CN117615774A

CN117615774A - PD-L1 binding peptides and peptide complexes and methods of use thereof

Info

Publication number: CN117615774A
Application number: CN202180092404.6A
Authority: CN
Inventors: Z·克鲁克; J·奥尔森; N·W·奈恩; C·科伦蒂
Original assignee: Fred Hutchinson Cancer Research Center; Blaze Bioscience Inc
Current assignee: Blaze Bioscience Inc; Fred Hutchinson Cancer Center
Priority date: 2020-11-30
Filing date: 2021-11-29
Publication date: 2024-02-27

Abstract

Described herein are cystine dense peptides capable of binding to PD-L1. This peptide may act as a PD-L1 inhibitor by being able to bind to the PD-1 binding interface of PD-L1 and inhibit the interaction between PD-L1 and PD-1. Also described herein are methods of using PD-L1 peptide complexes to treat cancer, autoimmune diseases, or other disorders. This method involves delivering an active agent complexed with a PD-L1 peptide to PD-L1 positive cells or recruiting immune cells to PD-L1 positive cells using bispecific immune cell cement. Chimeric antigen receptors comprising PD-L1 peptide-binding are also described herein.

Description

PD-L1 binding peptides and peptide complexes and methods of use thereof

Cross reference

The present application claims the benefits of U.S. provisional application No.63/119,195 AND U.S. provisional application No.63/273,103 entitled "composition AND method for selective depletion of target molecules (COMPOSITIONS AND METHODS FOR SELECTIVE DEPLETION OF TARGET MOLECULES)" filed on 11/30/2020 AND U.S. provisional application No.63/273,103 entitled "PD-L1 BINDING peptide AND peptide complex AND method for use thereof (PD-L1 BINDING peptide AND peptide complex AND METHODS OF USE THEREOF)" filed on 10/2021, each of which is incorporated herein by reference in its entirety for all purposes.

Sequence listing

The present application contains a sequence listing that has been electronically submitted in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy created at 2021, 11/23 is named 108406-709288_sl. Txt and is 627,485 bytes in size.

Background

Programmed death ligand 1 (PD-L1) is a transmembrane protein that binds programmed cell death protein 1 (PD-1) and is believed to play a role in immunosuppression and T cell inactivation. Cancer cells expressing PD-L1 can evade the host immune response and inactivate T cells by binding to PD-1 receptors on host T cells, thereby preventing the host immune system from destroying the cancer cells. anti-PD-L1 antibodies may block the binding of PD-L1 to PD-1. However, there is a need for other PD-L1 binding agents that can be used to target PD-L1.

Disclosure of Invention

In various aspects, the disclosure provides PD-L1 binding peptides, the PD-L1 binding peptides comprising a first PD-L1 binding motif, the first PD-L1 binding motif comprising the following sequence: (a) X is X ¹ X ² X ³ X ⁴ X ⁵ X ⁶ CX ⁷ X ⁸ X ⁹ C (SEQ ID NO: 361), wherein X ¹ D, E, H, K, N, Q, S, T, L, V, F, Y or P; x is X ² G, E, Q or F; x is X ³ Is D or K; x is X ⁴ G, V or P; x is X ⁵ G, H, R, V, F, W or P; x is X ⁶ A, D or K; x is X ⁷ E, H, Q, L or F; x is X ⁸ D, E, R, S, T, M, L or F; and X is ⁹ G, A, D, E, H, K, R, M, L or P; or (b) X ¹ FX ² VFX ² CLX ³ X ³ C (SEQ ID NO: 363) wherein X ¹ Is K or P; x is X ² Independently D or K; and X is ³ Independently any non-cysteine amino acid.

In some aspects, the PD-L1 binding peptide comprises at least six cysteine residues. In some aspects, at least six cysteine residues are located at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, where n corresponds to the position of the first cysteine residue of the at least six cysteine residues. In some aspects, amino acid position n corresponds to amino acid position 4 such that at least six cysteine amino acid residues are located at amino acid positions 4, 8, 18, 32, 42, and 46.

In some aspects, the PD-L1 binding peptide further comprises at least three disulfide bonds linking at least six cysteine residues. In some aspects, at least three disulfide bonds will: the first cysteine residue of the at least six cysteine residues is linked to the sixth cysteine residue of the at least six cysteine residues, the second cysteine residue of the at least six cysteine residues is linked to the fifth cysteine residue of the at least six cysteine residues, and the third cysteine residue of the at least six cysteine residues is linked to the fourth cysteine residue of the at least six cysteine residues. In some aspects, the first cysteine residue is at amino acid position n, the second cysteine residue is at amino acid position n+4, the third cysteine residue is at amino acid position n+14, the fourth cysteine residue is at amino acid position n+28, the fifth cysteine residue is at amino acid position n+38, and the sixth cysteine residue is at amino acid position n+42. In some aspects, the first cysteine residue is at amino acid position 4, the second cysteine residue is at amino acid position 8, the third cysteine residue is at amino acid position 18, the fourth cysteine residue is at amino acid position 32, the fifth cysteine residue is at amino acid position 42, and the sixth cysteine residue is at amino acid position 46.

In some aspects, the PD-L1 binding peptide further comprises a first alpha helix comprising residues n to n+20, wherein n corresponds to the amino acid position of the first cysteine residue. In some aspects, the PD-L1 binding peptide further comprises a second alpha helix comprising residues n+34 to n+44, wherein n corresponds to the amino acid position of the first cysteine residue. In some aspects, the second alpha helix comprises residues n+29 to n+44.

In some aspects, the N-terminal amino acid residue of the first PD-L1 binding motif is at amino acid residue position n+32, wherein N corresponds to the amino acid position of the first cysteine residue. In some aspects, the C-terminal amino acid residue of the first PD-L1 binding motif is at amino acid position n+42, wherein n corresponds to the amino acid position of the first cysteine residue. In some aspects, the first PD-L1 binding motif comprises the sequence of KFDVFKCLDHC (SEQ ID NO: 365).

In some aspects, the PD-L1 binding peptide further comprises a second PD-L1 binding motif, the second PD-L1 binding motif comprising the sequence: (a) CX (CX) ¹ X ² X ³ CX ⁴ X ⁵ X ⁶ X ⁷ X ⁸ X ⁹ X ¹⁰ X ¹¹ X ¹² C (SEQ ID NO: 360), wherein X ¹ K, R or V; x is X ² E, Q, S, M, L or V; x is X ³ D, E, H, K, R, N is a,Q, S or Y; x is X ⁴ D, M or V; x is X ⁵ A, K, R, Q, S or T; x is X ⁶ A, D, E, H, Q, S, T, M, I, L, V or W; x is X ⁷ A, E, R, Q, S, T, W or P; x is X ⁸ A, E, K, R, N, Q, T, M, I, L, V or W; x is X ⁹ G, A, E, K, N, T or Y; x is X ¹⁰ G, A, D, E, H, K, R, N, Q, S, T, M, I, L, V, W, Y or P; x is X ¹¹ D, K, R, N, L or V; and X is ¹² G, A, D, T, L, W or P; or (b) CKVX ¹ CVX ¹ X ¹ X ¹ X ¹ X ² X ³ KX ¹ C (SEQ ID NO: 362), wherein X ¹ Independently any non-cysteine amino acid; x is X ² M, I, L or V; and X is ³ Y, A, H, K, R, N, Q, S or T. In some aspects, the N-terminal amino acid residue of the second PD-L1 binding motif is located at amino acid residue position N, wherein N corresponds to the amino acid position of the first cysteine residue. In some aspects, the C-terminal amino acid residue of the first PD-L1 binding motif is at amino acid position n+14, wherein n corresponds to the amino acid position of the first cysteine residue. In some aspects, the second PD-L1 binding motif comprises the sequence of CKVHCVKEWMAGKAC (SEQ ID NO: 364).

In some aspects, the PD-L1 binding peptide comprises the sequence of SEQ ID NO 358 or SEQ ID NO 359. In some aspects, the PD-L1 binding peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% sequence identity to SEQ ID NO. 1. In some aspects, the PD-L1 binding peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% sequence identity to SEQ ID NO. 2. In some aspects, the PD-L1 binding peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% sequence identity to SEQ ID NO. 3. In some aspects, the PD-L1 binding peptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% sequence identity to SEQ ID NO. 4.

In various aspects, the disclosure provides a PD-L1 binding peptide comprising at least six cysteine residues at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, wherein n corresponds to the position of the first cysteine residue of the at least six cysteine residues and has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity to SEQ ID NO:57 or SEQ ID NO: 59.

In various aspects, the disclosure provides a PD-L1 binding peptide comprising at least eight cysteine residues at amino acid positions n, n+11, n+17, n+21, n+31, n+38, n+40, or n+44, wherein n corresponds to the position of the first cysteine residue of the at least six cysteine residues and has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity to SEQ ID NO: 58.

In various aspects, the disclosure provides a PD-L1 binding peptide comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity to any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437, or SEQ ID NO:554-SEQ ID NO: 567.

In some aspects, the PD-L1 binding peptide comprises the sequence of any one of SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436, SEQ ID NO:437 or SEQ ID NO: 554-567.

In some aspects, the PD-L1 binding peptide is capable of having an equilibrium dissociation constant (K) of no greater than 100nM, no greater than 50nM, no greater than 1nM, no greater than 500pM, no greater than 300pM, no greater than 250pM, or no greater than 200pM _D ) Binds to PD-L1. In some aspects, the PD-L1 binding peptide is capable of having an equilibrium dissociation constant (K _D ) Binds to PD-L1. In some aspects, the PD-L1 binding peptide is capable of having an equilibrium dissociation constant (K) that differs by no more than 1.5-fold, no more than 2-fold, no more than 5-fold, or no more than 10-fold _D ) Binding to human PD-L1 and cynomolgus PD-L1.

In some aspects, the PD-L1 binding peptide comprises at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, or at least 49 amino acid residues.

The PD-L1 binding peptide of any one of claims 1-29, wherein the PD-L1 binding peptide comprises 43 to 51 amino acid residues. In some aspects, the PD-L1 binding peptide comprises no more than 50 amino acid residues. In some aspects, the PD-L1 binding peptide comprises 43 to 49 amino acid residues.

In some aspects, the PD-L1 binding peptide further comprises a half-life modulator. In some aspects, the half-life modulator is selected from the group consisting of: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly (amino acids), water-soluble polymers of proline, alanine and serine, water-soluble polymers containing glycine, glutamic acid and serine, fc regions, fatty acids, palmitic acid, albumin and molecules bound to albumin. In some aspects, the half-life modulator is an albumin binding peptide. In some aspects, the half-life modulator is an Fc domain. In some aspects, the half-life modulator is polyethylene glycol. In some aspects, the half-life modulator is a fatty acid.

In various aspects, the present disclosure provides a peptide complex comprising a PD-L1 binding peptide complexed with an active agent, wherein the PD-L1 binding peptide comprises: at least six cysteine residues at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, wherein n corresponds to the amino acid position of the first of the at least six cysteine residues; at least three disulfide bonds linking the first cysteine residue to a sixth cysteine residue of the at least six cysteine residues, the second cysteine residue of the at least six cysteine residues to a fifth cysteine residue of the at least six cysteine residues, and the third cysteine residue of the at least six cysteine residues to a fourth cysteine residue of the at least six cysteine residues.

In some aspects, amino acid position n corresponds to amino acid position 4 such that at least six cysteine amino acid residues are located at amino acid positions 4, 8, 18, 32, 42, and 46. In some aspects, the PD-L1 binding peptide further comprises a first alpha helix comprising residues n to n+20, wherein n corresponds to the amino acid position of the first cysteine residue. In some aspects, the PD-L1 binding peptide further comprises a second alpha helix comprising residues n+34 to n+44, wherein n corresponds to the amino acid position of the first cysteine residue. In some aspects, the second alpha helix comprises residues n+29 to n+44.

In some aspects, the PD-L1 binding peptide comprises a first PD-L1 binding motif, the first PD-L1 binding motif comprising the sequence: (a) X is X ¹ X ² X ³ X ⁴ X ⁵ X ⁶ CX ⁷ X ⁸ X ⁹ C (SEQ ID NO: 361), wherein X ¹ D, E, H, K, N, Q, S, T, L, V, F, Y or P; x is X ² G, E, Q or F; x is X ³ Is D or K; x is X ⁴ G, V or P; x is X ⁵ G, H, R, V, F, W or P; x is X ⁶ A, D or K; x is X ⁷ E, H, Q, L or F; x is X ⁸ D, E, R, S, T, M, L or F; and X is ⁹ G, A, D, E, H, K, R, M, L or P; or (b) X ¹ FX ² VFX ² CLX ³ X ³ C (SEQ ID NO: 363) wherein X ¹ Is K or P; x is X ² Independently D or K; and X is ³ Independently any non-cysteine amino acid. In some aspects, the N-terminal amino acid residue of the first PD-L1 binding motif is located at amino acid residue position n+32. In some aspects, the C-terminal amino acid residue of the first PD-L1 binding motif is located at amino acid position n+42. In some aspects, the first PD-L1 binding motif comprises the sequence of KFDVFKCLDHC (SEQ ID NO: 365).

In some aspects, the PD-L1 binding peptide further comprises a second PD-L1 binding motif, the second PD-L1 binding motif comprising the sequence: (a) CX (CX) ¹ X ² X ³ CX ⁴ X ⁵ X ⁶ X ⁷ X ⁸ X ⁹ X ¹⁰ X ¹¹ X ¹² C (SEQ ID NO: 360), wherein X ¹ K, R or V; x is X ² E, Q, S, M, L or V; x is X ³ D, E, H, K, R, N, Q, S or Y; x is X ⁴ D, M or V; x is X ⁵ A, K, R, Q, S or T; x is X ⁶ A, D, E, H, Q, S, T is a,M, I, L, V or W; x is X ⁷ A, E, R, Q, S, T, W or P; x is X ⁸ A, E, K, R, N, Q, T, M, I, L, V or W; x is X ⁹ G, A, E, K, N, T or Y; x is X ¹⁰ G, A, D, E, H, K, R, N, Q, S, T, M, I, L, V, W, Y or P; x is X ¹¹ D, K, R, N, L or V; and X is ¹² G, A, D, T, L, W or P; or (b) CKVX ¹ CVX ¹ X ¹ X ¹ X ¹ X ² X ³ KX ¹ C (SEQ ID NO: 362), wherein X ¹ Independently any non-cysteine amino acid; x is X ² M, I, L or V; and X is ³ Y, A, H, K, R, N, Q, S or T. In some aspects, the N-terminal amino acid residue of the second PD-L1 binding motif is located at amino acid residue position N. In some aspects, the C-terminal amino acid residue of the first PD-L1 binding motif is located at amino acid position n+14. In some aspects, the second PD-L1 binding motif comprises the sequence of CKVHCVKEWMAGKAC (SEQ ID NO: 364). In some aspects, amino acid position n corresponds to amino acid position 4 of the PD-L1 binding peptide, such that at least six cysteine amino acid residues are located at amino acid positions 4, 8, 18, 32, 42, and 46 of the PD-L1 binding peptide.

In some aspects, the PD-L1 binding peptide is capable of having an equilibrium dissociation constant (K) of no greater than 100nM, no greater than 50nM, no greater than 30nM, no greater than 20nM, no greater than 1nM, no greater than 500pM, no greater than 300pM, no greater than 250pM, or no greater than 200pM _D ) Binds to PD-L1. In some aspects, the PD-L1 binding peptide comprises at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, or at least 49 amino acid residues. In some aspects, the PD-L1 binding peptide comprises 43 to 51 amino acid residues. In some aspects, the PD-L1 binding peptide comprises 43 to 49 amino acid residues.

In various aspects, the present disclosure provides a peptide complex comprising any PD-L1 binding peptide described herein complexed with an active agent.

In some aspects, the active agent comprises an immune cell targeting agent. In some aspects, the immune cell targeting agent is an immune cell targeting peptide. In some aspects, the immune cell targeting agent comprises a single chain variable fragment (scFv), a cysteine dense peptide, a high affinity multimer, a Kong Nici domain, an affibody, an adestin protein (adnectin), a nano-phenanthridine Ding Danbai (nanofittin), a fenomer (fynomer), a β -hairpin, a staple peptide, a bicyclic peptide, an antibody fragment, a protein, a peptide fragment, a binding domain, a small molecule, or a nanobody capable of binding to an immune cell. In some aspects, the immune cell targeting agent is capable of binding T cells, B cells, macrophages, natural killer cells, fibroblasts, regulatory T cells, regulatory immune cells, neural stem cells, or mesenchymal stem cells. In some aspects, the immune cell targeting agent is capable of binding T cells. In some aspects, the immune cell targeting agent is capable of binding regulatory T cells. In some aspects, the immune cell targeting agent is capable of binding to CD3, 4-1BB, CD28, CD137, CD89, CD16, CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX, PD-1, CTLA-4, or STRO-1. In some aspects, the immune cell targeting agent is capable of binding CD3. In some aspects, the immune cell targeting agent is capable of binding CD25. In some aspects, the immune cell targeting agent is capable of binding 4-1BB. In some aspects, the immune cell targeting agent is capable of binding CD28. In some aspects, the immune cell targeting agent comprises a sequence having at least 90% sequence identity to any one of SEQ ID NO. 122 or SEQ ID NO. 442-SEQ ID NO. 491.

In some aspects, the immune cell targeting agent is fused to a first heterodimerization domain and the PD-L1 binding peptide is fused to a second heterodimerization domain. In some aspects, the first heterodimerization domain is complexed with the second heterodimerization domain to form a heterodimer. In some aspects, the first heterodimerization domain, the second dimerization domain, or both comprise an Fc domain. In some aspects, the first heterodimerization domain, the second dimerization domain, or both comprise the sequence of any one of SEQ ID NOS: 124-153. In some aspects, the first heterodimerization domain comprises the sequence of any of SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, or SEQ ID NO:152 and the second heterodimerization domain comprises the sequence of any of SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO: 153. In some aspects, the first heterodimerization domain comprises chain 1 of the heterodimerization pair provided in table 3. In some aspects, the second heterodimerization domain comprises chain 2 of the heterodimerization pair provided in table 3. In some aspects, the second heterodimerization domain comprises the sequence of any of SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, or SEQ ID NO:152 and the first heterodimerization domain comprises the sequence of any of SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO: 153. In some aspects, the first heterodimerization domain comprises chain 2 of the heterodimerization pair provided in table 3. In some aspects, the second heterodimerization domain comprises chain 1 of the heterodimerization pair provided in table 3. In some aspects, the peptide complex comprises a sequence having at least 90% sequence identity to SEQ ID NO. 119 or SEQ ID NO. 120. In some aspects, the peptide complex comprises a sequence having at least 90% sequence identity to SEQ ID NO. 123.

In some aspects, the immune cell targeting agent and the PD-L1 binding peptide are fused to a homodimerization domain. In some aspects, the immune cell targeting agent and the PD-L1 binding peptide form a single polypeptide chain. In some aspects, the peptide complex comprises a sequence having at least 90% sequence identity to any one of SEQ ID NO:121 or SEQ ID NO:438-SEQ ID NO: 441.

In some aspects, the immune cell targeting agent is linked to the PD-L1 binding peptide via a linker. In some aspects, the linker comprises a peptide linker. In some aspects, the linker comprises a small molecule linker. In some aspects, the linker comprises an Fc domain. In some aspects, the peptide complex further comprises an albumin binding domain, polyethylene glycol, or both.

In some aspects, the active agent comprises a transmembrane domain, an intracytoplasmic domain, or a combination thereof. In some aspects, the active agent comprises a chimeric antigen receptor. In some aspects, the peptide complex further comprises T cells.

In some aspects, the active agent comprises a therapeutic agent, a detectable agent, or a combination thereof. In some aspects, the detectable agent comprises a fluorophore, a near infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radionuclide, or a radionuclide chelator. In some aspects, the therapeutic agent comprises an anticancer agent, a chemotherapeutic agent, a radiotherapeutic agent, an anti-inflammatory agent, a pro-inflammatory cytokine, an oligonucleotide, an immune tumor agent, or a combination thereof. In some aspects, the active agent comprises a radioisotope. In some aspects, the radioisotope comprises an alpha emitter, a beta emitter, a positron emitter, a gamma emitter, a metal, an actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, yttrium, actinium-225, lead-212, ¹¹ C or ¹⁴ C、 ¹³ N、 ¹⁸ F、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁸⁹ Zr、 ¹⁷⁷ Lu, indium-111, technetium-99 m, yttrium-90, iodine-131, iodine-123, or astatine-211.

In some aspects, the oligonucleotide comprises DNA, RNA, antisense oligonucleotide, aptamer, miRNA, siRNA, alternative splicing regulator, mRNA binding sequence, miRNA binding sequence, siRNA binding sequence, RNaseH1 binding oligonucleotide, RISC binding oligonucleotide, polyadenylation regulator, or a combination thereof. In some aspects, the oligonucleotide comprises the sequence of any one of SEQ ID NO 366-396, 492-545, or 552. In some aspects, the oligonucleotide junctionAnd a target sequence comprising any one of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO: 549. In some aspects, the peptide complex remains intact after incubation in human serum. In some aspects, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% remain intact after incubation in human serum. In some aspects, the PD-L1 binding peptide has an equilibrium dissociation constant (K) for PD-L1 upon complexing with an oligonucleotide _D ) No greater than 10nM, 5nM, 1nM, 800pM, 600pM, 500pM, 400pM, 300pM, 250pM or 200pM. In some aspects, the PD-L1 binding peptide has a lower affinity for PD-L1 at pH 5.5, 6.0, or 6.5 than at pH 7.4.

