDY (SEQ ID NO: 15).

[0728] C36. The anti-TLR7 agent of any one of embodiments C2 to C35, wherein CDRL1 comprises an amino acid sequence selected from RASEDIYNVLA (SEQ ID NO: 16), RASEDIYNELA (SEQ ID NO: 17), LPSEDIYNNLA (SEQ ID NO: 18) and RASQSLSTSIH (SEQ ID NO: 19).

[0729] C37. The anti-TLR7 agent of any one of embodiments C2 to C36, wherein CDRL2 comprises an amino acid sequence selected from NANRLHN (SEQ ID NO: 20), NANNLHT (SEQ ID NO: 21), NANSLHT (SEQ ID NO: 22), YASNLQD (SEQ ID NO: 23) and YASQPIS (SEQ ID NO: 24).

[0730] C38. The anti-TLR7 agent of any one of embodiments C2 to C37, wherein CDRL3 comprises an amino acid sequence selected from the group consisting of QQYYDYPNT (SEQ ID NO: 25), QQYYDYPHT (SEQ ID NO: 26), QQYYDYPWT (SEQ ID NO: 27), LQDSDYPFT (SEQ ID NO: 28) and QQSYSSPYT (SEQ ID NO: 29).

[0731] C39. The anti-TLR7 agent of any one of embodiments C1 to C38, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.

[0732] C40. The anti-TLR7 agent of embodiment C39, wherein the two immunoglobulin heavy chain variable domains each comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences.

[0733] C41. The anti-TLR7 agent of embodiment C39 or C40, wherein the two immunoglobulin light chain variable domains each comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences.

[0734] C42. The anti-TLR7 agent of any one of embodiments C2 to C41, wherein each immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2 and CDRH3 amino acid sequences, and each immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2 and CDRL3 amino acid sequences, and the amino acid sequences are selected from the following groups 1 to 6:

[0735]

[0736] C43. The anti-TLR7 agent of embodiment C42, wherein all CDR sequences are from the same group.

[0737] C44. The anti-TLR7 agent of any one of embodiments C1 to C43, wherein a significant level of TLR7 is detected in plasmacytoid dendritic cells.

[0738] C45. The anti-TLR7 agent of embodiment C44, wherein TLR7 is detected in plasmacytoid dendritic cells using a flow cytometry assay.

[0739] C46. The anti-TLR7 agent of embodiment C44, wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining.

[0740] C47. The anti-TLR7 agent of embodiment C46, ​​wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining with a signal-to-noise ratio of about 5 or greater.

[0741] C48. The anti-TLR7 agent of embodiment C46, ​​wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining with a signal-to-noise ratio of about 10 or greater.

[0742] C49. The anti-TLR7 agent of embodiment C46, ​​wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining with a signal-to-noise ratio of about 14 or greater.

[0743] C50. The anti-TLR7 agent of any one of embodiments C1 to C49, wherein a significant level of TLR7 is detected in B cells.

[0744] C50a. The anti-TLR7 agent of embodiment C50, wherein TLR7 is detected in B cells using a flow cytometry assay.

[0745] C51. The anti-TLR7 agent of embodiment C50, wherein TLR7 is detected in B cells by intracellular staining.

[0746] C52. The anti-TLR7 agent of embodiment C51, wherein TLR7 is detected in B cells by intracellular staining with a signal-to-noise ratio of about 2 or greater.

[0747] C53. The anti-TLR7 agent of embodiment C51, wherein TLR7 is detected in B cells by intracellular staining with a signal-to-noise ratio of about 3 or greater.

[0748] C54. The anti-TLR7 agent of embodiment C51, wherein TLR7 is detected in B cells by intracellular staining with a signal-to-noise ratio of about 4 or greater.

[0749] C54a. The anti-TLR7 agent of any one of embodiments C1 to C54, wherein a significant level of TLR7 is detected in monocytes.

[0750] C54b. The anti-TLR7 agent of embodiment C54a, wherein TLR7 is detected in monocytes using a flow cytometry assay.

[0751] C54c. The anti-TLR7 agent of embodiment C54a, wherein TLR7 is detected in monocytes by intracellular staining.

[0752] C54d. The anti-TLR7 agent of embodiment C54c, wherein TLR7 is detected in monocytes by intracellular staining with a signal-to-noise ratio of about 2 or greater.

[0753] C54e. The anti-TLR7 agent of embodiment C54c, wherein TLR7 is detected in monocytes by intracellular staining with a signal-to-noise ratio of about 3 or greater.