In some aspects, the anti-inflammatory agent comprises an anti-inflammatory cytokine, a steroid, a glucocorticoid, a corticosteroid, a cytokine inhibitor, a rory inhibitor, a JAK inhibitor, a tyrosine kinase inhibitor, or a non-steroidal anti-inflammatory drug. In some aspects, the anti-cancer agent comprises an anti-tumor agent, a cytotoxic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, a retinoid, an inhibitor of microtubule polymerization, a pyrrolobenzodiazepine dimer, or an anti-cancer antibody. In some aspects, the proinflammatory cytokine comprises TNF alpha, IL-2, IL-6, IL-12, IL-15, IL-21, or IFN gamma. In some aspects, the therapeutic agent comprises an oncolytic viral vector.

In some aspects, the peptide complex further comprises a half-life modulator. In some aspects, the half-life modulator is selected from the group consisting of: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly (amino acids), water-soluble polymers of proline, alanine and serine, water-soluble polymers containing glycine, glutamic acid and serine, fc regions, fatty acids, palmitic acid and molecules bound to albumin. In some aspects, the albumin-binding molecule is a serum albumin-binding peptide.

In some aspects, the peptide complex further comprises a cell penetrating peptide. In some aspects, the cell penetrating peptide comprises the sequence of any one of SEQ ID NOS 249-341.

In various aspects, the present disclosure provides a pharmaceutical composition comprising any PD-L1 binding peptide described herein, or any peptide complex described herein, and a pharmaceutically acceptable carrier.

In various aspects, the present disclosure provides a method of inhibiting PD-L1 in a subject, the method comprising: administering to the subject a composition comprising a PD-L1 binding peptide, the PD-L1 binding peptide comprising at least six cysteine residues, and at least three disulfide bonds connecting the at least six cysteine residues; binding the PD-L1 binding peptide to PD-L1 on a PD-L1 positive cell; and inhibiting the PD-L1.

In some aspects, at least six cysteine residues are located at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, where n corresponds to the amino acid position of the first cysteine residue of the at least six cysteine residues. In some aspects, amino acid position n corresponds to amino acid position 4 such that at least six cysteine amino acid residues are located at amino acid positions 4, 8, 18, 32, 42, and 46.

In various aspects, the present disclosure provides a method of inhibiting PD-L1 in a subject, the method comprising: administering to the subject a composition comprising any PD-L1 binding peptide described herein; binding the PD-L1 binding peptide to PD-L1 on a PD-L1 positive cell; and inhibiting the PD-L1.

In some aspects, inhibiting PD-L1 comprises inhibiting PD-1 from binding to PD-L1. In some aspects, the method further comprises reducing immunosuppression, reducing T cell depletion, restoring immune function, or a combination thereof. In some aspects, the method further comprises treating the subject for a disorder. In some aspects, the disorder is cancer, and wherein the PD-L1 positive cells are cancer cells. In some aspects, treating the cancer includes enhancing an immune response against the cancer cells.

In various aspects, the present disclosure provides a method of delivering an active agent to PD-L1 positive cells of a subject, the method comprising: administering to the subject a peptide complex comprising a PD-L1 binding peptide complexed with an active agent, the PD-L1 binding peptide comprising at least six cysteine residues, and at least three disulfide bonds linking the at least six cysteine residues; binding the PD-L1 binding peptide to a PD-L1 positive cell; and delivering the active agent to the PD-L1 positive cells.

In various aspects, the present disclosure provides a method of delivering an active agent to PD-L1 positive cells of a subject, the method comprising: administering to the subject a peptide complex comprising any PD-L1 binding peptide described herein complexed with an active agent, or the peptide complex of any one of claims 39-107; binding the PD-L1 binding peptide to a PD-L1 positive cell; and delivering the active agent to the PD-L1 positive cells.

In some aspects, the active agent comprises an anticancer agent, a chemotherapeutic agent, a radiotherapeutic agent, or a pro-inflammatory cytokine. In some aspects, the active agent comprises an oligonucleotide. In some aspects, the peptide complex remains intact after incubation in human serum. In some aspects, the PD-L1 binding peptide, when complexed with the oligonucleotide, has an equilibrium dissociation constant (K _D ) Binds to PD-L1.

In some aspects, the methods further comprise binding the oligonucleotide to a target sequence after delivery to the PD-L1 positive cell. In some aspects, the method further comprises modulating alternative splicing of the target sequence, indicating the location of a polyadenylation site of the target sequence, inhibiting translation of the target sequence, inhibiting binding of the target sequence to a secondary target sequence, recruiting RISC to the target sequence, recruiting RNaseH1 to the target sequence, inducing cleavage of the target sequence, or modulating the target sequence upon binding of an oligonucleotide to the target sequence. In some aspects, the active agent comprises an anti-inflammatory cytokine, a steroid, a glucocorticoid, a corticosteroid, or a non-steroidal anti-inflammatory drug. In some aspects, the active agent comprises an immune cell targeting agent.

In some aspects, the methods further comprise binding an immune cell targeting agent to the immune cell and recruiting the immune cell to the PD-L1 positive cell. In some aspects, recruiting immune cells to PD-L1 positive cells comprises forming immune synapses. In some aspects, the width of the immune synapse is 3nm to 25nm, 5nm to 20nm, or 10nm to 15nm. In some aspects, the width of the immune synapse is no greater than 3nm, no greater than 5nm, no greater than 8nm, no greater than 10nm, no greater than 13nm, no greater than 15nm, no greater than 18nm, no greater than 20nm, no greater than 23nm, no greater than 25nm, no greater than 30nm, no greater than 35nm, no greater than 40nm, no greater than 45nm, or no greater than 50nm.

In some aspects, the immune cells comprise T cells, B cells, macrophages, natural killer cells, fibroblasts, regulatory T cells, regulatory immune cells, neural stem cells, or mesenchymal stem cells. In some aspects, the immune cell targeting agent binds to CD3, 4-1BB, CD28, CD137, CD89, CD16, CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX, PD-1, CTLA-4, or STRO-1. In some aspects, the immune cell targeting agent binds CD3. In some aspects, the immune cell targeting agent binds CD25. In some aspects, the immune cell targeting agent binds 4-1BB. In some aspects, the immune cell targeting agent binds CD28.

In some aspects, the method further comprises killing the PD-L1 positive cells after the immune cells are delivered to the PD-L1 positive cells. In some aspects, the method further comprises inhibiting the PD-L1 positive cells after the immune cells are delivered to the PD-L1 positive cells. In some aspects, the immune cell targeting agent comprises a single chain variable fragment (scFv), a cysteine dense peptide, a high affinity multimer, a Kong Nici domain, an affibody, an adestin protein, a nano-phenanthrene Ding Danbai, a fenobody, a β -hairpin, a staple peptide, a bicyclic peptide, an antibody fragment, a protein, a peptide fragment, a binding domain, a small molecule, or a nanobody.

In some aspects, the immune cell targeting agent is fused to a first heterodimerization domain and the PD-L1 binding peptide is fused to a second heterodimerization domain. In some aspects, the first heterodimerization domain is complexed with the second heterodimerization domain to form a heterodimer. In some aspects, the first heterodimerization domain, the second dimerization domain, or both comprise an Fc domain.

In some aspects, the immune cell targeting agent is linked to the PD-L1 binding peptide via a linker. In some aspects, the immune cell targeting agent is linked to the PD-L1 binding peptide via an Fc domain. In some aspects, the immune cell targeting agent and the PD-L1 binding peptide form a single polypeptide chain.

In some aspects, the peptide complex comprises a chimeric antigen receptor. In some aspects, the active agent comprises a transmembrane domain, an intracytoplasmic domain, or a combination thereof. In some aspects, the peptide complex further comprises T cells. In some aspects, the method further comprises delivering the T cell to a PD-L1 positive cell. In some aspects, the method further comprises killing PD-L1 positive cells.

In some aspects, the method further comprises treating the subject for a disorder. In some aspects, the disorder is cancer. In some aspects, the PD-L1 positive cell is a cancer cell. In some aspects, the cancer comprises melanoma, skin cancer, non-small cell lung cancer, kidney cancer, esophageal cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, colon cancer, rectal cancer, head and neck cancer, lymphoma, bladder cancer, liver cancer, gastric cancer, stomach cancer, breast cancer, triple negative breast cancer, pancreatic cancer, prostate cancer, merck cell cancer, mesothelioma, brain cancer, or PD-L1 expressing cancer. In some aspects, the brain cancer comprises glioblastoma, astrocytoma, meningioma, primary brain cancer, metastatic brain cancer, PDL1 expressing cancer, or metastatic brain cancer.

In some aspects, the disorder is hyperglycemia, type 1 diabetes, or type 2 diabetes. In some aspects, the PD-L1 positive cells comprise pancreatic β cells. In some aspects, the immune cells are regulatory T cells, and wherein recruitment of regulatory T cells to pancreatic beta cells protects pancreatic beta cells and prevents hyperglycemia, type 1 diabetes, or type 2 diabetes, reduces their effects, reduces their symptoms, slows their onset, and thereby treats hyperglycemia, type 1 diabetes, or type 2 diabetes in the subject.

In some aspects, the disorder is an autoimmune or inflammatory disorder. In some aspects, the PD-L1 positive cells comprise pancreatic β cells. In some aspects, the immune cells comprise regulatory T cells or mesenchymal stem cells. In some aspects, the immune cell targeting agent binds CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, or STRO-1. In some aspects, the immune cells inhibit an autoimmune or inflammatory response upon recruitment to PD-L1 positive cells, thereby treating an autoimmune or inflammatory disorder. In some aspects, the autoimmune or inflammatory disorder comprises rheumatoid arthritis, atherosclerosis, ischemia reperfusion injury, colitis, psoriasis, lupus, inflammatory bowel disease, crohn's disease, ulcerative colitis, multiple sclerosis, type 1 diabetes, type 2 diabetes, or neuroinflammation.

In some aspects, the PD-L1 binding peptide has an equilibrium dissociation constant (K) of no greater than 100nM, no greater than 50nM, no greater than 1nM, no greater than 500pM, no greater than 300pM, no greater than 250pM, or no greater than 200pM _D ) Binds to PD-L1.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIGS. 1A-1D illustrate mammalian display and library screening methods for selecting a Cystine Dense Peptide (CDP) scaffold for a PD-L1 binding peptide.

Fig. 1A schematically illustrates a general mammalian surface display screening method using lentiviral vectors to screen for target binding properties or to evaluate peptide quality (right panel, "vector SDPR"). Mammalian cells, such as HEK 293F cells, express and display GFP-tagged cystine compact peptides (CDPs) from a CDP library. In the first assay (left panel, "vector SDGF"), CDP is screened for binding to a fluorescently labeled target protein (e.g., biotinylated protein labeled with fluorescent streptavidin). Cells were classified according to co-stained fluorescence to select CDPs that bound to the target protein. In the second assay, quality assessment was performed on labeled CDP from the CDP library. The complete labeled CPD (e.g., CDP with a 6XHis tag (SEQ ID NO: 248)) is labeled with a co-stain (e.g., a fluorescently labeled anti-6 XHis antibody). Cells were classified according to co-stained fluorescence to select for CDPs that were expressed and displayed intact.

FIG. 1B shows a histogram distribution of surface folding scores of CDPs from a taxonomically diverse CDP library. The highest scoring CDPs (n=953) corresponding to cystine scaffolds predicted to have high surface expression, protease resistance, or both were selected for further analysis.

FIG. 1C schematically illustrates a system for identifying members of a new CDP library. About 100,000 planned CDP sequences were searched for CDPs with sequence homology or cysteine pattern homology to the highest scoring CDP identified in fig. 1B. The second generation optimized library ("Gen 2 library") thus generated contained 8,893 CDPs.

FIG. 1D shows a flow-based comparison of CDP stability between a first generation diverse CDP library and a second generation optimized CDP library identified in FIG. 1C and containing peptides having homology to predicted high stability CDP scaffolds. Cells cloned for SDPR using CDP from a diverse (left) or optimized (right) library with a 6XHis tag (SEQ ID NO: 248) were treated with 5. Mu.g/mL trypsin (thin solid line), 20. Mu.g/mL trypsin (thick solid line) or PBS (dotted line, "trypsin free") for 5 min. Cells were then treated with DTT for 5 min and stained with fluorescent anti-6 xHis antibody. The histogram distribution of CDP is normalized to mode. The optimized library exhibits improved surface expression and protease resistance compared to the variegated library.

FIGS. 2A-2C illustrate the selection of various PD-L1 binding peptides that match three-dimensional structure, computational model and phylogenetic development, with a high confidence in the selection of PD-L1 binding peptides having PD-L1 binding activity.

FIG. 2A schematically illustrates a structurally modeled pipeline for predicting three-dimensional structure and disulfide bond pairing of optimized CDP library members. The structural model was created from the CDP sequence using modeling software version 5.1 of I-TASSER. The most likely disulfide pairing is determined by minimizing the average pairing distance between the bonded cysteine sulfur atoms in the structural model. The Rosetta ForceDislufides procedure relaxes the disulfide structure by means of a full atom finishing and reassembly algorithm to minimize spatial conflicts.

Fig. 2B shows a representative alignment of the crystal structure obtained as described in fig. 2A with the computational model for 20 CDPs from the optimized CDP library. The crystal structure of these CDPs is not included in the structural database for computational modeling. The crystal structure obtained from the RCSB protein database is referenced with PDB ID number. Root Mean Square Deviation (RMSD) of the alignment between crystal structure and modeled structure is provided below PDB ID number or SEQ ID NO. For crystal structures having multiple polypeptide chains in an asymmetric unit, RMSD represents an average alignment between the modeled structure and each polypeptide in the asymmetric unit.

FIG. 2C shows a graphical comparison of Root Mean Square Deviation (RMSD) of the twenty CDP comparisons shown in FIG. 2B. Comparing the calculation model with an Asymmetric Unit (AU) of a crystal structure, and obtaining an average RMSD of(left circle). The different asymmetric units of the crystals are aligned with each other and the average RMSD is +.>(right circle) provides a quantitative assessment of the accuracy of the models used in fig. 2A and 2B.

FIG. 3 shows a structural model of the centroid cluster (netpen indicated by arrow) of CDP library peptide docked to programmed death ligand 1 (PD-L1). PD-1, which binds to PD-L1, is shown as a band structure based on its crystal structure (PDB ID NO:4ZQK; zak et al, structure23,2341-2348 (2015)). These centroid clusters are examples of the potential binding of PD-L1 binding peptides to PD-L1. The centroid cluster located near the PD-1 binding site is indicated by the open arrow. Any of these PD-L1 binding peptides may modulate the activity of PD-1 at its receptor PD-L1, including by steric or direct effects, and those peptides that bind at or near the PD-1 binding site may directly inhibit the binding of PD-1 at the PD-L1 active site.

FIGS. 4A and 4B illustrate the selection of various PD-L1 binding peptides matched to crystal structure, computational model and phylogenetic development, with high confidence that PD-L1 binding-active, PD-L1 binding-binding, cystine-dense peptides. The cystine dense peptide binding to PD-L1 was tested as shown.

FIG. 4A shows Rosetta energy docking score plots for various docked PD-L1-binding cystine compact peptides plotted against Solvent Accessible Surface Area (SASA) of CDP scaffolds. Each dot represents a peptide that interfaces with the PD-L1 structure, color coded according to the main structural element in the scaffold (e.g., coil, alpha-helix or beta-sheet). Gray dots correspond to coil-rich CDPs, dark dots and solid triangles correspond to spiral-rich CDPs, and light dots and open triangles correspond to fold-rich CDPs. Triangles represent CDPs identified as binding hits, as determined by staining with fluorescent-labeled PD-L1 and flow sorting, enriched for peptides that bind well to PD-L1. Four triangles are shown, two of which almost overlap. The light grey shading corresponds to a PD-L1 binding peptide cluster with a docking cluster score at the PD-1 binding interface of less than about 26, representing scaffolds from the optimized library, for generating a docking-enriched Met/Tyr scan (DEMYS) library. Dark grey shading corresponds to peptide clusters with a cluster score of about 50% of the median score, indicating scaffolds that are not expected to bind well to PD-L1, as determined by fluorescent-labeled PD-L1 staining and flow sorting, in fact do not bind well. These results demonstrate that PD-L1 binding peptides selected and enriched for binding have increased potential to bind PD-L1 and PD-L1 binding peptide utility, as described herein.

FIG. 4B illustrates a phylogenetic tree showing the taxonomic diversity of CDPs that predict high-score binding PD-L1 that interfaces with PD-L1 near the PD-1 binding interface, with centroid clusters of PD-L1 binding peptides located near the PD-1 binding site, and with high utility for binding PD-L1 and disrupting PD-1 interactions.

FIGS. 5A-5F illustrate the enrichment and binding of PD-L1 by PD-L1 binding peptides of PD-L1 binding peptides expressed in a mammalian display system.

FIG. 5A illustrates the workflow (top) of protein interactions using a rich docking methionine (M) -tyrosine (Y) scan (DEMYS) to inoculate new proteins. This shows how the DEMYS enrichment in the reference sequence further diversifies the optimized library and changes the residues from hydrophilic to hydrophobic residues, in this case methionine (M) and tyrosine (Y) examples. A sample holder (bottom) using DEMYS is also shown. The sample holder is color coded in hydrophobicity such that the lightest shading indicates that the carbon atom is not in contact with a polar atom, the middle shading indicates an acidic atom, the darker shading indicates a basic atom, and the remaining atoms appear white. The reference sequence scaffold was not expected to bind well to PD-L1 but showed a way how to diversify the library, which was shown in its parent form (SEQ ID NO: 356), and three examples of Met or Tyr mutations corresponding to D18M (SEQ ID NO: 5), R26Y (SEQ ID NO: 6) and R36M (SEQ ID NO: 7).

FIG. 5B shows the results of a mammalian display screen based on optimizing CDP library fluorescence. The results are plotted as binding PD-L1 with CDP expression. Cells showing CDP binding to PD-L1 are indicated by boxes.

FIG. 5C shows the results of screening further diverse optimized libraries using a fluorescence-based mammalian display enriched in docked methionine-tyrosine scanning (DEMYS) CDP library. The results are plotted as binding PD-L1 with CDP expression. Cells showing CDP binding to PD-L1 are indicated by boxes.

FIG. 5D illustrates a structural model of the CDP scaffold parent of SEQ ID NO. 4, which was identified in the optimized CDP library mammalian display screen shown in FIG. 5B.

FIG. 5E shows a flow chart of CDP expression versus co-staining fluorescence for CDP of transiently transfected SDGF clones identified in the PD-L1 mammal display screen shown in FIG. 5B corresponding to SEQ ID NO. 4. The dark grey line corresponds to the staining in the presence of PD-L1. The light grey line corresponds to the staining in the absence of PD-L1.

FIG. 5F shows a flow chart of CDP expression versus co-stained fluorescence for CDPs corresponding to four transiently transfected SDGF clones of SEQ ID NO:4 (top left), SEQ ID NO:57 (top right), SEQ ID NO:58 (bottom left) and SEQ ID NO:59 (bottom right) identified in the PD-L1 DEYS library screen shown in FIG. 5C. The dark grey line corresponds to the staining in the presence of PD-L1. The light grey line corresponds to the staining in the absence of PD-L1. These data show that SEQ ID NO:4, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59 bind to PD-L1 and that the PD-L1 DEMYS library produces more molecules that the optimized library produces.

FIGS. 6A-6C illustrate structural and competitive binding aspects of PD-L1 binding peptides that bind to PD-L1. This data shows that SEQ ID NO 4 competes with PD-1 and that it binds to human and cynomolgus monkey PD-L1.

FIG. 6A shows a structural model of the binding interface between the first 200 PD-L1 binding peptides (grids) identified in the optimized CDP library screen shown in FIG. 5B and PD-L1 docking. Surface reproduction color coded for cynomolgus monkey (left) and mouse (right) homology. PD-1, which binds to PD-L1, is shown as a band structure.

FIG. 6B illustrates the results of a competition assay evaluating the ability of a CDP (corresponding to SEQ ID NO: 4) that binds PD-L1 to disrupt the interaction of PD-1 with PD-L1. HEK 293F cells expressing SEQ ID NO. 4 by SDGF surface were stained with fluorescent-labeled PD-L1. The staining was compared in the presence of 50nM, 150nM, 500nM, 1.5. Mu.M, 5. Mu.M or 15. Mu.M PD-1 competitive Fc fusion protein (striped bars) or in the presence of 50nM, 150nM, 500nM, 1.5. Mu.M, 5. Mu.M or 15. Mu.M control Fc fusion protein (dashed bars).

FIG. 6C shows flow-staining assays of cells expressing SEQ ID NO:4 in combination with human (darkest gray line), cynomolgus monkey (lightest gray line, "Cyno") or mouse (middle gray line) PD-L1.

FIGS. 7A-7C illustrate the use of site-directed saturation mutagenesis and flow sorting for binding to PD-L1 to identify and enrich high affinity binders for PD-L1 binding peptides.

FIG. 7A shows a first round-robin sorting of site-directed saturation mutagenesis (SSM) variants of SEQ ID NO. 4. The cell surface displaying the CDP variant of SEQ ID NO. 4 was stained with fluorescent PD-L1 and flow sorted based on binding to PD-L1.

FIG. 7B shows a second round robin sorting of site-directed saturation mutagenesis (SSM) variants of SEQ ID NO. 4. The cell surface displaying the CDP variant of SEQ ID NO. 4 was stained with fluorescent PD-L1 and flow sorted based on binding to PD-L1. The first 7% of PD-L1 binding agent is further analyzed in fig. 8.

FIG. 7C shows the flow sorting of site-directed saturation mutagenesis (SSM) variants of SEQ ID NO. 4 stained with co-stain only in the absence of PD-L1.

FIG. 8 shows an exemplary affinity maturation heat map of a variant of SEQ ID NO. 4 for identifying high performance residues and point mutations within PD-L1 binding peptides. The heat map was used to identify PD-L1 binding peptides, including SEQ ID NO 3, with high affinity for PD-L1. Shading corresponds to the relative enrichment of each amino acid point mutation (vertical axis) relative to SEQ ID NO:4 (horizontal axis). Lighter shaded boxes indicate more (positive) enrichment, while darker shaded boxes indicate depletion. That is, lighter shaded boxes indicate sequences with increased relative abundance after two rounds of selection for PD-L1 binding, indicating improved binding relative to SEQ ID NO:4, while darker shaded boxes indicate sequences with decreased relative abundance after two rounds of selection for PD-L1 binding, indicating disruption of binding relative to SEQ ID NO: 4. A binary color plot of this data indicating residues with positive enrichment is provided in fig. 21, and quantification of the enrichment data is provided in table 12. Thermogram shows log of each variant after two rounds of sorting and regrowth ₂ Conversion enrichment, initial variant abundance and performance normalization relative to SEQ ID NO 4 itself. Positive enrichment scores represent increased relative abundance, thus improving binding. The sequences of the parental scaffolds (SEQ ID NO: 353), primary hits (SEQ ID NO: 4) and affinity matured variants (SEQ ID NO: 3) are provided below the heat map. Abrupt changes are made according to benefits or possible passenger statesColor coding. Asterisks indicate novel N-linked glycosylation sites. "beneficial in SEQ ID NO: 3" means those point mutations that are combined and included in SEQ ID NO:3 to increase the affinity of SEQ ID NO:3 for PD-L1 relative to SEQ ID NO: 4. "omitted from SEQ ID NO: 3" means a point mutation that appears to be beneficial alone, but when combined with other beneficial point mutations, disrupts binding. This point mutation is omitted in SEQ ID NO. 3. "destructive reversion" means point mutations with reduced binding when reverted to the parent amino acid, indicating that those point mutations in SEQ ID NO. 4 favor PD-L1 binding. "neutral reversion" means that reversion to the parent amino acid present in SEQ ID NO:353 does not affect binding to PD-L1, indicating that these mutations were obtained during library generation, but does not contribute to the binding of SEQ ID NO:4 to PD-L1. This neutral reversion is also known as a passenger break.

FIG. 9 shows a comparison of PD-L1 binding of six amino acid-substituted, dense peptides of cystine that bind PD-L1 enriched in the affinity maturation assay shown in FIG. 8. The cystine compact peptide contains all six of the amino acid substitutions E11W, A13M, Y15G, I22N, Y K and W40F (SEQ ID NO:8, "all 6"), or five of the six substitutions (SEQ ID NO:9-SEQ ID NO:14, corresponding to the revertant mutants W11E, M13A, G15Y, N22I, K Y and F40W, respectively) relative to SEQ ID NO: 4. The dyeing is carried out in one step (solid bars) or in two steps (striped bars). Revertants W11E (SEQ ID NO: 9), G15Y (SEQ ID NO: 11) and K36Y (SEQ ID NO: 13) showed increased affinity for PD-L1 relative to SEQ ID NO:8, indicating that corresponding revertants favor PD-L1 binding relative to SEQ ID NO:8 ("all 6").

FIGS. 10A-10D illustrate the production, purity, purification, stability and PD-L1 binding affinity of a dense peptide of cystine that binds PD-L1.

FIG. 10A shows reverse phase high performance liquid chromatography (RP-HPLC) chromatograms (upper panel) and SDS-PAGE gels (lower panel) of three recombinant PD-L1 binding cystine compact peptides (SEQ ID NO:4, SEQ ID NO:3 and SEQ ID NO: 1). RP-HPLC and SDS-PAGE determinations are performed under non-reducing (NR) conditions or in the presence of 10mM DTT. The arrow in the RP-HPLC chromatogram represents a minor substance that may represent glycosylated SEQ ID NO: 3. This data demonstrates the successful production, purity, purification and disulfide bond formation of SEQ ID NO. 4, SEQ ID NO. 3 and SEQ ID NO. 1. RP-HPLC data also indicated that mature SEQ ID NO:3 and SEQ ID NO:1 undergo less unfolding when exposed to DTT than originally hit SEQ ID NO: 4.

FIG. 10B shows liquid chromatography-mass spectrometry (LC-MS) data for three recombinant PD-L1-binding cystine compact peptides (SEQ ID NO:4, SEQ ID NO:3, and SEQ ID NO: 1). This data verifies the identity of the above PD-L1 binding peptide based on predicted CDP molar mass and forms a cystine disulfide bond indicative of CDP structure, as DTT-treated (reducing conditions) peptides showed an increase in mass of about 6Da.

FIG. 10C shows Surface Plasmon Resonance (SPR) plots of three recombinant PD-L1-binding cystine compact peptides (SEQ ID NO:4, SEQ ID NO:3, and SEQ ID NO: 1). SEQ ID NO. 4 shows equilibrium dissociation constant (K) at 39.6.+ -. 0.3nM _D ) PD-L1 binding to SEQ ID NO 3 at K _D ＝160±1pM(k _a ＝1.25±0.01×10 ⁸ M ^-1 s，k _d ＝2.00±0.01×10 ^-2 s ^-1 ) Binds PD-L1 and SEQ ID NO 1 at K _D ＝202±2pM(k _a ＝9.73±0.06×10 ⁷ M ^-1 s，k _d ＝1.96±0.01×10 ^-2 s ^-1 ) Binds to PD-L1. This data demonstrates that SEQ ID NO. 4, SEQ ID NO. 3 and SEQ ID NO. 1 bind PD-L1 with high affinity. These data also indicate that mature variant binders SEQ ID NO. 1 and SEQ ID NO. 3 bind PD-L1 with higher affinity than their source SEQ ID NO. 4.

FIG. 10D shows SPR plots of SEQ ID NO 1 competing with PD-1 for binding to PD-L1. The binding of PD-1 (SEQ ID NO: 349) to PD-L1 in the absence of SEQ ID NO:1 or in the presence of 0.6. Mu.M or 3. Mu.M SEQ ID NO:1 was compared. This data demonstrates that SEQ ID NO 1 is able to compete with PD-1 for binding to PD-L1.

FIG. 11 schematically illustrates two exemplary bispecific immunocyte cement (BiICE) complexes capable of binding T cells by bispecific binding to PD-L1 and CD 3. CDP-based BiICE ("CS-BiICE") is formed from a first fusion protein (SEQ ID NO: 342) containing a PD-L1-binding CDP (SEQ ID NO: 2) having an N-terminal signal peptide (SEQ ID NO:247; "SP") and a FLAG tag (DYKDEGGS; SEQ ID NO: 246) fused via a linker to an Fc "mortar" sequence and a second fusion protein (SEQ ID NO: 347) containing an anti-CD 3 single chain variable fragment (scFv) fused to an Fc "pestle" sequence having an N-terminal signal peptide and a C-terminal 6XHis tag (SEQ ID NO: 248). The "mortar" sequence heterodimerizes with the "pestle" sequence. scFv-based biece ("SS-biece") is formed from a first fusion protein (SEQ ID NO: 346) containing an anti-PD-L1 scFc having an N-terminal signal peptide and a FLAG tag fused to an Fc "mortar" sequence, and a second fusion protein (SEQ ID NO: 347) containing an anti-CD 3 single chain variable fragment (scFv) fused to an Fc "pestle" sequence having an N-terminal signal peptide and a C-terminal 6xHis tag (SEQ ID NO: 248). The sequence organization of the CS-BiICE and SS-BiICE components is shown in the above figures. The structural organization of heterodimerized CS-BiICE and SS-BiICE is shown in the following figure.

FIG. 12 shows the results of a fluorescence-based binding assay to evaluate cross-reactivity of various PD-L1 binding moieties with different PD-L1 orthologs. Cells expressing the surface tethered PD-L1 binding moiety, including a cystine dense peptide (SEQ ID NO: 3) that binds PD-L1, a single chain variable fragment (scFv) derived from an anti-PD-L1 antibody (SEQ ID NO: 345) or a scFv (SEQ ID NO: 348) derived from the anti-PD-L1 drug, alezolizumab, were stained for human, cynomolgus monkey ("Cyno") or mouse His-labeled PD-L1 orthologs and co-stained with an anti-His-iFluor 647 antibody. As a comparison, an anti-His-iFluor 647 antibody alone ("co-stain only") was used. Co-stained fluorescence of the cells is shown on the horizontal axis. This data shows that SEQ ID NO:3 binds human and cynomolgus monkey PD-L1 but not mouse PD-L1, SEQ ID NO:345 binds human PD-L1 but not cynomolgus monkey or mouse PD-L1, and SEQ ID NO:348 binds human, cynomolgus monkey and mouse PD-L1 to varying degrees.

FIGS. 13A-13D illustrate the production, purity, purification and binding to PD-L1 of BiICE complexes containing a cystine dense peptide that binds to PD-L1 or an scFv that binds to PD-L1.

FIG. 13A shows an SDS-PAGE gel of purified CS-BiICE molecules (heterodimerized with SEQ ID NO: 347) SEQ ID NO: 342. Separate bands corresponding to heterodimer (H) and anti-CD 3-scFv-Fc monomer (M) were seen under non-reducing (NR) conditions. Separate bands corresponding to CDP-Fc and scFv-Fc species were seen under reducing conditions (DTT). This data suggests successful generation, purity, purification, heterodimer formation, and disulfide bonding of CDP-based biece.

FIG. 13B shows SPR measurements of CS-BiICE binding to PD-L1. CS-BiICE with a K of 11.2nM _D (k _a ＝5.42×10 ⁵ M ^-1 s，k _d ＝6.07±0.01×10 ^-3 s ^-1 ) Binds to PD-L1. This data suggests that CDP-based biece binds PD-L1 with high affinity.

FIG. 13C shows an SDS-PAGE gel of purified SS-BiICE molecules (heterodimerized with SEQ ID NO:347 and SEQ ID NO: 346). Separate bands corresponding to heterodimer (H) and anti-CD 3-scFv-Fc monomer (M) were seen under non-reducing (NR) conditions. A single band corresponding to the scFv-Fc species was seen under reducing conditions (DTT). This data suggests successful production, purity, dimer formation and purification of scFv-based biece.

FIG. 13D shows SPR measurements of SS-BiICE binding to PD-L1. SS-BiICE with a K of 65nM _D Binds to PD-L1. This data suggests that scFv biece binds to PD-L1 with lower affinity than CDP-based biece shown in fig. 13B.

FIGS. 14A-14E illustrate validation of PD-L1 binding BiICE complexes by binding to T cells and inducing tumor cell death.

FIG. 14A shows the flow staining of human primary T cells from peripheral blood mononuclear cells (PMBC) from patient sources using CS-BiICE and SS-BiICE molecules. Cells were co-stained with fluorescent anti-6 xHis antibody. Co-staining alone served as a control. Both CS-BiICE and SS-BiICE showed increased staining over co-staining alone, indicating T cell binding. This data suggests that CS-BiICE and SS-BiICE molecules that bind PD-L1/CD3 are capable of binding to T cells via CD 3.

FIG. 14B shows the results of human T cell killing assays at increased concentrations of CS-BiICE or SS-BiICE in PC3 cancer cell lines. CS-BiICE induced T cell killing in PC3 cells expressing PD-L1 ("WT") at a half maximal effective concentration (EC 50) of 28 pM. SS-BiICE induced T cell killing at an EC50 of 97pM in PC3 cells expressing PD-L1 ("WT"). T cell killing assays were also performed in PD-L1 Knockout (KO) PC3 cells. T cell killing was lower in PD-L1 KO cells than in WT cells for CS-BiICE and SS-BiICE. This data indicates that CS-biece and SS-biece molecules are able to recruit T cells to cancer cells and induce cancer cell death in PC3 cells in a PD-L1 dependent manner. This data also shows that CS-BiICE is more effective at inducing cancer cell death than SS-BiICE.

FIG. 14C shows the results of human T cell killing assays at increased concentrations of CS-BiICE or SS-BiICE in MDA-MB-231 cancer cell lines. CS-BiICE induced T cell killing with an EC50 of 142 pM. SS-BiICE induced T cell killing at an EC50 of 333 pM. This data indicates that CS-BiICE and SS-BiICE molecules are capable of recruiting T cells to cancer cells and inducing cancer cell death in MDA-MB-231 cells. This data also shows that CS-BiICE is more effective at inducing cancer cell death than SS-BiICE.

FIG. 14D shows the results of human T cell killing assays at increased concentrations of CS-BiICE or SS-BiICE in a PBT-05 cancer cell line. CS-BiICE induced T cell killing with an EC50 of 2.4 pM. SS-BiICE induced T cell killing with an EC50 of 7.7 pM. This data indicates that CS-BiICE and SS-BiICE molecules are capable of recruiting T cells to cancer cells and inducing cancer cell death in PBT-05 cells. This data also shows that CS-BiICE is more effective at inducing cancer cell death than SS-BiICE.

Fig. 14E schematically illustrates that representative biece promotes contact between T cells and cancer cells, enabling T cells to kill cancer cells. The immune synapse is sufficiently narrow to allow signal exchange between a targeted cell engaged by the PD-L1 binding peptide and a targeted immune cell engaged by an immune cell binding moiety (e.g., an anti-CD 3 moiety), thereby generating an immune response against a cancer cell. In this example, SEQ ID NO. 2 is used. It will be appreciated that any PD-L1 binding peptide (e.g., any of SEQ ID NOS: 1-118) can be used with an immune cell binding moiety (e.g., any of SEQ ID NOS: 122 or 422-491) to make BiICE to generate an immune response against the targeted cancer.

FIGS. 15A and 15B illustrate additional purification of SS-BiICE and CS-BiICE complexes and induction of tumor cell death by T cell recruitment.

FIG. 15A shows SDS-PAGE gels of BiICE preparations of IMAC purification and additional purification (IMAC and FLAG). The purified SS-BiICE formulation is shown on the left and the purified CS-BiICE formulation is shown on the right. This data shows that additional FLAG purification removes impurities, anti-CD 3-scFv-Fc monomers, present in the SS-BiICE and CS-BiICE formulations after IMAC purification.

FIG. 15B shows the results of human T cell killing assays at different concentrations of standard purified (IMAC) or additionally purified (IMAG+FLAG) CS-BiICE or SS-BiICE in PC3 cancer cell lines. In addition to IMAC purification, the additionally purified BiICE preparation was FLAG purified. After an additional FLAG purification step, T cell killing activity was retained. This data shows that after removal of impurities by additional purification steps, T cell recruitment and tumor cell killing properties of SS-biece and CS-biece complexes are preserved and tumor cell killing is due to the function of the heterodimeric biece molecule and not due to impurities.

FIGS. 16A-16H illustrate the ability of BiICE complexes based on PD-L1 binding CDP to shrink tumors and increase cancer survival in vivo.

Fig. 16A shows an experimental timeline of in vivo tumor shrinkage assays. Nude mice were implanted with PC3 or MDA-MB-231 tumors. Setting the tumor size to 100-200mm on the zeroth day ² Is the day of the year. Mice received four SS-biece or CS-biece treatments on days 1, 4, 8 and 11 and two activated human T-cell treatments on days 2 and 7. Tumor size and mortality were tracked to day 95 and compared to control groups that received vehicle alone or T cells alone.

Fig. 16B shows the probability of survival of the first group of mice implanted with PC3 tumors and treated as described in fig. 16A. This data shows that CS-biece complexes significantly increase the survival probability in mice implanted with PC3 tumors compared to SS-biece complexes or T cells or vehicle alone. ns: has no statistical significance. * : p <0.05.* *: p <0.01.* **: p <0.001. Unlabeled: p <0.0001. The Kaplan-Meier curve (Kaplan-Meier curve) P value was determined by a log rank (Mantel-Cox) test.

Fig. 16C shows tumor sizes of mice implanted with PC3 tumors and treated with vehicle only (no T cells, no BiICE, upper left), T cells only (no BiICE, upper right), T cells and SS-BiICE (lower left) or T cells and CS-BiICE (lower right). This data shows that while both SS-BiICE and CS-BiICE complexes show the ability to shrink tumors in vivo, tumors recover in mice treated with SS-BiICE but not in mice treated with CS-BiICE.

Fig. 16D shows the probability of survival of a second group of mice implanted with PC3 tumors and treated as described in fig. 16A, except that both arms were treated with the clinical anti-PD-L1 antibody, dulvalumab, in an amount of 1nmol or 0.1 nmol. In addition to repeating 1nmol of CS-BiICE, an additional arm of 0.1nmol of CS-BiICE treatment was included. This data shows that the CS-biece complex increases the probability of survival in mice implanted with PC3 tumors at doses of 1nmol or 0.1nmol compared to either the dulcis You Shan antibody or the vehicle alone. 1nmol and 0.1nmol CS-BiICE compared to vehicle and two Duvalli You Shan antibody groups, P <0.0001.ns: has no statistical significance. The kaplan-mel curve P value is determined by a log rank (mantel-cox) test.

FIG. 16E shows tumor sizes of mice implanted with PC3 tumors and treated with vehicle alone (no T cells, no BiICE, upper left), T cells and 1nmol of Kovalli You Shan antibody (upper center, "Durva (1)"), T cells and 0.1nmol of Kovali You Shan antibody (upper right, "Durva (0.1)"), T cells and 1nmol of CS-BiICE (lower left, "CS-BiICE (1)") or T cells and 0.1nmol of CS-BiICE (lower center, "CS-BiICE (0.1)"). This data indicates tumor shrinkage and no recovery in mice treated with either 1nmol or 0.1nmol of CS-BiICE.

FIG. 16F shows the probability of survival of mice implanted with MDA-MB-231 tumors and treated as described in FIG. 16A. This data shows that the CS-BiICE complex increases the probability of survival in mice implanted with MDA-MB-231 tumors compared to T cells or vector alone. P <0.0001 for CS-BiITE compared to vehicle or T cells alone. ns: has no statistical significance. The kaplan-mel curve P value is determined by a log rank (mantel-cox) test.

Figure 16G shows the number of days in which tumor volume was increased three times in mice implanted with MDA-MB-231 tumors and treated as described in figure 16A. This data shows that treatment with the CS-BiICE complex slows the growth of MDA-MB-231 tumors in vivo compared to treatment with the SS-BiICE complex or with T cells or vehicle alone. ns: has no statistical significance. * : p <0.05.* *: p <0.01.* **: p <0.001. Unlabeled: p <0.0001. The kaplan-mel curve P value is determined by a log rank (mantel-cox) test.

FIG. 16H shows tumor sizes of mice implanted with MDA-MB-231 tumors and treated with vehicle only (no T cells, no BiICE, upper left), T cells only (no BiICE, upper right), T cells and SS-BiICE (lower left) or T cells and CS-BiICE (lower right). This data shows that treatment with the CS-BiICE complex slows the growth of MDA-MB-231 tumors in vivo compared to treatment with the SS-BiICE complex or with T cells or vehicle alone.

Figure 17 shows the weight of mice implanted with PC3 tumors and treated as described in figure 16A throughout the experiment. This data shows that mice treated with CS-BiICE maintain healthy weight throughout the course of the study. ns: has no statistical significance. The kaplan-mel curve P value is determined by a log rank (mantel-cox) test.

FIG. 18 shows a complete SDS-PAGE gel of recombinant proteins described herein. PBS or 10mM DTT was included in the sample buffer prior to boiling and loading. All Fc-containing proteins, including the mortar and pestle bispecific molecules SS-biece and CS-biece, contain an IgG1 hinge region between the conjugate of interest (PD-1, control target, CDP, or scFv) and the Fc domain, including two cysteines, which form a pair of inter-domain disulfide bonds between the paired hinge regions. This further stabilizes the dimer between Fc domains in an SDS stable manner. The self-expressed anti-CD 3scFv Fc pestle protein (bottom right) is not a disulfide stabilized dimer, because the pestle mutation does not allow stable dimerization and thus disulfide formation. ", and" # "indicate SDS-PAGE mobility of non-BiICE contaminants in SS-BiICE and CS-BiICE corresponding to monomeric anti-CD 3scFv Fc pestle.

Fig. 19 shows an example of histidine substitution scanning for introducing pH-dependent binding affinity into target binding peptides. Histidine substitution scans of CDP (SEQ ID NO: 1) binding PD-L1 are shown. The peptide sequence is provided above and sideways, and each black box represents a first site and a second site in which His may be substituted. Those positions falling down the diagonal from top left to right represent single His substitutions.

FIG. 20A shows the eutectic structure of a high affinity PD-L1-binding CDP (SEQ ID NO:1, sketch) that binds or interfaces with PD-L1 (surface).

FIG. 20B shows the relative binding enrichment (shown as absolute value of average SSM enrichment) of CDP variants that bind PD-L1 containing amino acid substitutions in resolved (R) residues or Unresolved (UR) residues, as seen in the eutectic structure of FIG. 20A. Substitution at resolved residues had a greater (positive or negative) effect on binding than substitution at non-resolved residues (: p=0.0055), indicating that resolved residues play a greater role in interactions with PD-L1 than non-resolved residues.