[0754] C54f. The anti-TLR7 agent of embodiment C54c, wherein TLR7 is detected in monocytes by intracellular staining with a signal-to-noise ratio of about 4 or greater.

[0755] C55. The anti-TLR7 agent of any one of embodiments C1 to C54f, wherein TLR7 is not significantly detected in other immune cells of the cell population, wherein the other immune cells include CD3 - CD19 - Lymphocytes and CD3 + CD19 - One or more T cells.

[0756] C56. The anti-TLR7 agent of any one of embodiments C1 to C55, wherein the agent is isolated.

[0757] C57. The anti-TLR7 agent of any one of embodiments C1 to C56, wherein the agent is non-naturally occurring.

[0758] C58. The anti-TLR7 agent of any one of embodiments C1 to C57, wherein the agent is an antibody or an antigen-binding fragment thereof.

[0759] C59. The anti-TLR7 agent of any one of embodiments C1 to C57, wherein the agent is an antibody or a derivative thereof.

[0760] C60. The anti-TLR7 agent of any one of embodiments C1 to C59, wherein the agent is a humanized antibody or an antigen-binding fragment thereof.

[0761] C61. The anti-TLR7 agent of any one of embodiments C1 to C59, wherein the agent is a derivative of a humanized antibody that binds to TLR7.

[0762] C62. The anti-TLR7 agent of any one of embodiments C1 to C61, wherein the agent comprises a detectable marker or label.

[0763] C63. The anti-TLR7 agent of any one of embodiments C1 to C62, wherein the agent is conjugated to a detectable marker or label.

[0764] C64. The anti-TLR7 agent of any one of embodiments C1 to C63, wherein the agent is non-diffusively immobilized on a solid support.

[0765] C65. A diagnostic reagent comprising the anti-TLR7 agent of any one of embodiments C1 to C64.

[0766] C66. A kit comprising the anti-TLR7 agent of any one of embodiments C1 to C64 or the diagnostic agent of embodiment C65.

[0767] C67. A diagnostic kit configured to detect Toll-like receptor 7 (TLR7) in a biological sample, wherein the kit comprises the anti-TLR7 agent of any one of embodiments C1 to C64 or the diagnostic reagent of embodiment C65.

[0768] C68. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the immunoglobulin heavy chain variable domain of the anti-TLR7 agent of any one of embodiments C1 to C64.

[0769] C69. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the immunoglobulin light chain variable domain of the anti-TLR7 agent of any one of embodiments C1 to C64.

[0770] C70. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an anti-TLR7 agent of any one of embodiments C1 to C64, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of an anti-TLR7 agent of any one of embodiments C1 to C64.

[0771] C71. A recombinant host cell transfected with the recombinant expression vector of embodiment C70.

[0772] C72. A method for detecting TLR7, comprising contacting a sample known or suspected to contain TLR7 with the anti-TLR7 agent of any one of embodiments C1 to C64, and if the sample contains TLR7, detecting a TLR7:anti-TLR7 complex.

[0773] C73. A method for detecting TLR7 in a heterogeneous immune cell population, comprising contacting the cell population with an anti-TLR7 agent of any one of embodiments C1 to C64, and if the cell population contains cells expressing TLR7, detecting a TLR7:anti-TLR7 complex.

[0774] ***

[0775] The entire contents of each patent, patent application, publication, and document cited herein are incorporated herein by reference. Citation of the above patents, patent applications, publications, and documents does not admit that any of the above is relevant prior art, nor does it constitute any admission of the contents or dates of these publications or documents. These citations do not imply a search for relevant publications. All statements regarding the dates or contents of documents are based on the available information and do not constitute an admission of their accuracy or correctness.

[0776] The foregoing may be modified without departing from the basic aspects of the technology. Although the technology has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, but these modifications and improvements are still within the scope and spirit of the technology of the present invention.

[0777] The technology described in this paper example can be implemented without any element not specifically disclosed herein. Therefore, for example, in each example herein, any term in the term "comprising", "consisting essentially of ... " and "consisting of ... " can be replaced by any of the other two terms. The terms and expressions used are used as descriptive terms rather than restrictive, and the use of these terms and expressions does not exclude any equivalent of the features shown and described or its parts, and various modifications can be made within the scope of the claims of this technology. The term "one" or "a kind of" can refer to one or more / kinds of elements (such as "a reagent" can refer to one or more reagents) modified by it, unless the context clearly states that it is a kind of element or multiple elements described. As used herein, the term "about" refers to a value within 10% of the basic parameter (i.e., plus or minus 10%), and the use of the term "about" at the beginning of a string of values ​​can modify each value (i.e., "about 1, 2 and 3" refers to about 1, about 2 and about 3). For example, the weight of "about 100g" can include the weight between 90g and 110g. In addition, when a list of values ​​is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values ​​and fractional values ​​thereof (e.g., 54%, 85.4%). Therefore, it should be understood that although the present technology has been specifically disclosed through representative embodiments and optional features, those skilled in the art may make modifications and variations to the concepts disclosed herein, and these modifications and variations are considered to be within the scope of the present technology.