FIG. 20C shows the superposition of PD-1 (grid) and SEQ ID NO 1 (sketch) at the binding interface with PD-L1 (surface). The PD-1 binding site overlaps with SEQ ID NO. 1, indicating that SEQ ID NO. 1 is expected to compete with PD-1 for binding to PD-L1.

FIG. 20D shows an enlarged view of the eutectic structure of SEQ ID NO:1PD-L1 of FIG. 20A from two different angles. Residues of SEQ ID NO. 1 (including K5, V9, W12, M13, K16, V39, F40, L43 and D44) that interact with PD-L1 are shown as bars. Residues of PD-L1, including Y56, Q66, R113, M115, A121 and Y123, which interact with SEQ ID NO. 1 are also labeled.

FIG. 20E shows isolated side chains of selected residues in SEQ ID NO. 1 (grey) at the PD-L1 binding interface relative to the parent CDP (SEQ ID NO:353, black, minimal impinging rotamers). The tag residues of SEQ ID NO. 1 (including M13, V39, F40 and L43) correspond to substitutions relative to SEQ ID NO. 353 which improve binding to PD-L1.

FIG. 20F shows an enlarged view of the binding interface between SEQ ID NO:1 (sketch) and PD-L1 (surface). The PD-L1 surface is color coded for human (Hs) versus murine (Mm) homology, where white corresponds to the same residues, dark shades correspond to similar residues, and light shades correspond to different residues. These differences in the binding interface between human and murine PD-L1 are consistent with the lack of murine PD-L1 cross-reactivity seen in the case of SEQ ID NO: 1. In addition, the data presented in fig. 6B, 6C, and 8 are consistent with a crystal structure.

FIG. 20G shows the eutectic structure of SEQ ID NO. 1 and PD-L1, wherein SEQ ID NO. 1 is shown as a line graph, wherein the side chain of interest is shown with bold bars (upper panel). The binding of PD-1 to PD-L1 is shown in the following graph for comparison. The overlap of the binding interfaces of SEQ ID NO 1 and PD-1 on PD-L1 is consistent with the observed competition data shown in FIGS. 6B, 6C and 8.

FIG. 21 shows binary recolouring of the enrichment fraction map shown in FIG. 8. Amino acid substitutions determined to have a neutral or beneficial effect relative to SEQ ID NO. 4 are grey. The original residues of the SEQ ID NO. 4 sequence are colored black. Grey or black shaded amino acids are believed to contribute to binding to PD-L1.

FIG. 22 illustrates the mechanism of action of oligonucleotides. Oligonucleotides that target specific sequences for modulation complex with CDPs that bind PD-L1, enter cells via PD-L1 binding and natural endocytosis of PD-L1, allowing the oligonucleotides to compartmentalize into endosomes. Oligonucleotides are released from the endocytic compartment into the cytoplasm where they can move freely between the nucleus and the cytoplasm. Upon entry into the nucleus, the oligonucleotides may (1) modulate alternative splicing of the targeting sequence, (2) indicate the location of the polyadenylation (polyA) site of the targeting sequence, and (3) recruit RNaseH1 to induce cleavage of the targeting sequence. Oligonucleotides in the cytoplasm can be designed to (4) bind directly to microrna (miRNA) or messenger (mRNA) sequences. Alternatively, sirnas that target specific sequences for modulation may be used to (5) bind and modulate targeting sequences in the cytosol, in conjunction with an RNA-induced silencing complex (RISC), a multiprotein complex incorporating single-stranded small interfering RNAs (sirnas) or micrornas (mirnas), using the sirnas or mirnas as templates to recognize complementary mrnas of the targeting sequences. When it finds the complementary strand, the RISC complex cleaves the targeting sequence. Alternatively, aptamers that target specific sequences for modulation can be used for (6) binding and modulation of target molecules. The aptamer binds directly to and inhibits its intracellular or extracellular target.

FIG. 23 illustrates examples of structures of various peptide oligonucleotide complexes (e.g., CDP-oligonucleotide complexes, wherein the peptide portion comprises CDP) containing alternative and non-conventional bases, as shown by single-stranded, double-stranded, and hairpin structures. Examples of oligonucleotides include aptamers, empty-mers, anti-miR, siRNA, splice-blocking ASO, and U1 adaptors. The CDP portion of the CDP-oligonucleotide complex can be used to direct the oligonucleotide sequence to a particular tissue, target, or cell, or to cause endocytosis of the oligonucleotide sequence by the cell. The legend is as follows: gray black = 2' -H (DNA); white circle black = 2' -OH (RNA); horizontal stripes and black circles = 2' -O-ME; vertical striped circle = 2' -O-MOE; black grey = 2' -F; spot circle gray = LNA; shaded circle grey = morpholino (unique phosphorodiamidate linkages not shown); gray angle = PO bond; black angle = PS bond.

FIGS. 24A-24E illustrate the incorporation of the indicated groups on RNA or DNA.

FIG. 24A illustrates the structure of oligonucleotides containing 5 '-thiol (thiol; C6) modifications (left) and 3' -thiol (C3) modifications (right).

FIG. 24B illustrates MMT-hexyl amino linker phosphoramidate.

FIG. 24C illustrates TFA-amyl amino linker phosphoramidate.

FIG. 24D illustrates RNA residues incorporating amine or thiol residues.

FIG. 24E illustrates oligonucleotides with aminohexyl modifications at the 5 'end (left) and 3' end (right).

FIG. 25 illustrates the formation of cleavable disulfide bonds between a peptide (e.g., PD-L1 binding peptide of SEQ ID NO: 1) and a cyclic dinucleotide.

FIG. 26 shows flow sort data illustrating enrichment of peptides with binding to PD-L1 that are pH dependent. This data suggests that pH-dependent binding peptides can be generated by flow sorting. Peptides were screened for those that showed stronger PD-L1 binding at neutral pH (7.4) and weaker binding at acidic pH (5.5) in histidine-doped libraries based on PD-L1 binding peptide (SEQ ID NO: 1) prepared as described in FIG. 19. The input library was initially screened for high PD-L1 binding at pH 7.4. The second and third rounds of screening (respectively "sort 1" and "sort 2") were performed at pH 5.5 to mimic endosomal pH, enriching for poor PD-L1 binding at that pH. The last round of screening ("sort 3") was performed at pH 7.4. Differential binding at pH 7.4 and pH 5.5 was observed after screening ("sort 4"). The area covered by the 5-sided polygon in each figure represents the population selected during sorting. Darker topography density maps indicated staining with PD-L1 at pH 7.4, and lighter topography density maps indicated staining with PD-L1 at pH 5.5.

FIG. 27 shows the binding data for the pH-dependent PD-L1 binding peptide variants identified in FIG. 26 at pH7.4 (left bar) and pH 5.5 (right bar). Variants of SEQ ID NO. 1 with E2H, M H and K16H substitutions alone or in combination were screened for pH dependent binding to PD-L1. Peptide variants containing substitutions at E2H (SEQ ID NO: 234), M13H (SEQ ID NO: 235), K16H (SEQ ID NO: 236), E2H and M13H (SEQ ID NO: 237), E2H and K16H (SEQ ID NO: 233), M13H and K16H (SEQ ID NO: 238) or E2H, M H and K16H (SEQ ID NO: 239) exhibit varying degrees of pH dependent binding to PD-L1. "UTF" indicates untransfected cells (negative control). The parent peptide (SEQ ID NO: 187) showed a degree of pH dependent binding to PD-L1. Some variants of SEQ ID NO. 187 exhibit greater pH dependence in PD-L1 binding than the parent, while some variants of SEQ ID NO. 187 exhibit less pH dependence in PD-L1 binding than the parent. The peptide of SEQ ID NO 234 shows a large difference in binding at pH7.4 versus at pH 5.5, indicating that binding is higher at pH7.4 than at pH 5.5. The peptide of SEQ ID NO. 233 (black arrow) shows that the difference between binding at pH7.4 and binding at pH 5.5 is particularly large, indicating that binding at pH7.4 is higher than binding at pH 5.5. This data demonstrates the production of peptides that bind to PD-L1 at a higher level at pH7.4 and to PD-L1 at a lower level at pH 5.5.

Detailed Description

Programmed death ligand 1 (PD-L1) has been demonstrated to be a valuable therapeutic target for small molecule therapeutics and anti-PD-L1 antibodies that act as immune checkpoint inhibitors and are useful as anti-cancer therapies. However, small molecule therapeutics, typically less than 1000Da in size, and antibody therapeutics, typically greater than 140kDa in size, have significant limitations in their efficacy and utility. Small molecule therapeutics often lack selectivity for their intended targets, resulting in severe off-target effects and limited therapeutic window (i.e., a range of drug doses that may be therapeutically effective without toxicity). Furthermore, small target binding interfaces may be susceptible to mutation selection, where individual amino acid changes of the target at the binding interface may greatly alter the binding of the small molecule to the target. On the other hand, large sized antibodies may prevent them from approaching central nervous system targets or penetrating through solid tumors. Furthermore, antibodies may require extensive engineering, e.g., humanization, to render them suitable for pharmaceutical use. Furthermore, PD-L1 targeting provides an opportunity for cancer targeting by bispecific immunocyte cement. Bispecific immune cell cement (biece) may have a moiety that binds to cancer cells (e.g., via PD-L1) and immune cells, thereby bringing the immune cells directly adjacent to the cancer cells to direct cell killing. However, the particular geometry of the immune synapse (e.g., the distance between a cancer cell and an immune cell) may be important for effective cell killing. Antibodies and antibody fragments, such as scFv or nanobodies with molecular weights around 15-27kDa, may not produce sufficiently close immune synapses to drive optimal cell killing.

Described herein are small, typically less than about 10kDa in size and typically less than 6kDa in size, drug-like proteins engineered to bind to therapeutic targets, including PD-L1 and exert therapeutic effects. These small proteins or "mini-proteins" themselves may provide therapeutic effects, for example, by binding and inhibiting a target protein or by delivering additional active agents (e.g., detectable agents, small molecule or protein drugs, immune cell-engaging moieties, or additional active agents) to a target region. For example, because some cancer cells overexpress PD-L1, the PD-L1 binding peptides described herein can be used to target therapeutic moieties to cancer cells and tissues, promote cell killing of cancer cells, or both. The present disclosure provides PD-L1 binding peptides, also referred to herein as mini-proteins, that bind to PD-L1. These proteins may be cysteine compact peptides (CDPs) or cystine compact mini proteins, which are stabilized by disulfide bridges formed between cysteine amino acid residues. Cystine dense peptides may have additional benefits of thermostability, protease resistance, low immunogenicity, smaller size, and tissue penetration. Also described herein are methods of using the PD-L1 binding peptides (also referred to as CDPs that bind PD-L1) as therapeutic or diagnostic agents.

The present disclosure utilizes peptide design methods based on 3D protein structure to select peptides or proteins that are capable of binding to PD-L1, and protein-based 3D structures designed to bind at specific interfaces of proteins (e.g., PD-L1 proteins). For example, the peptide may be designed to bind at the PD-1 binding interface of PD-L1.

As used herein, abbreviations for the natural L-enantiomer amino acids are conventional and are as follows: alanine (a, ala); arginine (R, arg); asparagine (N, asn); aspartic acid (D, asp); cysteine (C, cys); glutamic acid (E, glu); glutamine (Q, gin); glycine (G, gly); histidine (H, his); isoleucine (I, ile); leucine (L, leu); lysine (K, lys); methionine (M, met); phenylalanine (F, phe); proline (P, pro); serine (S, ser); threonine (T, thr); tryptophan (W, trp); tyrosine (Y, tyr); valine (V, val). In general, xaa may indicate any amino acid. In some embodiments, X may be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).

Some embodiments of the present disclosure contemplate D-amino acid residues of any standard or non-standard amino acid or analog thereof. When an amino acid sequence is expressed as a series of three-letter or one-letter amino acid abbreviations, the left hand direction is the amino-terminal direction and the right hand direction is the carboxy-terminal direction, according to standard usage and convention.

The terms "peptide", "polypeptide", "mini-protein", "collectin", "cystine compact peptide", "knotted peptide" or "CDP" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" may be a chain of amino acids in which the alpha carbons are linked by peptide bonds. Thus, a terminal amino acid at one end of the chain (e.g., the amino-terminus or N-terminus) may have a free amino group, while a terminal amino acid at the other end of the chain (e.g., the carboxyl-terminus or C-terminus) may have a free carboxyl group. As used herein, the term "amino-terminal" (e.g., abbreviated as N-terminal) may refer to a free α -amino group on an amino acid at the amino-terminal end of a peptide or an α -amino group of an amino acid at any other position within a peptide (e.g., imino when involved in a peptide bond). Similarly, the term "carboxy terminus" may refer to the free carboxy group on the carboxy terminus of a peptide or the carboxy group of an amino acid at any other position within the peptide. Peptides also include essentially any polyamino acid, including but not limited to peptidomimetics, such as amino acids joined by ether or thioether bonds, rather than amide bonds.

As used herein, the term "peptide construct" or "peptide complex" may refer to a molecule comprising one or more peptides of the present disclosure that may be conjugated, linked or fused to one or more peptides or carrier molecules. In some cases, the cargo molecule is an active agent. The term "active agent" may refer to any molecule, e.g., any molecule capable of eliciting a biological effect and/or which may allow for the localization, detection or visualization of the physical effect (e.g., emission of radiation) of the corresponding peptide construct. In various embodiments, the term "active agent" refers to a therapeutic and/or diagnostic agent. The peptide constructs of the present disclosure may comprise a PD-L1 binding peptide linked to one or more active agents through one or more linker moieties (e.g., cleavable linkers or stable linkers) as described herein. As used herein, the term "peptide complex" may also refer to one or more peptides of the present disclosure that are fused, linked, conjugated or otherwise linked to form a complex. In some cases, the one or more peptides may include a PD-L1 binding peptide, another peptide active agent, a peptide that binds immune cells (e.g., T cells), a half-life modulating peptide, a peptide that improves pharmacodynamic and/or pharmacokinetic properties, or a combination thereof.

The terms "nucleotide", "oligonucleotide", "polynucleotide", "polynucleic acid" or "nucleic acid" may refer to any molecule comprising a nucleic acid, such as a short single-or double-stranded DNA or RNA molecule. The nucleotides may comprise deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides or ribonucleotides, derivatives of deoxyribonucleotides or ribonucleotides, synthetic nucleotides, other nucleotides comprising various nucleobases or various sugars, or combinations thereof. As used herein, "nucleotide," "oligonucleotide," "polynucleotide," "polynucleic acid," or "nucleic acid" includes any single strand (ssDNA, ssRNA) or double strand (dsDNA, dsRNA) or combination of single and double strands (e.g., having a mismatched sequence, hairpin or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), oligonucleotides complementary to the sequence of a Natural Antisense Transcript (NATs), siRNA, snRNA, aptamer, empty mer, anti-miR, splice-blocking ASO, or U1 adaptor. In the peptide oligonucleotide complexes described herein, "nucleotide," "oligonucleotide," "polynucleotide," "polynucleic acid," or "nucleic acid" may be intended for modulating gene or protein expression, or for modulating intermolecular or intramolecular interactions, and each may be considered a target binding agent capable of binding a target molecule. The target may be a protein, nucleic acid or other non-nucleic acid molecule. When the target is a nucleic acid, the target molecule sequence may be derived from an RNA (e.g., mRNA or pre-mRNA) or Open Reading Frame (ORF) of a gene or protein coding sequence. The sequence of the target molecule may be found in or derived from the coding or non-coding region of the gene, or it may be found in or derived from mature mRNA (e.g., mRNA that has been spliced, polyadenylation, capped, and exported into the cytosol for translation) or immature pre-mRNA. The target binding agent may be a complement of such a target molecule sequence (e.g., an open reading frame, a non-coding sequence, or RNA).

As used herein, the term "complement" or "reverse complement" can refer to a nucleotide sequence that is fully or partially reverse-complementary to a target or reference sequence. The term "complementary" is used interchangeably with "reverse complement" or "antisense" to describe a nucleotide sequence that forms a base pairing interaction (e.g., a/T, A/U or C/G interaction) with a target or reference nucleotide sequence.

As used herein, the term "antisense oligonucleotide" includes small, non-coding and diffusible molecules containing about 15-35 nucleotides that form the reverse complement of a nucleic acid target sequence (e.g., a transcript or mRNA molecule). In some embodiments, the antisense molecule can be fully reverse-complementary to the target sequence. In some embodiments, the antisense molecule can comprise one or more base mismatches relative to the target sequence. As used herein, "antisense" can refer to nucleotides of different chemical nature, whether natural (RNA and/or DNA) or synthetic (e.g., 2' pentose modification, 2' f, 2' ome, LNA, PNA, and/or morpholino)), having natural or synthetic linkages (e.g., phosphodiester, phosphorothioate, phosphorodiamidate, or phosphorothioate), depending on the context, and can comprise oligonucleotides, ribonucleotides, ribonucleosides, deoxyribonucleotides, deoxyribonucleosides, either single-stranded or double-stranded in whole or in part, or any combination, and in modified form and any combination, to form any of the foregoing polynucleic acids. Similarly, phosphorothioate linkages may be used. Such polynucleic acids may further contain modified bases (e.g., synthetic purines or pyrimidines that differ chemically from adenine, cytosine, guanine, thymine or uracil) or contain other atypical elements or chemistries. In various embodiments, the antisense RNA contains 19-23 nucleotides (nt) or 15-35nt, complementary to the target RNA. The antisense RNA is about 5 to 30nt in length, 10 to 25nt in length, 15 to 25nt in length, 19 to 23nt in length, or at least 10nt in length, at least 15nt in length, at least 20nt in length, at least 25nt in length, or at least 30nt in length, at least 50nt in length, at least 100 nucleotides in length. Non-limiting examples of antisense oligonucleotides (ASOs) include aptamers, vacancy mers, anti-miR, siRNA, miRNA, snRNA, splice-blocking ASOs, and U1 adaptors.

As used herein, the terms "interfering RNA" or "inhibitory RNA" are used interchangeably and include RNA molecules that are involved in sequence-specific inhibition of gene expression by forming double stranded RNA. As used herein, an "interfering RNA" or "inhibitory RNA" may comprise ribonucleotides, ribonucleosides, deoxyribonucleotides, deoxyribonucleosides, be single-stranded or double-stranded in whole or in part, or any combination, and form any of the foregoing in modified form and in any combination of polynucleic acids. Such polynucleic acids may further comprise modified bases or comprise other atypical elements or chemistries. Common forms of "interfering RNAs" or "inhibitory RNAs" include small inhibitory RNAs (siRNA or RNAi), as well as dsRNA, ssRNA, hairpin RNAs, and other known structures. In various embodiments, the inhibitory RNA is about 5 to 30nt in length, 10 to 25nt in length, 15 to 25nt in length, 19 to 23nt in length, or at least 10nt in length, at least 15nt in length, at least 20nt in length, at least 25nt in length, or at least 30nt in length, at least 50nt in length, at least 100 nucleotides in length.

As used herein, the term "nuclear RNA" includes any RNA molecule present in the nucleus of a cell. As used herein, "nuclear RNA" can include small nuclear RNA (snRNA), spliceosome RNA, and other known structures.

As used herein, the term "U1 adapter" includes bifunctional oligonucleotides having a target domain complementary to a site near the polyadenylation site of the target gene and a U1 domain of a U1 nuclear microrna component that binds to a U1 nuclear microribonucleoprotein (U1 snRNP). The U1 adaptors may be used as synthetic oligonucleotides to recruit endogenous U1snRNP to a target sequence or site. As used herein, a U1 adapter may comprise any nucleotide sequence that is complementary to the ssRNA component of a U1 ribonucleoprotein (U1 snRNP). In various embodiments, the U1 adapter is about 5 to 30nt in length, 10 to 25nt in length, 15 to 25nt in length, 19 to 23nt in length, or at least 10nt in length, at least 15nt in length, at least 20nt in length, at least 25nt in length, or at least 30nt in length, at least 50nt in length, at least 100 nucleotides in length, the nucleotide length complementary to any sequence along the U1 domain or U1 ribonucleoprotein (U1 snRNP) splicing factor.

As used herein, the terms "comprising" and "having" are used interchangeably. For example, the terms "peptide comprising the amino acid sequence of SEQ ID NO. 1" and "peptide having the amino acid sequence of SEQ ID NO. 1" are used interchangeably.

As used herein, and unless otherwise indicated, the term "PD-L1" or "programmed death ligand 1" is a class of proteins used herein, which may refer to PD-L1 from any species (e.g., human or murine PD-L1 or any human or non-human animal PD-L1). In some cases, and as used herein, the term "PD-L1" or "programmed death ligand 1" refers to human PD-L1, which may include PD-L1 or any combination or fragment thereof (e.g., extracellular domain). In some cases, PD-L1 may also be referred to as "CD274", "B7-H", "B7H1", "PDCD1L1", "PDCD1LG1" or "PDL1".

The term "engineered" when applied to a polynucleotide means that the polynucleotide has been removed from its natural genetic environment and is therefore free of other foreign or undesired coding sequences and in a form suitable for use within a genetically engineered protein production system. Such engineered molecules are those isolated from their natural environment and include cDNA and genomic clones (i.e., prokaryotic or eukaryotic cells having vectors containing DNA fragments from different organisms). The engineered DNA molecules of the invention are free of other genes with which they are typically associated, but may include naturally occurring or non-naturally occurring 5 'and 3' untranslated regions such as enhancers, promoters, and terminators.

An "engineered" polypeptide or protein is one that is found under conditions other than its natural environment, e.g., away from blood and animal tissue. In a preferred form, the engineered polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptide in a highly purified form, e.g., greater than 90% pure, greater than 95% pure, more preferably greater than 98% pure or greater than 99% pure. The term "engineered" when used in this context does not exclude the presence of the same polypeptide in alternative entity forms, such as dimers, heterodimers and multimers, heteromultimers, or alternatively glycosylated, carboxylated, modified or derivatized forms.

An "engineered" peptide or protein is a polypeptide that differs from the structure, sequence, or composition of a naturally occurring polypeptide. Engineered peptides include non-naturally occurring, artificial, isolated, synthetic, designed, modified, or recombinantly expressed peptides. Provided herein are engineered PD-L1 binding peptides, variants or fragments thereof. These engineered PD-L1 binding peptides may be further linked to an active agent or half-life extending moiety, or may be further linked to an active agent or detectable agent, or any combination of the foregoing.

Polypeptides of the disclosure include polypeptides that have been modified in any manner, for example, to achieve the following: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinity, (5) altered binding affinity at certain pH values, and (6) imparts or improves other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) are made in a naturally occurring sequence (e.g., in a portion of a polypeptide that is external to one or more domains that form intermolecular contacts). "conservative amino acid substitution" may refer to the substitution of an amino acid in a polypeptide with a functionally similar amino acid. Each of the following six groups contains amino acids that can be conservatively substituted with each other: i) Alanine (a), serine (S) and threonine (T); ii) aspartic acid (D) and glutamic acid (E); iii) Asparagine (N) and glutamine (Q); iv) arginine (R) and lysine (K); v) isoleucine (I), leucine (L), methionine (M) and valine (V); vi) phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments, conservative amino acid substitutions may comprise unnatural amino acids. For example, a non-natural derivative in which the same amino acid is substituted with an amino acid may be a conservative substitution.

The terms "polypeptide fragment" and "truncated polypeptide" as used herein may refer to a polypeptide having an amino-terminal and/or carboxy-terminal deletion as compared to the corresponding full-length peptide or protein. In various embodiments, the fragment is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids in length. In various embodiments, the length of the fragment may also be, for example, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids. A fragment may also comprise one or more additional amino acids at either or both of its ends, such as an amino acid sequence (e.g., fc or leucine zipper domain) or an artificial amino acid sequence (e.g., artificial linker sequence) from a different naturally occurring protein.

As used herein, the term "peptide" or "polypeptide" when combined with "variation", "mutation" or "enrichment mutation" or "alteration enrichment mutation" may refer to a peptide or polypeptide that may comprise an amino acid sequence into which one or more amino acid residues are inserted, deleted from, and/or substituted into, relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted or substituted is at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Variants of the disclosure include peptide conjugates or fusion molecules (e.g., peptide constructs or peptide complexes).

A "derivative" of a peptide or polypeptide may be a peptide or polypeptide that may be chemically modified, e.g., conjugated, phosphorylated and glycosylated with another chemical moiety such as polyethylene glycol, albumin (e.g., human serum albumin).

The term "percent sequence identity" is used interchangeably herein with the term "percent identity" and may refer to the level of amino acid sequence identity between two or more peptide sequences, or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity determined by a determination algorithm, and means that a given sequence is at least 80% identical to another sequence of another length. In various embodiments, the% identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more percent up to 100% sequence identity to a given sequence. In various embodiments, the% identity is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

The terms "percent sequence homology" or "percent sequence identity" are used interchangeably herein with the terms "percent homology", "percent sequence identity" or "percent identity" and may refer to the level of amino acid sequence homology between two or more peptide sequences, or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, 80% homology, as used herein, means the same as 80% sequence homology determined by the determination algorithm, and thus, a homolog of a given sequence has greater than 80% sequence homology over a certain length of the given sequence. In various embodiments, the% homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or more percent up to 100% sequence homology to a given sequence. In various embodiments, the% homology is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

A protein or polypeptide may be "substantially pure," "substantially homogenous," or "substantially purified" when at least about 60% to 75% of the sample exhibits a single polypeptide species. The polypeptide or protein may be a monomer or a multimer. The substantially pure polypeptide or protein may typically comprise about 50%, 60%, 70%, 80% or 90% W/W of the protein sample, more typically about 95%, and for example will be more than 98% or 99% pure. Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visual inspection of the single polypeptide band after staining the gel with a stain well known in the art. For some purposes, higher resolution is provided by using high pressure liquid chromatography (e.g., HPLC) or other high resolution analysis techniques (e.g., LC-mass spectrometry).

As used herein, the term "pharmaceutical composition" may generally refer to a composition suitable for pharmaceutical use in a subject, such as an animal (e.g., human or mouse). The pharmaceutical composition may comprise a pharmacologically effective amount of the active agent and a pharmaceutically acceptable carrier. The term "pharmacologically effective amount" may refer to an amount of an agent effective to produce a desired biological or pharmacological result.

As used herein, the term "pharmaceutically acceptable carrier" may refer to any standard pharmaceutical carrier, vehicle, buffer and excipient, such as phosphate buffered saline solution or buffered saline solution, 5% dextrose in water solution and emulsion, such as oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington' sPharmaceutical Sciences, 21 st edition 2005,Mack Publishing Co,Easton. A "pharmaceutically acceptable salt" may be a salt of a compound that can be formulated for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) as well as salts of ammonia or organic amines.

As used herein, the terms "treatment", "treatment" and "treatment" may refer to a method of alleviating or eliminating a biological disorder and/or at least one concomitant symptom thereof. As used herein, "alleviating" a disease, disorder or condition means, for example, reducing the severity and/or frequency of symptoms of the disease, disorder or condition. Furthermore, references herein to "treatment" may include references to curative, palliative and prophylactic or diagnostic treatments.

In general, the cells of the present disclosure may be eukaryotic cells or prokaryotic cells. The cell may be an epithelial cell, a cancer cell or a cell of the immune system. The cells may be microbial, bacterial, yeast, fungal or algal cells. The cells may be animal cells or plant cells. Animal cells may include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. Mammalian cells may be obtained from primates, apes, equines, bovine, porcine, canine, feline, or rodents. The mammal may be a primate, ape, canine, feline, rabbit, ferret, or the like. The rodent may be a mouse, rat, hamster, gerbil, hamster, chinchilla or guinea pig. Bird cells may be from canary, long tail parrots or parrots. The reptile cells may be from a turtle, lizard or snake. The fish cells may be derived from hairtail. For example, the fish cells may be from zebra fish (e.g., zebra fish (Danino rerio)). The helminth cells may be from nematodes (e.g. caenorhabditis elegans (c.elegans)). The amphibian cells may be from a frog. The arthropod cells may be from a spider or a living crab.

Mammalian cells may also include cells obtained from primates (e.g., humans or non-human primates). Mammalian cells may include blood cells, stem cells, epithelial cells, connective tissue cells, hormone-secreting cells, nerve cells, skeletal muscle cells, or cells of the immune system.

As used herein, the term "vector" generally refers to a DNA molecule that is capable of replication in a host cell and/or to which another DNA segment may be operatively linked so as to replicate the linked segment. Plasmids are one exemplary vector.

As used herein, the term "subject" generally refers to a human or another animal. The subject may be of any age, for example the subject may be an infant, toddler, child, pre-pubertal teenager, adolescent, adult or senior individual.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will also be understood that the endpoints of each of the ranges are relative to the other endpoint, and independent of the other endpoint. The term "about" as used herein refers to a range of plus or minus 15% from the stated value in the context of a particular use. For example, about 10 may include a range from 8.5 to 11.5.

Peptides

The PD-L1 binding peptides described herein may comprise a Cystine Dense Peptide (CDP) that binds to PD-L1. The PD-L1 binding peptide can be engineered to bind to PD-L1 (e.g., human PD-L1). In some cases, the PD-L1 binding peptide may be engineered to bind at a particular interface of PD-L1 (e.g., at a PD-1 binding interface). The PD-L1 binding peptides and PD-L1-binding peptide complexes of the present disclosure may comprise one or more peptides. For example, the PD-L1 binding peptide complex may comprise a PD-L1 binding peptide and another peptide therapeutic agent. The PD-L1 binding peptides of the present disclosure can be engineered to remain bound to PD-L1 upon complexing with another active agent. In some cases, the PD-L1 binding peptide may be engineered to contain one or more amino acid residues that are capable of modification (e.g., have a linker).

In some cases, a peptide as disclosed herein may contain only one lysine residue, or no lysine residue. In some cases, one or more or all of the lysine residues in the peptide are replaced with arginine residues. In some cases, one or more or all of the methionine residues in the peptide are replaced with leucine or isoleucine. One or more or all of the tryptophan residues in the peptide may be replaced with phenylalanine or tyrosine. In some cases, one or more or all of the asparagine residues in the peptide are replaced with glutamine. In some embodiments, one or more or all of the aspartic acid residues may be replaced with a glutamic acid residue. In some cases, one or more or all of the lysine residues in the peptide are replaced with alanine or arginine. In some embodiments, the N-terminus of the peptide is blocked or protected, for example, by acetyl or t-butyloxycarbonyl. Alternatively or in combination, the C-terminus of the peptide may be blocked or protected, for example, by an amide group or by formation of an ester (e.g., butyl or benzyl ester). In some embodiments, the peptide is modified by methylation on a free amine. For example, complete methylation is achieved by using reductive methylation with formaldehyde and sodium cyanoborohydride.

In some embodiments, the N-terminal dipeptide may be absent, as shown in SEQ ID NO: 1-59, SEQ ID NO:435, or SEQ ID NO: 554-560, or the dipeptide GS may be added as the first two N-terminal amino acids, as shown in SEQ ID NO: 60-118, SEQ ID NO:436, or SEQ ID NO: 561-567, or may be substituted with any other one or two amino acids. In some embodiments, the dipeptide GS is used as a linker, or is used to couple with a linker to form a peptide conjugate or fusion molecule, such as a peptide construct or peptide complex. In some embodiments, the linker comprises G _x S _y (SEQ ID NO: 155) peptide, wherein x and y are independently any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16 or 20, and the G and S residues are arranged in any order. In some embodiments, the peptide linker comprises (GS) x (SEQ ID NO: 156), where x can be any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 157), GGGGG (SEQ ID NO: 158), GSGSGSGS (SEQ ID NO: 159), GSGG (SEQ ID NO: 160), GGGGS (SEQ ID NO: 161), GGGS (SEQ ID NO: 154), GGS (SEQ ID NO: 162), GGGSGGGSGGGS (SEQ ID NO: 163), or variants or fragments thereof, or any number of repetitions and combinations thereof. In addition, KKYKPY from DkTx VPVTTN (SEQ ID NO: 166) and from human IgG ₃ EPKSSDKTHT (SEQ ID NO: 167) may be used as peptide linkers or any number of repeats and combinations thereof. In some embodiments, the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 164) or a variant or fragment thereof, or any number of repeats and combinations thereof. In some embodiments, the peptide linker comprises any one of SEQ ID NO:154-SEQ ID NO:241 or SEQ ID NO: 433. Additional linkers that can be linked, fused or conjugated to the PD-L1 binding peptides of the present disclosure are provided in table 9. It will be appreciated that any of the foregoing linkers, or variants or fragments thereof, may be used with any number of repetitions or any combination thereof. It is also understood that other peptide linkers of the art, or variants or fragments thereof, may be used with any number of repetitions or any combination thereof. The length of the linker can be adjusted to maximize the binding of the PD-L1 binding peptide complex to both PD-L1 and another target (e.g., a target on an immune cell) at the same time, including taking into account spatial entry. In some embodiments, the linker between the PD-L1 binding peptide and the immune cell binding peptide is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 62, at least 60, at least 61, at least 65 residues up to at least 65 residues.

In some embodiments of the present disclosure, a peptide or peptide complex as described herein comprises an amino acid sequence set forth in any one of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567. The peptide as disclosed herein may be a peptide comprising SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO: a fragment of a contiguous fragment of any of 435, 436 or 554-567 of SEQ ID NOs, which is at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 61, at least 64, at least 65, at least 61, at least 65 peptide residues. In some embodiments, the peptide sequence is flanked by additional amino acids. One or more additional amino acids, for example, confer a specific in vivo charge, isoelectric point, chemical conjugation site, stability or physiological property to the peptide.

In some cases, CDPs described herein that are capable of targeting and binding to PD-L1 comprise a length of no more than 80 amino acids or a length of no more than 70, no more than 65, no more than 60, no more than 55, no more than 50, no more than 49, no more than 45, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, or no more than 10 amino acids. In some cases, a PD-L1 binding moiety (e.g., scFv) described herein that is capable of targeting and binding to PD-L1 comprises a length of about 100 to about 400, about 200 to about 300, or about 240 to about 250 amino acids in length.

In other embodiments, the peptide may be conjugated, linked or fused to a carrier or molecule that targets a targeting or homing function for a cell of interest or target cell (e.g., immune cell). In other embodiments, the peptide may be conjugated, linked or fused to a molecule, such as an Fc region or polyethylene glycol, that extends the half-life of the peptide or improves the pharmacodynamic and/or pharmacokinetic properties of the peptide or any combination thereof.

In some cases, the peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged residues, such as Arg or Lys, or any combination thereof. In some cases, one or more lysine residues in the peptide are replaced with an arginine residue. In some embodiments, the peptide comprises one or more Arg patches. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more Arg or Lys residues on the peptide are exposed to the solvent. In some cases, the peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 histidine residues.

The peptides of the present disclosure may also comprise neutral amino acid residues. In some embodiments, the peptide has 35 or fewer neutral amino acid residues. In other embodiments, the peptide has 81 or less neutral amino acid residues, 70 or less neutral amino acid residues, 60 or less neutral amino acid residues, 50 or less neutral amino acid residues, 40 or less neutral amino acid residues, 36 or less neutral amino acid residues, 33 or less neutral amino acid residues, 30 or less neutral amino acid residues, 25 or less neutral amino acid residues, or 10 or less neutral amino acid residues.

The peptides of the present disclosure may also comprise negative amino acid residues. In some embodiments, the peptide has 10 or less, 9 or less, 8 or less, 7 or less, 6 or less negative amino acid residues, 5 or less negative amino acid residues, 4 or less negative amino acid residues, 3 or less negative amino acid residues, 2 or less negative amino acid residues, or 1 or less negative amino acid residues. Although the negative amino acid residue may be selected from any negatively charged amino acid residue, in some embodiments the negative amino acid residue is E, or D, or a combination of both E and D.

In some embodiments of the present disclosure, the three-dimensional or tertiary structure of the peptide comprises predominantly β -sheet and/or α -helical structures. In some embodiments, the engineered or engineered PD-L1 binding peptide or peptide complex of the present disclosure is a small compact peptide or polypeptide stabilized by an intrachain disulfide bond (e.g., mediated by cysteine) to form a cystine bond. In some embodiments, the engineered PD-L1 binding peptide has a structure comprising a helix-turn-helix motif, with at least one disulfide bond between each alpha helix, thereby stabilizing the peptide. In some embodiments, the engineered PD-L1 binding peptide or peptide complex comprises a structure having an alpha helix, one or more beta sheets, one or more alpha helices, or one or more intrachain disulfide bonds. In some embodiments, the engineered PD-L1 binding peptide or peptide complex does not contain a hydrophobic core.

Cystine compact peptide

In some embodiments, a PD-L1 binding peptide or peptide complex of the present disclosure comprises one or more cysteine (Cys) amino acid residues or one or more disulfide bonds. In some embodiments, the PD-L1 binding peptide or peptide complex is derived from a Cystine Dense Peptide (CDP), knotted peptide, or a collectin. In some embodiments, the CDP contains at least 3 intramolecular cystine bonds. As used herein, the term "peptide" is considered interchangeable with the terms "knotted peptide", "cystine dense peptide", "CDP", "knotting element" and "hitching element" (see, e.g., correnti et al Screening, large-scale production, and structure-based classification for cystine-dense peptides. Nat structure Mol biol.2018, month 3; 25 (3): 270-278).

The PD-L1 binding peptides of the present disclosure, or derivatives, fragments or variants thereof, may have affinity and selectivity for PD-L1, or derivatives or analogs thereof. In some cases, the PD-L1 binding peptides of the present disclosure can be engineered using site-saturation mutagenesis (SSM) to exhibit improved PD-L1-binding properties or alter the properties of the binding interface with PD-L1. In some cases, the peptides of the present disclosure are Cysteine Dense Peptides (CDPs) associated with knotted peptides or noosin-derived peptides or knotted-in-derived peptides. The PD-L1 binding peptide may be a cystine compact peptide (CDP). The PD-L1-bound CDP may have a "floating bridge" structure characterized by a pair of alpha helices separated by an unstructured loop and stabilized with disulfide bonds, similar to the bottom of a floating bridge vessel, with example disulfide bond connectivity of 1-6, 2-5, 3-4 for a tri-disulfide bridge scaffold. CDP binding to PD-L1 may also have a structure resembling a noosin. The collectin may be a subclass of CDP in which six cysteine residues form disulfide bonds with the fourth residue according to the connectivity [1-4], 2-5, 3-6, which indicates that the first cysteine residue forms disulfide bonds with the fourth residue, the second residue forms disulfide bonds with the fifth residue, and the third cysteine residue forms disulfide bonds with the sixth residue. Brackets in this nomenclature indicate that cysteine residues form knotted disulfide bonds. (see, e.g., correnti et al Screening, large-scale production, and structure-based classification for cystine-dense peptides, nat structure Mol biol.2018, month 3; 25 (3): 270-278). Knotting elements can be a subclass of CDP in which six cysteine residues form disulfide bonds according to the connectivity 1-4, 2-5, [3-6 ]. Knottins are a class of peptides typically ranging in length from about 20 to about 80 amino acids, which are often folded into a tight structure. Knottins are generally assembled into complex tertiary structures characterized by having many intramolecular disulfide crosslinks and may contain β chains and other secondary structures. The presence of disulfide bonds imparts significant environmental stability to CDPs including knottins, floating bridges, and nootoxins, allowing them to withstand extreme temperatures and pH, and to resist proteolytic enzymes and reducing molecules of the blood stream. In some cases, the peptides described herein can be derived from knotted peptides. The amino acid sequence of a peptide as disclosed herein may comprise a plurality of cysteine residues. In some cases, at least a cysteine residue of a plurality of cysteine residues present within the amino acid sequence of the peptide is involved in disulfide bond formation. In some cases, all of the plurality of cysteine residues present within the amino acid sequence of the peptide participate in disulfide bond formation. As used herein, the term "knotted peptide" may be used interchangeably with the terms "cystine dense peptide", "CDP" or "peptide".

Provided herein are methods of identifying, maturing, characterizing, and utilizing CDPs that bind to PD-L1 and allow selection, optimization, and characterization of PD-L1-binding CDPs that can be used alone or in peptide complexes, including as therapeutically relevant concentrations of bioactive molecules in a subject (human or non-human animal). The present disclosure demonstrates the utility of CDP as a diverse scaffold family that can be screened for applicability with respect to modern drug discovery strategies. CDPs comprise alternatives to existing biological agents, mainly antibodies, which can bypass some of the disadvantages of immunoglobulin scaffolds, including poor tissue penetration, immunogenicity, larger size, and long serum half-life that can become problematic if toxicity occurs. Peptides of the present disclosure in the range of 20-80 amino acids represent mid-sized medically relevant therapeutic agents, have many of the favorable binding specificity and affinity characteristics of antibodies, but have improved stability, reduced immunogenicity, and simpler manufacturing methods. The intramolecular disulfide structure of CDPs provides a particularly high measure of stability, thereby reducing fragmentation and immunogenicity, while their smaller size can improve tissue penetration or cell penetration, and contribute to a tunable serum half-life. Disclosed herein are peptides representing candidate peptides that can bind and inhibit PD-L1 or act as carriers for delivering an active agent to PD-L1 positive cells.

In some embodiments, the PD-L1 binding peptide may be an engineered peptide. The engineered peptide may be a non-naturally occurring, artificial, isolated, synthetic, designed, or recombinantly expressed peptide. In some embodiments, the PD-L1 binding peptides of the present disclosure comprise one or more properties of CDP, knotted peptide, or noosin, such as stability, resistance to proteolysis, resistance to reducing conditions, and/or the ability to cross the blood-brain barrier.