[0778] Certain embodiments of the present technology are set forth in the following claims.

Claims

1. An anti-TLR7 agent that binds to Toll-like receptor 7 (TLR7) under laboratory or physiological conditions, wherein the agent comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain, wherein: The immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2 and CDRH3 amino acid sequences and the immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2 and CDRL3 amino acid sequences, which are selected from the following groups 1 to 6: And wherein all CDR sequences are from the same group.

2. The anti-TLR7 agent according to claim 1, wherein the agent is isolated.

3. The anti-TLR7 agent according to any one of claims 1 to 2, wherein the agent is non-naturally occurring.

4. The anti-TLR7 agent according to any one of claims 1 to 2, wherein the agent is an antibody or an antigen-binding fragment thereof.

5. The anti-TLR7 agent according to any one of claims 1 to 2, wherein the agent is a humanized antibody or an antigen-binding fragment thereof.

6. The anti-TLR7 agent according to any one of claims 1 to 2, wherein the agent comprises a detectable marker or label.

7. The anti-TLR7 agent according to any one of claims 1 to 2, wherein the agent is conjugated to a detectable marker or label.

8. The anti-TLR7 agent according to any one of claims 1 to 2, wherein the agent is non-diffusively immobilized on a solid support.

9. A diagnostic agent comprising the anti-TLR7 agent according to any one of claims 1 to 8. 10 . A kit comprising the anti-TLR7 agent according to claim 1 or the diagnostic agent according to claim 9 . 11 . A diagnostic kit configured to detect Toll-like receptor 7 (TLR7) in a biological sample, wherein the kit comprises the anti-TLR7 agent of any one of claims 1 to 8 or the diagnostic agent of claim 9.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the anti-TLR7 agent of any one of claims 1 to 8.

13. An isolated first nucleic acid molecule and an isolated second nucleic acid molecule, wherein the isolated first nucleic acid molecule comprises a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an anti-TLR7 agent of any one of claims 1 to 8, and the isolated second nucleic acid molecule comprises a nucleotide sequence encoding an immunoglobulin light chain variable domain of an anti-TLR7 agent of any one of claims 1 to 8.

14. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an anti-TLR7 agent of any one of claims 1 to 8, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of an anti-TLR7 agent of any one of claims 1 to 8.

15. A recombinant host cell transfected with the recombinant expression vector of claim 14.

16. A non-diagnostic method for detecting TLR7, comprising contacting a sample known or suspected to contain TLR7 with an anti-TLR7 agent according to any one of claims 1 to 8, and if the sample contains TLR7, detecting a TLR7:anti-TLR7 complex.

17. A non-diagnostic method for detecting TLR7 in a heterogeneous population of immune cells comprising contacting the population with an anti-TLR7 agent according to any one of claims 1 to 8 and detecting a TLR7:anti-TLR7 complex if the population contains cells expressing TLR7.

18. An anti-TLR7 agent for detecting TLR7 in a heterogeneous immune cell population, wherein significant levels of TLR7 are detected in plasmacytoid dendritic cells, monocytes and / or B cells in the population, while TLR7 is not significantly detected in other immune cells in the population, wherein the agent comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain, wherein the immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2 and CDRH3 amino acid sequences and the immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2 and CDRL3 amino acid sequences, which are selected from the following 1 to 6 groups: And wherein all CDR sequences are from the same group.

19. The anti-TLR7 agent according to claim 18, wherein TLR7 is detected at a significant level in plasmacytoid dendritic cells.

20. The anti-TLR7 agent according to claim 19, wherein TLR7 is detected in plasmacytoid dendritic cells using a flow cytometry assay.

21. The anti-TLR7 agent according to claim 19, wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining.

22. The anti-TLR7 agent according to claim 21, wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining at a signal-to-noise ratio of 5 or more.

23. The anti-TLR7 agent according to claim 21, wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining at a signal-to-noise ratio of 10 or more. 24 . The anti-TLR7 agent according to claim 21 , wherein TLR7 is detected in plasmacytoid dendritic cells by intracellular staining at a signal-to-noise ratio of 15 or more.

25. The anti-TLR7 agent according to any one of claims 18 to 24, wherein TLR7 is detected at a significant level in B cells.

26. The anti-TLR7 agent according to claim 25, wherein TLR7 is detected in B cells using a flow cytometry assay.

27. The anti-TLR7 agent according to claim 25, wherein TLR7 is detected in B cells by intracellular staining.

28. The anti-TLR7 agent according to claim 27, wherein TLR7 is detected in B cells by intracellular staining at a signal-to-noise ratio of 2 or more.

29. The anti-TLR7 agent according to claim 27, wherein TLR7 is detected in B cells by intracellular staining at a signal-to-noise ratio of 3 or more.

30. The anti-TLR7 agent according to claim 27, wherein TLR7 is detected in B cells by intracellular staining at a signal-to-noise ratio of 4 or more.

31. The anti-TLR7 agent according to any one of claims 18 to 24, wherein TLR7 is detected at a significant level in monocytes.

32. The anti-TLR7 agent according to claim 31, wherein TLR7 is detected in monocytes using a flow cytometry assay.

33. The anti-TLR7 agent according to claim 31, wherein TLR7 is detected in monocytes by intracellular staining.

34. The anti-TLR7 agent according to claim 33, wherein TLR7 is detected in monocytes by intracellular staining at a signal-to-noise ratio of 2 or more.

35. The anti-TLR7 agent according to claim 33, wherein TLR7 is detected in monocytes by intracellular staining at a signal-to-noise ratio of 3 or more.

36. The anti-TLR7 agent according to claim 33, wherein TLR7 is detected in monocytes by intracellular staining at a signal-to-noise ratio of 4 or more.

37. The anti-TLR7 agent according to any one of claims 18 to 24, wherein TLR7 is not significantly detected in other immune cells in the population, wherein the other immune cells include CD3 - CD19 - Lymphocytes and CD3 + CD19 - One or more T cells.

38. An anti-TLR7 agent according to any one of claims 18 to 24, wherein the agent is isolated.

39. An anti-TLR7 agent according to any one of claims 18 to 24, wherein the agent is non-naturally occurring.

40. An anti-TLR7 agent according to any one of claims 18 to 24, wherein the agent is an antibody or an antigen-binding fragment thereof.

41. An anti-TLR7 agent according to any one of claims 18 to 24, wherein the agent is a humanized antibody or an antigen-binding fragment thereof.

42. An anti-TLR7 agent according to any one of claims 18 to 24, wherein the agent comprises a detectable marker or label.

43. An anti-TLR7 agent according to any one of claims 18 to 24, wherein the agent is conjugated to a detectable marker or label.

44. An anti-TLR7 agent according to any one of claims 18 to 24, wherein the agent is non-diffusively immobilized on a solid support.

45. A diagnostic reagent comprising the anti-TLR7 agent of any one of claims 18 to 44.

46. ​​A kit comprising the anti-TLR7 agent of any one of claims 18 to 44 or the diagnostic agent of claim 45.

47. A diagnostic kit configured to detect Toll-like receptor 7 (TLR7) in a biological sample, wherein the kit comprises the anti-TLR7 agent of any one of claims 18 to 44 or the diagnostic reagent of claim 45.

48. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the anti-TLR7 agent of any one of claims 18 to 44.

49. An isolated first nucleic acid molecule and an isolated second nucleic acid molecule, wherein the isolated first nucleic acid molecule comprises a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an anti-TLR7 agent of any one of claims 18 to 44, and the isolated second nucleic acid molecule comprises a nucleotide sequence encoding an immunoglobulin light chain variable domain of an anti-TLR7 agent of any one of claims 18 to 44.

50. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of an anti-TLR7 agent of any one of claims 18 to 44, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of an anti-TLR7 agent of any one of claims 18 to 44.

51. A recombinant host cell transfected with the recombinant expression vector of claim 50.

52. A non-diagnostic method for detecting TLR7 comprising contacting a sample known or suspected to contain TLR7 with an anti-TLR7 agent according to any one of claims 18 to 44, and if the sample contains TLR7, detecting a TLR7:anti-TLR7 complex.

53. A non-diagnostic method for detecting TLR7 in a heterogeneous population of immune cells comprising contacting the population with an anti-TLR7 agent according to any one of claims 18 to 44, and if the population comprises cells expressing TLR7, detecting a TLR7:anti-TLR7 complex.

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