CDP is advantageous for intratumoral delivery, intracellular delivery or delivery to the CNS due to smaller size, greater tissue or cell penetration, lack of Fc function, and faster clearance from serum compared to other molecules such as antibodies, and due to resistance to proteases (both stability and reduced immunogenicity) compared to smaller peptides. In some embodiments, a PD-L1 binding peptide or engineered PD-L1 binding complex of the present disclosure (e.g., comprising one or more PD-L1 binding peptides and one or more additional active agents) may have properties that are superior to a PD-L1 binding antibody or target binding antibody (e.g., a bispecific antibody or chimeric antigen receptor). For example, the peptides and complexes described herein can provide superior, deeper, and/or faster tissue or cell penetration (e.g., brain parenchymal penetration, solid tumor penetration) of cells and targeted tissue, as well as faster clearance from non-targeted tissue and serum. The PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure may have a lower molecular weight than the PD-L1 binding antibodies. Lower molecular weights may confer advantageous properties to the PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure as compared to PD-L1 binding antibodies. For example, the PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure may penetrate cells or tissues more readily than anti-PD-L1 antibodies, or may have lower molar dose toxicity than anti-PD-L1 antibodies. In addition, the PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure can form an immune synapse between an immune cell and a cancer cell that has a better geometry to induce cancer cell killing. The PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure are advantageous due to lack of Fc function of the antibodies. The PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure are advantageous in that they allow for the use of higher concentrations of the formulation on a molar basis. The PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure have a higher affinity or faster association rate for PD-L1 than the antibody or antibody fragment. The PD-L1 binding peptides or PD-L1 binding peptide complexes of the present disclosure may also target cancer cells in the CNS or brain through a Blood Brain Barrier (BBB) penetrating moiety (e.g., BBB penetrating CDP) to better access CNS tumors that are inaccessible to antibodies.

CDPs (e.g., knotted peptides or collectins) are a class of peptides typically ranging in length from about 11 to about 81 amino acids, which are often folded into a tight structure. Knotted peptides are generally assembled into complex tertiary structures characterized by having many intramolecular disulfide crosslinks and may contain β chains, α helices and other secondary structures. The presence of disulfide bonds imparts significant environmental stability to knotted peptides, allowing them to withstand extreme temperatures and pH, and to resist proteolytic enzymes of the blood stream. The presence of disulfide bonds may provide resistance to reduction by a reducing agent. The rigidity of knotted peptides also allows them to bind to the target without being subject to the "entropy penalty" that the relaxed peptides produce after binding to the target. For example, binding is adversely affected by entropy loss that occurs when a peptide binds to a target to form a complex. Thus, the "entropy penalty" is the adverse effect on the binding, and the greater the entropy loss that occurs after this binding, the greater the "entropy penalty". Furthermore, because the flexible is lost when tethered in the complex, the flexible unbound molecules lose more entropy when forming the complex than the rigid structured molecules. However, rigidity in unbound molecules also typically increases specificity by limiting the number of complexes that the molecule can form. Peptides may bind to targets with an affinity comparable to or higher than antibodies or with nanomolar or picomolar affinities. A more extensive examination of the sequence structure and sequence identity or homology of knotted peptides reveals that they have emerged through convergent evolution in all species of animals and plants. In animals, they are often found in venom of venom such as spiders and scorpions, and have been involved in the regulation of ion channels. Plant knotting proteins can inhibit proteolytic enzymes or have antimicrobial activity in animals, suggesting that knotting peptides may play a role in the molecular defense system found in plants.

The peptides of the disclosure (e.g., PD-L1 binding peptides) can comprise cysteine amino acid residues. In some embodiments, the peptide has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cysteine amino acid residues. In some embodiments, the peptide has at least 6 cysteine amino acid residues. In some embodiments, the peptide has at least 8 cysteine amino acid residues. In other embodiments, the peptide has at least 10 cysteine amino acid residues, at least 12 cysteine amino acid residues, at least 14 cysteine amino acid residues, or at least 16 cysteine amino acid residues. In some embodiments, the peptides of the disclosure have an even number of cysteine residues. In some embodiments, all cysteines in the peptides of the present disclosure are engaged within cystine disulfide bonds.

The knotted-peptide may comprise a disulfide bridge. The knotted peptide may be a peptide in which 5% or more of the residues are cysteines forming intramolecular disulfide bonds. The disulfide-linked peptide may be a drug stent. In some embodiments, the disulfide bridge forms a junction. Disulfide bridges may be formed between cysteine residues, for example between cysteines 1 and 4, 2 and 5, or 3 and 6. In some embodiments, one disulfide bridge passes through the loop formed by the other two disulfide bridges, e.g., to form a junction. In other embodiments, a disulfide bridge may be formed between any two cysteine residues.

Some peptides of the disclosure may comprise at least one amino acid residue in the L configuration. The peptide may comprise at least one amino acid residue in the D configuration. In some embodiments, the peptide is 15-75 amino acid residues in length. In other embodiments, the peptide is 11-55 amino acid residues in length. In other embodiments, the peptide is 11-65 amino acid residues in length. In other embodiments, the peptide is at least 20 amino acid residues in length.

Some CDPs may be derived or isolated from a class of proteins known to be present in or associated with toxins or venom. In some cases, the peptide may be derived from a toxin or venom associated with scorpions or spiders. Peptides can be derived from venom and toxins of spiders and scorpions of various genera and species. For example, the processing steps may be performed, the peptide can be derived from Buthus martensii (Leiurus quinquestriatus hebraeus), buthus martensii (Huang Xie) in Raney, buthus martensii (Hottentotta judaicus), buthus martensii (Mesobuthus eupeus), buthus martensii (Huang Xie) in Raney, kyowa (Hadrurus gertschi), buthus martensii (Androctonus australis), leptodermus mex (Androctonus australis), latifolia (Androctonus australis), african Androctonus australis (Androctonus australis), ornithogalum kanehira (Androctonus australis), buthus martensii (Androctonus australis), ornithogali (Androctonus australis), hainan Karsch (Androctonus australis), ornithogali China the method comprises the steps of (1) a American funnel spider (Androctonus australis), a blue mountain funnel spider (Androctonus australis), a tiger-vein bird-catching spider (Androctonus australis), a white-head high-foot spider (Androctonus australis), a Chile red rose spider (Androctonus australis), a tiger-vein bird-catching spider (Androctonus australis), a blue mountain funnel spider, a snow pear funnel spider (atlax robustus), a American funnel spider (Androctonus australis), a groundsel spider tail (Androctonus australis), a funnel spider (Androctonus australis), a lacuna spider (Androctonus australis) and a jingzhaozhaoma spider (Androctonus australis), and venom or toxin of another scorpion or spider of a suitable genus or species. In some cases, the peptide may be derived from an east asian scorpion (Buthus martensii Karsh) (scorpion) toxin. In certain instances, the CDP may be derived from the 6 th subunit of cytochrome BC1 oxidoreductase or from an antimicrobial defensin.

In some embodiments, a peptide of the disclosure (e.g., a PD-L1 binding peptide) can comprise a sequence having a cysteine residue at one or more of the corresponding positions 4, 8, 18, 32, 42, and 46, for example, with reference to SEQ ID No. 1. For example, in certain embodiments, a peptide may comprise a sequence having a cysteine residue at position 4. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 8. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 18. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 32. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at corresponding position 42. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 46. In some embodiments, the peptide comprises a cysteine at a corresponding position n, n+4±2, n+14±2, n+28±2, n+38±2, or n+42±2, or any combination thereof, wherein n corresponds to the amino acid position of the first cysteine residue (e.g., position 4 of SEQ ID NO: 1). In some embodiments, the peptide comprises a cysteine at a corresponding position n, n+4, n+14, n+28, n+38, or n+42, or any combination thereof, wherein n corresponds to the amino acid position of the first cysteine residue (e.g., position 4 of SEQ ID NO: 1). For example, the peptides of the present disclosure may comprise a sequence having cysteines positioned such that the second cysteine residue is located at 4 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the third cysteine residue is located at 14 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the fourth cysteine residue is located at 28 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the fifth cysteine residue is located at 38 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the sixth cysteine residue is located at 42 amino acid residues from the first cysteine residue to the C-terminus of the peptide, or a combination thereof. In some embodiments, the peptides of the present disclosure may comprise sequences having cysteines that are spaced such that there are 3 amino acid residues between the first cysteine and the second cysteine, 9 amino acid residues between the second cysteine and the third cysteine, 13 amino acid residues between the third cysteine and the fourth cysteine, 9 amino acid residues between the fourth cysteine and the fifth cysteine, 3 amino acid residues between the fifth cysteine and the sixth cysteine, or a combination thereof.

In some embodiments, a peptide of the disclosure (e.g., a PD-L1 binding peptide) can comprise a sequence having a cysteine residue at one or more of the corresponding positions 4, 15, 21, 25, 35, 42, 44, 48, for example, with reference to SEQ ID No. 58. For example, in certain embodiments, a peptide may comprise a sequence having a cysteine residue at position 4. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 15. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 21. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 25. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 35. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at corresponding position 42. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at corresponding position 44. In certain embodiments, the peptide may comprise a sequence having a cysteine residue at position 48. In some embodiments, the peptide comprises a cysteine at a corresponding position n, n+11+2, n+17+2, n+21+2, n+31+2, n+38+2, n+40+2, or n+44+2, or any combination thereof, wherein n corresponds to the amino acid position of the first cysteine residue (e.g., position 4 of SEQ ID NO: 58). In some embodiments, the peptide comprises a cysteine at a corresponding position n, n+11, n+17, n+21, n+31, n+38, n+40, or n+44, or any combination thereof, wherein n corresponds to the amino acid position of the first cysteine residue (e.g., position 4 of SEQ ID NO: 58). For example, the peptides of the present disclosure can comprise a sequence having cysteines positioned such that the second cysteine residue is located at 11 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the third cysteine residue is located at 17 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the fourth cysteine residue is located at 21 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the fifth cysteine residue is located at 31 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the sixth cysteine residue is located at 38 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the seventh cysteine residue is located at 40 amino acid residues from the first cysteine residue to the C-terminus of the peptide, the eighth cysteine residue is located at the C-terminus of the peptide, or a combination thereof. In some embodiments, the peptides of the present disclosure can comprise sequences having cysteines that are spaced such that 10 amino acid residues exist between a first cysteine and a second cysteine, 5 amino acid residues exist between a second cysteine and a third cysteine, 3 amino acid residues exist between a third cysteine and a fourth cysteine, 9 amino acid residues exist between a fourth cysteine and a fifth cysteine, 6 amino acid residues exist between a fifth cysteine and a sixth cysteine, 1 amino acid residue exists between a sixth cysteine and a seventh cysteine, 3 amino acid residues exist between a seventh cysteine and an eighth cysteine, or a combination thereof.

In some embodiments, a peptide of the disclosure (e.g., a PD-L1 binding peptide) comprises at least one cysteine residue. In some embodiments, the peptides of the disclosure comprise at least two cysteine residues. In some embodiments, the peptides of the disclosure comprise at least three cysteine residues. In some embodiments, the peptides of the disclosure comprise at least four cysteine residues. In some embodiments, the peptides of the disclosure comprise at least five cysteine residues. In some embodiments, the peptides of the disclosure comprise at least six cysteine residues. In some embodiments, the peptides of the disclosure comprise at least eight cysteine residues. In some embodiments, the peptides of the disclosure comprise at least ten cysteine residues. In some embodiments, the peptides of the disclosure comprise six cysteine residues. In some embodiments, the peptides of the disclosure comprise seven cysteine residues. In some embodiments, the peptides of the disclosure comprise eight cysteine residues. In some embodiments, the peptides of the disclosure comprise nine cysteine residues.

In some embodiments, the first cysteine residue in the sequence may be disulfide bonded to the fourth cysteine residue in the sequence, the second cysteine residue in the sequence may be disulfide bonded to the fifth cysteine residue in the sequence, and the third cysteine residue in the sequence may be disulfide bonded to the sixth cysteine residue in the sequence. Optionally, the peptide may comprise one disulfide bridge through the loop formed by two other disulfide bridges, also known as a "two-and-through" structural system. In some embodiments, the peptides disclosed herein have one or more cysteines that may be mutated to serine. In some embodiments, a first cysteine residue in the sequence (e.g., at position 4 of SEQ ID NO: 1) may be disulfide-bonded to a sixth cysteine residue in the sequence (e.g., at position 46 of SEQ ID NO: 1), a second cysteine residue in the sequence (e.g., at position 8 of SEQ ID NO: 1) may be disulfide-bonded to a fifth cysteine residue in the sequence (e.g., at position 42 of SEQ ID NO: 1), and a third cysteine residue in the sequence (e.g., at position 18 of SEQ ID NO: 1) may be disulfide-bonded to a fourth cysteine residue in the sequence (e.g., at position 32 of SEQ ID NO: 1). In some embodiments, the disulfide bond structure may be present in the peptide of any of SEQ ID NO 1-4, SEQ ID NO 8-57, SEQ ID NO 59-63, SEQ ID NO 67-116, SEQ ID NO 118, SEQ ID NO 435, SEQ ID NO 436, or SEQ ID NO 554-567.

In some embodiments, the first cysteine residue in the sequence (e.g., at position 4 of SEQ ID NO: 58) may be disulfide-bonded to the eighth cysteine residue in the sequence (e.g., at position 48 of SEQ ID NO: 58), the second cysteine residue in the sequence (e.g., at position 15 of SEQ ID NO: 58) may be disulfide-bonded to the fifth cysteine residue in the sequence (e.g., at position 35 of SEQ ID NO: 58), the third cysteine residue in the sequence (e.g., at position 21 of SEQ ID NO: 58) may be disulfide-bonded to the sixth cysteine residue in the sequence (e.g., at position 42 of SEQ ID NO: 58), and the fourth cysteine residue in the sequence (e.g., at position 25 of SEQ ID NO: 58) may be disulfide-bonded to the seventh cysteine residue in the sequence (e.g., at position 44 of SEQ ID NO: 58).

In some embodiments, a peptide of the disclosure (e.g., a PD-L1 binding peptide) comprises, for example, a peptide of SEQ ID No. 1, having a cysteine at one or more positionsAmino acid sequence of amino acid residues. In some embodiments, one or more cysteine residues are located at any of the corresponding amino acid positions 4, 8, 18, 32, 42, 46, or any combination thereof. In some embodiments, one or more cysteine residues are located at any of the corresponding amino acid positions 4, 15, 21, 25, 35, 42, 44, 48, or any combination thereof. In some aspects of the disclosure, one or more cysteine (C) residues are present in various pairing modes (e.g., C ¹⁰ -C ²⁰ ) Participating in disulfide bonds. In some embodiments, a peptide as described herein comprises at least one, at least two, at least three, or at least four disulfide bonds. In some embodiments, the peptide as described herein comprises a peptide having a corresponding pairing mode C ⁴ -C ⁴⁶ 、C ⁸ -C ⁴² And C ¹⁸ -C ³² Is a disulfide bond of (c). In some embodiments, the peptide as described herein comprises a peptide having a corresponding pairing mode C ⁴ -C ⁴⁸ 、C ¹⁵ -C ³⁵ 、C ²¹ -C ⁴² And C ²⁵ -C ⁴⁴ Is a disulfide bond of (c). In some embodiments, the peptide as described herein comprises a peptide having a corresponding pairing mode C ¹⁵ -C ³⁵ 、C ²¹ -C ⁴² And C ²⁵ -C ⁴⁴ Is a disulfide bond of (c).

In some cases, one or more or all of the methionine residues in the peptide are replaced with leucine or isoleucine. In some cases, one or more or all of the tryptophan residues in the peptide are replaced with phenylalanine or tyrosine. In some cases, one or more or all of the asparagine residues in the peptide are replaced with glutamine. In some embodiments, the N-terminus of the peptide is blocked, for example, by an acetyl group. Alternatively or in combination, in some cases, the C-terminus of the peptide is blocked, for example, by an amide group. In some embodiments, the peptide is modified by methylation on a free amine. For example, complete methylation can be achieved by using reductive methylation with formaldehyde and sodium cyanoborohydride.

PD-L1 binding peptides

Disclosed herein are peptide sequences capable of binding to PD-L1 or any combination or fragment thereof (e.g., extracellular domain), such as those listed in table 1. Peptides capable of binding to PD-L1 may be referred to herein as PD-L1 binding peptides. In some embodiments, a peptide disclosed herein can penetrate, pass through, or enter a target cell or can be modified to penetrate, pass through, or enter a target cell (e.g., a PD-L1 positive cell). In some embodiments, the peptides disclosed herein may penetrate or cross the Blood Brain Barrier (BBB), or may be modified to penetrate or cross the Blood Brain Barrier (BBB). In some cases, the PD-L1 binding peptide may be part of a PD-L1 binding peptide complex that comprises a PD-L1 binding peptide conjugated, linked or fused to an additional active agent (e.g., a therapeutic agent, a detectable agent, or an immune cell targeting agent), such as a small molecule or peptide having affinity for an additional target protein (e.g., an immune cell surface protein). In some cases, the peptide complexes of the present disclosure exert biological effects mediated by PD-L1 binding peptides, additional active agents, or combinations thereof.

In some embodiments, the PD-L1 binding peptides of the disclosure, including peptides having the amino acid sequences set forth in SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO:567, and any derivatives or variants thereof, prevent or reduce binding of an endogenous PD-L1 binding agent (e.g., PD-1) to PD-L1. The PD-L1 binding peptides of the disclosure (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) can compete with PD-1 for binding to PD-L1. The PD-L1 binding peptide can translocate PD-1 from PD-L1. In some embodiments, the PD-L1 binding peptide can be present in a subject (e.g., a human subject) at about 1pM to about 10 μm, about 10pM to about 10 μm, about 100pM to about 10 μm, about 300pM to about 10 μm, about 500pM to about 10 μm, about 1pM to about 1 μm, about 10pM to about 1 μm, about 100pM to about 1 μm, about 300pM to about 1 μm, about 500pM to about 1 μm, about 1pM to about 100nM, about 10pM to about 100nM, about 100pM to about 100nM, about 300pM to about 100nM, about 1pM to about 10nM, about 10pM to about 10nM, about 100pM to about 10nM, about 300pM to about 10nM, about 500pM to about 10nM, about 1pM to about 1nM, about 10pM to about 1nM, about 1pM to about 1nM, about 100pM to about 100nM, about 1nM to about 1nM, about 1nM to about 100pM Half maximal Inhibitory Concentration (IC) of 500pM, about 10pM to about 500pM, or about 100pM to about 500pM ₅₀ ) Inhibit binding of PD-1 to PD-L1 or shift PD-1 from PD-L1. In some embodiments, the PD-L1 binding peptide can be present on a cell (e.g., a human cell) at a half-maximal Inhibitory Concentration (IC) of about 1pM to about 1 μm, about 10pM to about 1 μm, about 100pM to about 1 μm, about 300pM to about 1 μm, about 500pM to about 1 μm, about 1pM to about 100nM, about 10pM to about 100nM, about 100pM to about 100nM, about 300pM to about 100nM, about 500pM to about 100nM, about 1pM to about 10nM, about 10pM to about 10nM, about 100pM to about 10nM, about 300pM to about 10nM, about 500pM to about 10nM, about 1pM to about 1nM, about 10pM to about 1nM, about 100pM to about 1nM, about 1pM to about 500pM, about 10pM to about 500pM, or about 100 to about 500pM under physiological conditions ₅₀ ) Inhibit binding of PD-1 to PD-L1 or shift PD-1 from PD-L1.

In some embodiments, the peptides of the present disclosure comprise derivatives and variants having at least 40% homology, at least 50% homology, at least 60% homology, at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology or 100% homology to the amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO: 567. For example, the PD-L1 binding peptide may comprise a sequence having at least 40% homology, at least 50% homology, at least 60% homology, at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology or 100% homology to the amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO: 567.

In some embodiments, with an innerThe PD-L1 binding peptide has the same, similar or higher affinity (e.g., lower equilibrium dissociation constant K) than the source molecule (e.g., PD-1) or any other endogenous PD-L1 ligand) or other exogenous molecule (e.g., PD-L1-binding antibody or antibody fragment) _D ) Binds to PD-L1. In some embodiments, the peptide or peptide complex binds to PD-L1 at K _D May be no greater than 50 μM, no greater than 5 μM, no greater than 500nM, no greater than 100nM, no greater than 40nM, no greater than 30nM, no greater than 20nM, no greater than 15nM, no greater than 10nM, no greater than 5nM, no greater than 2nM, no greater than 1nM, no greater than 0.9nM, no greater than 0.8nM, no greater than 0.7nM, no greater than 0.6nM, no greater than 0.5nM, no greater than 0.4nM, no greater than 0.3nM, no greater than 0.2nM, or no greater than 0.1nM. In some embodiments, the PD-L1 binding peptide that exhibits improved PD-L1 binding exhibits improved recruitment to PD-L1 positive cells, improved inhibition of PD-L1 or PD-1 binding, improved delivery of an active agent, improved immune cell recruitment, improved cell killing, improved tumor regression, or a combination thereof. In some embodiments, the PD-L1 binds to K of the peptide _A 、K _D 、k _{Association with} 、k _{Dissociation of} The values or combinations thereof may be adjusted and optimized (e.g., by amino acid substitutions) to provide a preferred PD-L1 binding affinity ratio, binding rate to PD-L1, rate of release from PD-L1, or combinations thereof. In some embodiments, the PD-L1 binding peptide binds to a site of low homology between human and murine PD-L1, reducing cross-reactivity of the PD-L1 binding peptide to human and murine PD-L1. The binding kinetics of CDP are different from antibodies, so bispecific molecules comprising PD-L1 binding CDP and antibodies may have different behavior than antibody-based molecules. The association rate of PD-L1 with the herein described PD-L1 binding CDP may be faster compared to antibody-based molecules.

In some embodiments, a peptide disclosed herein or a variant thereof binds to PD-L1 at a residue found in the binding interface (e.g., binding domain or binding pocket) of PD-L1 with other exogenous or endogenous ligands (e.g., PD-1 derivatives, or PD-1-like peptides or proteins). In some embodiments, a peptide disclosed herein or variant thereof that binds to PD-L1 comprises at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology, or at least 100% homology to the sequence of residues that make up the binding pocket that bind to PD-L1. In some embodiments, a peptide disclosed herein or variant thereof that binds to PD-L1 comprises at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology, or at least 100% homology to an endogenous or exogenous polypeptide known to bind to PD-L1, e.g., endogenous PD-1 or any of the peptides listed in table 1. In other embodiments, the peptides described herein bind to a protein of interest that comprises at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology, or at least 100% homology to PD-L1, fragments, homologs, or variants thereof.

In other embodiments, the nucleic acid, vector, plasmid, or donor DNA comprises a sequence encoding a peptide, peptide construct, peptide complex, or variant or functional fragment thereof as described in the present disclosure. In other embodiments, portions or fragments of the PD-L1 binding motif (e.g., a conserved binding motif) may be grafted onto a peptide or peptide complex having the sequence of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567.

In some embodiments, peptides may be selected for further testing or use based on their ability to bind to an amino acid residue or amino acid residue motif. An amino acid residue or amino acid residue motif in PD-L1 can be identified from an amino acid residue or amino acid residue sequence involved in the binding of PD-L1 to PD-1. An amino acid residue or amino acid residue motif can be identified from the crystal structure of the PD-L1-PD-1 complex. In some embodiments, the peptide (e.g., CDP) exhibits resistance to heat, proteases (pepsin, trypsin, or others), and reduction.

Peptides and peptide complexes (e.g., peptide conjugates or fusion peptides) comprising one or more of the amino acid sequences set forth in SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO:567 may bind to a protein of interest. In some embodiments, the protein of interest is PD-L1. In some embodiments, the peptide and peptide complex (e.g., peptide conjugate or fusion peptide) that binds to PD-L1 comprises at least one of the amino acid sequences set forth in SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567. Table 1 lists exemplary peptide sequences for methods and compositions according to the present disclosure.

TABLE 1 exemplary PD-L1 binding peptides

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In some embodiments, a PD-L1 binding peptide disclosed herein comprises X ¹ X ² X ³ CX ⁴ X ⁵ X ⁶ CX ⁷ X ⁸ X ⁹ X ¹⁰ X ¹¹ X ¹² X ¹³ X ¹⁴ X ¹⁵ CX ¹⁶ X ¹⁷ X ¹⁸ X ¹⁹ X ²⁰ X ²¹ X ²² X ²³ X ²⁴ X ²⁵ X ²⁶ X ²⁷ X ²⁸ CX ²⁹ X ³⁰ X ³¹ X ³² X ³³ X ³⁴ X ³⁵ X ³⁶ X ³⁷ CX ³⁸ X ³⁹ X ⁴⁰ CX ⁴¹ X ⁴² X ⁴³ (SEQ ID NO: 358), wherein X ¹ May be independently selected from E, M, V or W; x is X ² May be independently selected from G, E, L or F; x is X ³ May be independently selected from D, E or S; x is X ⁴ May be independently selected from K, R or V; x is X ⁵ May be independently selected from E, Q, S, M, L or V; x is X ⁶ May be independently selected from D, E, H, K, R, N, Q, S or Y; x is X ⁷ May be independently selected from D, M or V; x is X ⁸ May be independently selected from A, K, R, Q, S or T; x is X ⁹ May be independently selected from A, D, E, H, Q, S, T, M, I, L, V or W; x is X ¹⁰ May be independently selected from A, E, R, Q, S, T, W or P; x is X ¹¹ May be independently selected from A, E, K, R, N, Q, T, M, I, L, V or W; x is X ¹² May be independently selected from G, A, E, K, N, T or Y; x is X ¹³ May be independently selected from G, A, D, E, H, K, R, N, Q, S, T, M, I, L, V, W, Y or P; x is X ¹⁴ May be independently selected from D, K, R, N, L or V; x is X ¹⁵ May be independently selected from G, A, D, T, L, W or P; x is X ¹⁶ May be independently selected from G, A, E, H, K, N, S, F or P; x is X ¹⁷ May be independently selected from G, A, D, E, N or P; x is X ¹⁸ May be independently selected from G, D, H, K, R, N, Q, S, T, V or Y; x is X ¹⁹ May be independently selected from G, D, E, H, K, N, Q, S, T, M, I, F, W, Y or P; x is X ²⁰ Can be independently selected from G, A, D, E, H, KR, N, Q, S, Y or P; x is X ²¹ May be independently selected from G, A, D, H, N, Q, S, V, F or P; x is X ²² May be independently selected from A, D, H, N, Q, S, T, M, I, V, Y or P; x is X ²³ May be independently selected from G, A, D, K, R, T, W or Y; x is X ²⁴ May be independently selected from G, A, E, N, Q, T, I, V or P; x is X ²⁵ May be independently selected from G, D, N, Q, T, L, V, F or P; x is X ²⁶ May be independently selected from G, A, E, K, R, N, Q, S, T, I, Y or P; x is X ²⁷ May be independently selected from A, D, N or I; x is X ²⁸ May be independently selected from G, D, E, H, N, F or W; x is X ²⁹ May be independently selected from G, A, E, N, S, Y or P; x is X ³⁰ May be independently selected from G, M or L; x is X ³¹ May be independently selected from G, A, D, K, N, Q or W; x is X ³² May be independently selected from D, E, H, K, N, Q, S, T, L, V, F, Y or P; x is X ³³ May be independently selected from G, E, Q or F; x is X ³⁴ May be independently selected from D or K; x is X ³⁵ May be independently selected from G, V or P; x is X ³⁶ May be independently selected from G, H, R, V, F, W or P; x is X ³⁷ May be independently selected from A, D or K; x is X ³⁸ May be independently selected from E, H, Q, L or F; x is X ³⁹ May be independently selected from D, E, R, S, T, M, L or F; x is X ⁴⁰ May be independently selected from G, A, D, E, H, K, R, M, L or P; x is X ⁴¹ May be independently selected from G, A, K, S, I or L; x is X ⁴² May be independently selected from G, A, D, E, R, Q, T or F; and X is ⁴³ May be independently selected from A, H, N, Q, S, F or P.

In some embodiments, the binding peptides disclosed herein comprise EEDCKVX ¹ CVX ¹ X ¹ X ¹ X ¹ X ² X ³ KX ¹ CX ¹ EX ¹ X ⁴ X ¹ X ¹ X ¹ X ¹ X ¹ X ¹ X ¹ AX ¹ CX ¹ GX ¹ X ⁵ FX ⁶ VFX ⁶ CLX ¹ X ¹ CX ¹ X ¹ X ¹ (SEQ ID NO: 359), wherein X ¹ May be independently selected from any non-cysteine amino acid; x is X ² Can be independently selected from M,I. L or V; x is X ³ May be independently selected from Y, A, H, K, R, N, Q, S or T; x is X ⁴ May be independently selected from D, E, N, Q or P; x is X ⁵ Independently selected from K or P; and X is ⁶ May be independently selected from D or K.

The PD-L1 binding peptide may comprise a PD-L1 binding motif that forms part or all of the binding interface with PD-L1. One or more residues of the PD-L1 binding motif may interact with one or more residues of PD-L1 at the binding interface between the PD-L1 binding peptide and PD-L1. In some embodiments, multiple PD-L1 binding motifs may be present in the PD-L1 binding peptide. The PD-L1 binding motif may comprise CX ¹ X ² X ³ CX ⁴ X ⁵ X ⁶ X ⁷ X ⁸ X ⁹ X ¹⁰ X ¹¹ X ¹² C (SEQ ID NO: 360), wherein X ¹ May be independently selected from K, R or V; x is X ² May be independently selected from E, Q, S, M, L or V; x is X ³ May be independently selected from D, E, H, K, R, N, Q, S or Y; x is X ⁴ May be independently selected from D, M or V; x is X ⁵ May be independently selected from A, K, R, Q, S or T; x is X ⁶ May be independently selected from A, D, E, H, Q, S, T, M, I, L, V or W; x is X ⁷ May be independently selected from A, E, R, Q, S, T, W or P; x is X ⁸ May be independently selected from A, E, K, R, N, Q, T, M, I, L, V or W; x is X ⁹ May be independently selected from G, A, E, K, N, T or Y; x is X ¹⁰ May be independently selected from G, A, D, E, H, K, R, N, Q, S, T, M, I, L, V, W, Y or P; x is X ¹¹ May be independently selected from D, K, R, N, L or V; and X is ¹² May be independently selected from G, A, D, T, L, W or P. In some embodiments, the PD-L1 binding motif may comprise CKVX ¹ CVX ¹ X ¹ X ¹ X ¹ X ² X ³ KX ¹ C (SEQ ID NO: 362) wherein X ¹ May be independently selected from any non-cysteine amino acid; x is X ² May be independently selected from M, I, L or V; and X is ³ May be independently selected from Y, A, H, K, R, N, Q, S or T. In some embodiments, the PD-L1 binding motif may comprise the sequence of CKVHCVKEWMAGKAC (SEQ ID NO: 364). In some embodimentsIn cases, the PD-L1 binding motif may comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity to SEQ ID NO 364.

The PD-L1 binding motif may comprise X ¹ X ² X ³ X ⁴ X ⁵ X ⁶ CX ⁷ X ⁸ X ⁹ C (SEQ ID NO: 361), wherein X ¹ May be independently selected from D, E, H, K, N, Q, S, T, L, V, F, Y or P; x is X ² May be independently selected from G, E, Q or F; x is X ³ May be independently selected from D or K; x is X ⁴ May be independently selected from G, V or P; x is X ⁵ May be independently selected from G, H, R, V, F, W or P; x is X ⁶ May be independently selected from A, D or K; x is X ⁷ May be independently selected from E, H, Q, L or F; x is X ⁸ May be independently selected from D, E, R, S, T, M, L or F; and X is ⁹ May be independently selected from G, A, D, E, H, K, R, M, L or P. In some embodiments, the PD-L1 binding motif may comprise X ¹ FX ² VFX ² CLX ³ X ³ C (SEQ ID NO: 363), wherein X ¹ Independently selected from K or P; x is X ² May be independently selected from D or K; and X is ³ May be independently selected from any non-cysteine amino acid. In some embodiments, the PD-L1 binding motif may comprise the sequence of KFDVFKCLDHC (SEQ ID NO: 365). In some embodiments, the PD-L1 binding motif may comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity to SEQ ID NO 365.

The PD-L1 binding peptides of the disclosure (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) may comprise one or more secondary structural elements. In some embodiments, the PD-L1 binding peptide may comprise an α -helix, a β -sheet, a loop, or a combination thereof. PD-L1 binding peptides (e.g., any of SEQ ID NO:1-SEQ ID NO:56 or SEQ ID NO:60-SEQ ID NO: 115) may include an alpha-helix comprising amino acid residues n to n+20, where n corresponds to the amino acid position of the first cysteine residue. For example, the PD-L1 binding peptide of SEQ ID NO. 3 may comprise an alpha-helix comprising amino acid residues C4 to S24. PD-L1 binding peptides (e.g., any of SEQ ID NO:1-SEQ ID NO:56 or SEQ ID NO:60-SEQ ID NO: 115) may include an alpha-helix comprising amino acid residues n+29 to n+44, where n corresponds to the amino acid position of the first cysteine residue. For example, the PD-L1 binding peptide of SEQ ID NO. 3 may comprise an alpha-helix comprising amino acid residues S33 to A48. PD-L1 binding peptides (e.g., any of SEQ ID NO:1-SEQ ID NO:56 or SEQ ID NO:60-SEQ ID NO: 115) may include an alpha-helix comprising amino acid residues n+34 to n+44, where n corresponds to the amino acid position of the first cysteine residue. For example, the PD-L1 binding peptide of SEQ ID NO. 3 may comprise an alpha-helix comprising amino acid residues D38 to A48. In some embodiments, the PD-L1 binding peptides of the present disclosure can bind to PD-L1 by forming a hydrophobic interaction with I54, Y56, R113, M115, or Y123 of PD-L1. For example, residues V9, W12, M13, V39 or F40 of SEQ ID NO. 1 may undergo hydrophobic interactions with PD-L1. In some embodiments, the PD-L1 binding peptides of the present disclosure can form a salt bridge with Q66, a121, and Y123 of PD-L1. For example, residues K5, K16, L43 and D44 of SEQ ID NO. 1 may form a salt bridge with PD-L1. In some embodiments, the PD-L1 binding peptides of the present disclosure can bind to PD-L1 in a similar manner to the use of the natural binding partners PD-1 on PD-L1 that interact with the same sites on K78, I126, L128, A132, I134 and E136 as those of SEQ ID NO. 1, K5, L43, V9, W12, F40 and D44, respectively. In some embodiments, any of SEQ ID NOS.358-365 may comprise a portion of a PD-L1 binding peptide that interacts with PD-L1 (e.g., forms a hydrophobic interaction or a salt bridge). For example, any of SEQ ID NOs 358-365 may include K5, V9, W12, M13, K16, V39, F40, L43, D44, or a combination thereof, with respect to SEQ ID NO 1.

In some embodiments, the PD-L1 binding peptides of the present disclosure can bind to PD-L1 with a pH-dependent affinity. For example, the PD-L1 binding peptide can bind PD-L1 with substantially the same affinity at extracellular pH (about pH 7.4) as at endocytic pH (e.g., about pH 5.5 or about pH 6.5). In some embodiments, the PD-L1 binding peptide can bind PD-L1 with an affinity at extracellular pH (about pH 7.4) that is lower than the binding affinity at endocytic pH (e.g., about pH 5.5 or about pH 6.5). In some embodiments, the PD-L1 binding peptide can bind PD-L1 with an affinity at extracellular pH (about pH 7.4) that is higher than the binding affinity at endocytic pH (e.g., about pH 5.5 or about pH 6.5). In some embodiments, the binding affinity of the PD-L1 binding peptide to PD-L1 at extracellular pH (about pH 7.4) may differ from the binding affinity of the PD-L1 binding peptide to PD-L1 at endocytic pH (about pH 5.5) by no more than about 1%, no more than about 2%, no more than about 3%, no more than about 4%, no more than about 5%, no more than about 6%, no more than about 7%, no more than about 8%, no more than about 9%, no more than about 10%, no more than about 12%, no more than about 15%, no more than about 17%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 35%, no more than about 40%, no more than about 45%, or no more than about 50%. In some embodiments, the affinity of the PD-L1 binding peptide for PD-L1 at pH7.4 and at pH 5.5 may differ by no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 40-fold, or no more than 50-fold. In some embodiments, the PD-L1 binding peptide (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) may be modified to remove one or more histidine amino acids in the PD-L1 binding interface, thereby reducing the pH dependence of the binding affinity of the PD-L1 binding peptide to PD-L1. In some embodiments, the PD-L1 binding peptide (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) may lack a histidine amino acid at the PD-L1 binding interface.

In some embodiments, the PD-L1 binding peptide can be cleaved at an extracellular pH (about pH 7.4) at an equilibrium dissociation constant of, for example, no greater than 50. Mu.M, no greater than 5. Mu.M, no greater than 500nM, no greater than 100nM, no greater than 40nM, no greater than 30nM, no greater than 20nM, no greater than 10nM, no greater than 5nM, no greater than 2nM, no greater than 1nM, no greater than 0.5nM, no greater than 0.4nM, no greater than 0.3nM, no greater than 0.25nM, no greater than 0.2nM, or no greater than 0.1nM(K _D ) Binds to PD-L1. In some embodiments, a PD-L1 binding peptide having pH-independent binding can be cleaved at an endosomal pH (about pH 5.5) at a dissociation constant (K) of no greater than 50. Mu.M, no greater than 5. Mu.M, no greater than 500nM, no greater than 100nM, no greater than 40nM, no greater than 30nM, no greater than 20nM, no greater than 10nM, no greater than 5nM, no greater than 2nM, no greater than 1nM, no greater than 0.5nM, no greater than 0.2nM, or no greater than 0.1nM _D ) Binds to PD-L1. In some embodiments, the PD-L1 binding peptide can bind to PD-L1 with a pH-dependent affinity. For example, a PD-L1-binding molecule can bind to PD-L1 with higher affinity at extracellular pH (about pH 7.4) and lower affinity at endosomal pH (about pH 5.5), releasing the peptide or peptide complex from PD-L1 upon entry and acidification of the endosomal compartment.

The PD-L1 binding peptides of the present disclosure may cross-react with PD-L1 of two or more species, or the PD-L1 binding peptides may be selective for PD-L1 of one or more species. For example, the PD-L1 binding peptide may be cross-reactive to both human and cynomolgus monkey PD-L1. If the PD-L1 binding peptide binds to two species with equilibrium dissociation constants (K _D ) A difference of no more than 1.5 times, no more than 2 times, no more than 5 times or no more than 10 times, it may be cross-reactive to both species. In some embodiments, the PD-L1 binding peptide may not cross-react with PD-L1 of one or more species. For example, the PD-L1 binding peptide may have an equilibrium dissociation constant (K _D ) Binds human PD-L1, but may have an equilibrium dissociation constant (K) that is at least 10-fold, 50-fold, or 100-fold greater _D ) Binding murine PD-L1.

Sequence identity and homology

Percent (%) sequence identity or homology is determined by conventional methods. (see, e.g., altschul et al (1986), bull. Math. Bio.48:603 (1986) and Henikoff (1992), proc. Natl. Acad. Sci. USA 89:10915). Briefly, two amino acid sequences can be aligned to optimize alignment using gap opening penalty 10, gap extension penalty 1, and the "BLOSUM62" scoring matrices of Henikoff and Henikoff (supra). Sequence identity or homology is then calculated as: ([ total number of identical matches ]/[ length of longer sequence plus number of gaps introduced into longer sequence to align two sequences ]) (100).

Various methods and software programs may be used to determine homology between two or more peptides, such as NCBI BLAST, clustal W, MAFFT, clustal Omega, alignMe, praline, or another suitable method or algorithm. A pairwise sequence alignment may be used to identify regions of similarity that may indicate a functional, structural, and/or evolutionary relationship between two biological sequences (e.g., amino acid or nucleic acid sequences). In addition, multiple Sequence Alignment (MSA) is an alignment of three or more biological sequences. From the output of the MSA application, homology can be implied and the evolutionary relationship between sequences evaluated. As used herein, "sequence homology" and "sequence identity" and "percent sequence identity (%)" and "percent sequence homology (%)" are used interchangeably to refer to sequence relatedness or variation with respect to a reference polynucleotide or amino acid sequence, as appropriate.

In addition, there are several algorithms that have been identified for aligning two amino acid sequences. For example, the Pearson and Lipman "FASTA" similarity search algorithm may be a protein alignment method suitable for examining the level of sequence identity or homology shared by the amino acid sequences of the peptides disclosed herein and the amino acid sequences of the peptide variants. FASTA algorithm is described, for example, by Pearson and Lipman, proc.Nat' l Acad.Sci.USA 85:2444 (1988) and by Pearson, meth.enzymol.183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying the region with the highest identity density (if ktup variable is 1) or identity pair (if ktup=2) shared by the query sequence (e.g., SEQ ID NO: 1) and the test sequence, without regard to conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest identity density were then rescaled by comparing the similarity of all paired amino acids using the amino acid substitution matrix, and the ends of the regions were "trimmed" to include only those residues contributing to the highest score. If there are several regions having a score greater than the "cutoff value (calculated by a predetermined formula based on the sequence length and ktup value), the pruned initial region is examined to determine if the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring region of the two amino acid sequences was aligned using the modification of Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J.mol. Biol.48:444 (1970); selmers, siam J.appl. Math.26:787 (1974)), which allowed amino acid insertions and deletions. For example, illustrative parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=blosum 62. These parameters can be introduced into the FASTA program by modifying the scoring matrix file ("smamix") as described in annex 2 of Pearson, meth. Enzymol.183:63 (1990).

FASTA can also be used to determine sequence identity or homology of a nucleic acid sequence or molecule using ratios as disclosed above. For nucleic acid sequence comparisons, ktup values can range between one and six, preferably three to six, most preferably three, with other parameters set as described herein.

Some examples of common amino acids as "conservative amino acid substitutions" are illustrated by substitutions between amino acids within each of the following groups: (1) Glycine, alanine, valine, leucine and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine. BLOSUM62 is a matrix of about 2,000 local multiple alignments of amino acid substitutions derived from a segment of a protein sequence, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, proc.Nat' l Acad.Sci.USA 89:10915 (1992)). Thus, the frequency of BLOSUM62 substitutions can be used to determine conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. Although amino acid substitutions can be designed based solely on chemical nature (as discussed above), the expression "conservative amino acid substitutions" preferably refers to substitutions represented by BLOSUM62 values greater than-1. For example, amino acid substitutions are conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, a preferred conservative amino acid substitution is characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2, or 3), while a more preferred conservative amino acid substitution is characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Amino acid residues within a region or domain critical for maintaining structural integrity may be determined. Within these regions, it was determined that changes could be tolerated more or less and specific residues of the overall tertiary structure of the molecule were maintained. Methods for analyzing sequence structure include, but are not limited to, computer analysis using available software (e.g., insight II. RTM. Browser and homology modeling tool; MSI, san Diego, calif.), secondary structure propensity, binary mode, complementary packing, and embedded polarity interactions (Barton, G.J., current Opin. Structure. Biol.5:372-6 (1995) and Cordes, M.H. et al, current Opin. Structure. Biol.6:3-10 (1996)). In general, when designing modifications to a molecule or identifying specific fragments, determination of structure may often be accompanied by assessing the activity of the modified molecule.

Peptide active agent complexes

In some embodiments, CDPs that bind PD-L1, such as those described in table 1, including engineered, non-naturally occurring CDPs and those found in nature, can be conjugated, linked or fused with additional active agents to selectively deliver the active agents to PD-L1 positive cells. The cells may be cancer cells, immune cells, pancreatic beta cells, or any combination thereof. The cell may be any cell expressing PD-L1. The engineered peptide may be a non-naturally occurring, artificial, synthetic, designed, or recombinantly expressed peptide. In some embodiments, a PD-L1 binding peptide complex comprising a PD-L1 binding peptide (e.g., a PD-L1 binding bispecific immune cell cement or a PD-L1 binding chimeric antigen receptor) is capable of delivering an additional active agent (e.g., a therapeutic agent, a detectable agent, or an immune cell) to a target cell under PD-L1 mediation. Target cells (e.g., PD-L1 positive target cells) may be associated with a disease or disorder. In some embodiments, delivery of an active agent to a target cell can treat a disease or disorder (e.g., prevent, reduce, eliminate, diagnose, or alleviate symptoms thereof). In some cases, the target cell is a cancer cell. The cancer may include melanoma, non-small cell lung cancer, kidney cancer, esophagus cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, lymphoma, bladder cancer, liver cancer, stomach cancer, breast cancer, pancreatic cancer, prostate cancer, merck cell cancer, mesothelioma, brain cancer, metastatic brain cancer, primary brain cancer, glioblastoma, or cancer that is overexpressed by PD-L1. In some cases, the PD-L1 binding peptide or peptide complex is capable of crossing the blood brain barrier, delivering the PD-L1 binding peptide or other active agent to target cells in the central nervous system.

The PD-L1 binding peptides of the present disclosure can be linked, fused, conjugated or otherwise complexed with additional active agents to form PD-L1 binding peptide complexes. In some embodiments, the PD-L1 binding peptide and the additional active agent can be complexed via a linker (e.g., a peptide linker or a small molecule linker). The activity (e.g., binding, inhibitory or activating activity) of the PD-L1 binding peptide and additional active agent may be retained after complex formation. In some embodiments, the appropriate linker is selected to retain activity. The active agent may be any agent capable of performing a function. Such functions may include binding, inhibition, activation, inactivation, recruitment, signaling, synthesis, disruption, or a combination thereof. In some embodiments, the active agent may be a therapeutic agent (e.g., a therapeutic small molecule or therapeutic peptide) or a detectable agent (e.g., a fluorophore or radioisotope).

The active agent may be complexed with the PD-L1 binding peptide such that it does not disrupt the binding to PD-L1. In some embodiments, the peptide active agent complex may have an equilibrium dissociation constant (K) of no greater than 100nM, no greater than 50nM, no greater than 30nM, no greater than 20nM, no greater than 1nM, no greater than 500pM, no greater than 300pM, no greater than 250pM, or no greater than 200pM _D ) Binds to PD-L1.

Peptide therapeutic agent complexes

The PD-L1 binding peptides of the disclosure (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) can be complexed with a therapeutic agent to form a peptide therapeutic agent complex. The therapeutic agent of the peptide complex may perform a therapeutic function upon delivery to a cell (e.g., a PD-L1 positive cell). Therapeutic functions may include activating or inhibiting a target (e.g., a target enzyme), recruiting additional components to a cell, or killing a cell (e.g., a PD-L1 positive cancer cell). Examples of therapeutic agents that can be complexed with the PD-L1 binding peptide include anticancer agents, chemotherapeutic agents, radiotherapeutic agents, anti-inflammatory agents, pro-inflammatory cytokines, oligonucleotides, or combinations thereof. In some embodiments, the active agent (e.g., fc domain or globular protein) may act as a steric blocker. For example, an Fc domain linked to a PD-L1 binding peptide can enhance disruption of PD-1 binding to PD-L1 by sterically blocking entry of PD-L1 by the PD-L1 binding peptide. In some embodiments, the PD-L1 binding peptide may be complexed with an oncolytic viral vector to deliver the viral vector to PD-L1 positive cells.

Chemotherapeutic or anti-cancer agents may act by killing or inhibiting proliferation of target cancer cells (e.g., PD-L1 positive cancer cells). Examples of chemotherapeutic or anti-cancer agents that may be complexed with the PD-L1 binding peptides of the present disclosure include anti-tumor agents, cytotoxic agents, tyrosine kinase inhibitors, mTOR inhibitors, retinoids, microtubule polymerization inhibitors, pyrrolobenzodiazepine dimers, or anti-cancer antibodies. Proinflammatory cytokines can act by stimulating an immune response against a target (e.g., PD-L1 positive cancer cells). Examples of pro-inflammatory cytokines that can be complexed with the PD-L1 binding peptides of the present disclosure include TNFα, IL-2, IL-6, IL-12, IL-15, IL-21, or IFNγ. Anti-inflammatory agents may act by inhibiting inflammatory responses within or around the target (e.g., by inhibiting cyclooxygenase or stimulating glucocorticoid receptors). Examples of anti-inflammatory agents that can be complexed with the PD-L1 binding peptides of the present disclosure include anti-inflammatory cytokines, steroids, glucocorticoids, corticosteroids, cytokine inhibitors, rory inhibitors, JAK inhibitors, casein kinase inhibitors, or non-steroidal anti-inflammatory drugs (NSAIDs).

In some embodiments, the active agent is an immunotherapeutic agent, an immunoneoplastic agent, a CTLA-4 targeting agent, a PD-1 targeting agent, a PDL-1 targeting agent, an IL15 agent, a fusion IL-15/IL-15Ra complex agent, an IFNgamma agent, an anti-CD 3 agent, an ion channel modulator, an auristatin, an MMAE, a maytansinoid, DM1, DM4, a doxorubicin (doxorubicin), a calicheamicin (calicheamicin), a platinum compound, cisplatin (cispratin), a taxane (taxane), a paclitaxel (paclitaxel), SN-38, a BACE inhibitor, a Bcl-xL inhibitor, WEHI-539, a vitamin E-tock (velocex), an ABT-199, a nanotock (navitocratin), an AT-101, an oxybutyrate (atoclax), a pyrrolobenzodiazepine or a benzodiazepine, a dol, an immunostatin (immunostatin), an immune cell targeting agent such as a tumor, GITR, 4-1BB, CD27, TIGIT, LAG3, TCR, CD40L, OX40, PD-1, CTLA-4, CD 28) or an agent that targets a tumor cell (e.g., GITRL, 4-1BBL, CD17, CD156/CD112/CD113, MHC11, CD40, OX40L, PD-L1/L2, CD 80/86).

In some embodiments, the PD-L1 binding peptide may direct an active agent (e.g., a target binding nucleotide, small molecule, peptide, or protein active agent) into a cell. In other embodiments, the PD-L1 binding peptide may direct an active agent into the nucleus of a cell. In some embodiments, the active agent has intrinsic tumor homing properties, or the active agent can be engineered to have tumor homing properties. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 active agents may be attached to a peptide or nucleotide. Multiple active agents (e.g., multiple target binding nucleotides) can be linked, e.g., conjugated to multiple lysine residues and/or the N-terminus, or by linking multiple active agents to a scaffold, e.g., a polymer or dendrimer, and then linking the agent-scaffold to a peptide (e.g., described in yurkovitskiy, a.v., cancer Res 75 (16): 3365-72 (2015). Examples of active agents include, but are not limited to: peptides, oligopeptides, polypeptides, peptidomimetics, polynucleotides, polyribonucleotides, DNA, cDNA, ssDNA, RNA, dsRNA, micrornas, oligonucleotides, antisense RNAs, complementary RNAs, inhibitory RNAs, interfering RNAs, nuclear RNAs, antisense oligonucleotides (ASOs), micrornas (mirnas), oligonucleotides complementary to Natural Antisense Transcript (NAT) sequences, siRNA, snrnas, aptamers, gapmers, anti-miR, splice-blocking ASOs or U1 adaptor antibodies, single-chain variable fragments (scFv), antibody fragments, aptamers, cytokines, interferons, hormones, enzymes, growth factors, checkpoint inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CD47 inhibitors, CTLA4 inhibitors, CD antigens, chemokines, ion channel inhibitors, ion channel activators, G protein-coupled receptor inhibitors, G protein-coupled receptor activators, chemicals radiosensitizers, radioprotectors, radionuclides, therapeutic small molecules, steroids, corticosteroids, anti-inflammatory agents, immunomodulators, immunotumoral agents, complement fixation peptides or proteins, tumor necrosis factor inhibitors, tumor necrosis factor activators, tumor necrosis factor receptor family agonists, tumor necrosis receptor antagonists, tim-3 inhibitors, protease inhibitors, aminosugars, chemotherapeutic agents, cytotoxic molecules, toxins, tyrosine kinase inhibitors, anti-infective agents, antibiotics, antivirals, antifungals, aminoglycosides, non-steroidal anti-inflammatory drugs (NSAIDs), statins, nanoparticles, liposomes, polymers, biopolymers, polysaccharides, proteoglycans, glycosaminoglycans, polyethylene glycols, lipids, dendrimers, fatty acids or Fc regions, or an active fragment or modification thereof.

Only a small fraction of the currently available drug molecules are applicable in CNS diseases due to poor BBB penetration. About 98% of small molecule drugs do not cross the BBB or only to a very limited extent. Furthermore, nearly 100% of macromolecular drug molecules (e.g., antibodies) do not exhibit significant BBB penetration capacity. (see, e.g., mikitsh et al Pathways for Small Molecule Delivery to the Central Nervous System Across the Blood-Brain Barrier, perspin medicine chem.2014; 6:11-24). PD-L1 binding antibodies may not cross the blood brain barrier at sufficient therapeutic levels. The PD-L1 binding peptides of the present disclosure are capable of crossing the BBB by virtue of CDP properties, PD-L1 binding properties, or other properties. The PD-L1 binding peptides of the present disclosure may also be complexed with other agents, such as transferrin receptor (TfR) binding agents, to enable the complex to cross the BBB. Cells expressing PD-L1, such as cancer cells in the brain (e.g., from primary or metastatic cancers), can be beneficially targeted by administering a PD-L1 binding molecule that can cross the BBB. Thus, the PD-L1 binding peptides of the present disclosure can be administered for therapeutic use in the CNS, e.g., blocking PD-L1 in the brain, delivering an active agent to the brain (e.g., T cell binding agent or oligonucleotide). The PD-L1 binding peptides of the present disclosure can also be complexed with TfR binding peptides (e.g., SEQ ID NO: 350) in order to deliver the PD-L1 binding peptides of the present disclosure across the BBB to the CNS. Brain tumors that may be treated or prevented using conjugates or fusion molecules comprising one or more PD-L1 binding peptides of the present disclosure may include glioblastomas, astrocytomas, gliomas, medulloblastomas, ependymomas, choroidal plexus cancers, midline gliomas, metastatic cancers (including but not limited to metastatic melanoma), breast and lung cancers, and diffuse endogenous pontic gliomas.

Active agents that may be used in combination with the PD-L1 binding peptides described herein include cytotoxic molecules. For example, cytotoxic molecules that may be used include Australian, MMAE, MMAF, duloxetine (dolostatin), australian F, monomethyl Australian D, DM, DM4, maytansinoids, maytansine, calicheamicin, N-acetyl-gamma-calicheamicin, pyrrolobenzodiazepine, PBD dimer, doxorubicin, vinca alkaloids (4-deacetylvinblastine), a cyclic octapeptide analog of duocarmycin, mushroom toxin (amatosetin), epothilones (epothilones) and anthracyclines (anthracyclines), CC-1065, taxanes, paclitaxel, cabazitaxel, docetaxel, SN-38, irinotecan, neomycin (vincristine), vinblastine, cisplatin, mitomycin (37-betacellulin), and mitomycin (37-beta), such as the protease, the mitomycin (bacitracin) and the mitomycin (37). Additional examples of active agents are described in McCombs, J.R., AAPS J,17 (2): 339-51 (2015); ducry, l., antibody Drug Conjugates (2013); and Singh, S.K., pharm Res.32 (11): 3541-3571 (2015). Additional examples of therapeutic payloads that may significantly improve therapeutic efficacy when used in combination with the compositions and methods of the present disclosure include Carmustine (Carmustine), cisplatin, cyclophosphamide, etoposide (Etoposide), irinotecan, lomustine (Lomustine), procarbazine (Procarbazine), temozolomide (Temozolomide), vincristine, and Bevacizumab. Additional examples of therapeutic payloads are compounds having therapeutic benefit in neurodegenerative diseases, such as BACE inhibitors or compounds having therapeutic benefit in autoimmune diseases.

Peptide detectable and peptide radiotherapeutic agent complexes

The PD-L1 binding peptides (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) or peptide complexes of the present disclosure can be complexed with a detectable or radiotherapeutic agent to form a peptide detectable or radiotherapeutic agent complex. The peptides or peptide complexes of the disclosure (e.g., PD-L1 binding peptide complexes) can be conjugated, linked or fused to a detectable or radiotherapeutic agent. In some embodiments, the detectable agent or therapeutic agent can be complexed with the PD-L1 binding peptide and an additional active agent (e.g., a therapeutic agent, oligonucleotide, or therapeutic oligonucleotide). For example, the detectable agent may be conjugated to a PD-L1 binding peptide oligonucleotide complex. In some embodiments, the detectable or radiotherapeutic agent may be directly or indirectly linked to the PD-L1 binding peptide. In some embodiments, the detectable or radiotherapeutic agent may be directly or indirectly linked to an active agent of the PD-L1 binding peptide complex (e.g., an oligonucleotide of the PD-L1 binding peptide oligonucleotide complex). The peptide complex comprising the detectable agent may be referred to as a detectable agent peptide conjugate or a detectable agent peptide complex. Peptides (e.g., PD-L1 binding peptides) can be conjugated, linked or fused with agents for use in imaging, research, therapy, theranostics, drugs, chemotherapy, chelation therapy, targeted drug delivery, and radiation therapy. In some embodiments, the peptide is conjugated, linked or fused with a detectable agent such as a fluorophore, near infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radionuclide chelator, or another suitable substance that can be used for imaging.

In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 detectable agents may be attached to a peptide or nucleotide. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of: actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinide-225 or lead-212. In some embodiments, near infrared dyes are not readily quenched by biological tissues and fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation at wavelengths between 650nm and 4000nm, such emission being used to detect the agent. Non-limiting examples of fluorescent dyes that can be used as conjugate molecules in the present disclosure include Dylight-680, dylight-750, vivoTag-750, dylight-800, IRDye-800, vivoTag-680, cy5.5, or indocyanine green (ICG). In some embodiments, the near infrared dye often includes cyanine dyes (e.g., cy7, cy5.5, and Cy 5). Additional non-limiting examples of fluorescent dyes for use as conjugate molecules in the present disclosure include acridine orange or acridine yellow, alexa Fluor (e.g., alexa Fluor 790, 750, 700, 680, 660 and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dye and any derivatives thereof, gold amine-rhodamine dye and any derivatives thereof, benzanthrone (bensantrhene), bi Maen (bimane), 9-10-bis (phenylethynyl) anthracene, 5, 12-bis (phenylethynyl) tetracene, bisbenzoyl imine, brain iride (brinbow), calcein, carboxyfluorescein and any derivatives thereof, 1-chloro-9, 10-bis (phenylethynyl) anthracene and any derivatives thereof, DAPI, diOC6, dyLight Fluor and any derivatives thereof, epicocconone, ethidium bromide, flAsH-EDT2 Fluo dye and any derivative thereof, fluoProbe and any derivative thereof, fluorescein and any derivative thereof, fura and any derivative thereof, gelGreen and any derivative thereof, gelRed and any derivative thereof, fluorescent protein and any derivative thereof, subtype m protein and any derivative thereof (such as mCherry, heptamethine dye and any derivative thereof, helst (hoeschst) stain, iminocoumarin, indian yellow, endo-1 and any derivative thereof, laudan (laurdan), fluorescein and any derivative thereof, fluorescein (luciferin) and any derivative thereof, luciferase and any derivative thereof, merocyanine (and any derivatives thereof), nile Luo Ranliao and any derivatives thereof, perylene, phloxine, phycocyanine and any derivatives thereof, propidium iodide (propidium iodide), hydroxypyrenesulfonic acid (pyraine), rhodamine and any derivatives thereof, ribogreen (ribogreen), rogp, rubrene, stilbene and any derivatives thereof, sulfonylrhodamine and any derivatives thereof, SYBR and any derivatives thereof, synapto-pHluorin, tetraphenylbutadiene, tris tetrasodium (tetrasodium tris), texas Red (Texas Red), titanium Yellow (Titan Yellow), TSQ, umbelliferone, anthrone violet, yellow fluorescent protein, yoyoyo-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5' -dichloro-2 ',7' -dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrins, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-Rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine GREEN, rhodamine RED, tetramethyl rhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), oregon GREEN dyes (e.g., oregon GREEN 488 Oregano GREEN 500, oregon GREEN 514, etc.), texas RED-X, spectrum RED (SPECTRUM RED), spectrum GREEN (SPECTRUM GREEN), cyanine dyes (e.g., CY-3, cy-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591 BODIPY 630/650, BODIPY 650/665, etc.), IRDye (e.g., IRD40, IRD 700, IRD 800, etc.), and so forth. Additional suitable detectable agents are described in PCT/US 14/56177.

In some embodiments, the peptides of the disclosure (e.g., PD-L1 binding peptides) may further comprise or be complexed with a radioisotope, a radiochelator, a radiosensitizer, or a photosensitizer. In some embodiments, the radioisotope, radiochelator, radiosensitizer, or photosensitizer may be incorporated into or directly or indirectly linked to the PD-L1 binding peptide. In some embodiments, the radioisotope, the radiochelator, the radiosensitizer, or the photosensitizer. In some embodiments, the radioisotope, radiochelator, radiosensitizer, or photosensitizer may be incorporated into or complexed with a PD-L1 binding peptide complex comprising an additional active agent (e.g., a therapeutic agent, oligonucleotide, or therapeutic oligonucleotide). For example, a radioisotope, a radiochelator, a radiosensitizer, or a photosensitizer may be incorporated into or directly or indirectly linked to an oligonucleotide of the PD-L1 binding peptide oligonucleotide complex. The radioisotope, the radiochelator, the radiosensitizer, the photosensitizer may act as a detectable agent, a therapeutic agent, or both. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of: actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinide-225 or lead-212. In addition, the following radionuclides may be used for diagnosis and/or therapy: the carbon (e.g., ¹¹ C or ¹⁴ C) Nitrogen (e.g., ¹³ n), fluorine (e.g., ¹⁸ f) Gallium (e.g., ⁶⁷ ga or ⁶⁸ Ga), copper (e.g., ⁶⁴ cu or ⁶⁷ Cu), zirconium (e.g., ⁸⁹ zr), lutetium (e.g., ¹⁷⁷ lu). In some embodiments, the radioisotope is indium-111, technetium-99 m, yttrium-90, iodine-131, iodine-123, or astatine-211.

The PD-L1 binding peptide or active agent of the PD-L1 binding peptide complex may be conjugated, linked or fused to a radiosensitizer or photosensitizer. Examples of radiosensitizers include, but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin (oxaliptin), gemcitabine (gemcitabine), etanidazole (etanidazole), misonazole (misonazole), tirapazamine (tirapazamine), and nucleobase derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers may include, but are not limited to: fluorescent molecules or beads, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorohydrin), bacteriochlorin (bacteriochlorin), isophthalocyanine (isophthalocyanine) and naphthalocyanine (naphthalocyanine)), metalloporphyrins, metallophthalocyanines, angelicins (angelicin), chalcopyrylium dyes (chalcogenapyrrillium dye), chlorophyll, coumarin, flavins and related compounds (e.g., alloxazine and riboflavin), fullerenes, pheophorbides (pheophorbides), pyropheophorbides (pyropheophorbides), cyanines (e.g., merocyanine 540), and the like) that generate heat upon irradiation pheophytin, thialin (sapphyllin), texaphyrin (texaphyrin), rhodopsin, porphyrinene (porphyrine), phenothiazinium, methylene blue derivatives, naphthalimide (naphthalimide), nile blue derivatives, quinones, perylenequinones (e.g. hypericin, hypocrellin and cercosporin), psoralens (psoralen), quinones, retinoids, rhodamine, thiophene, waldine (verdin), xanthene dyes (e.g. eosin, erythrosin, rose bengal), dimeric and oligomeric forms of porphyrin, such as 5-aminolevulinic acid. Advantageously, such a method may allow for highly specific targeting of diseased cells (e.g., cancer cells) using both therapeutic agents (e.g., drugs) and electromagnetic energy (e.g., radiation or light) in parallel. In some embodiments, the peptide is conjugated, linked, fused or covalently or non-covalently linked to the agent, e.g., directly or through a linker. Exemplary linkers suitable for use with embodiments herein are discussed in more detail below.

The PD-L1 binding peptide or active agent of the PD-L1 binding peptide complex may be conjugated, linked or fused to the radionuclide via a chelator. In some embodiments, the radionuclide may be linked to the peptide of the peptide oligonucleotide complex or the nucleotide of the peptide oligonucleotide complex via a chelator. In some aspects of the disclosure, the radionuclide is linked to a peptide oligonucleotide complex as described herein using a chelator. Exemplary chelator moieties may include 2,2',2 "- (3- (4- (3- (1- (4- (1, 2,4, 5-tetrazin-3-yl) phenyl) -1-oxo-5,8,11,14,17,20,23-heptaoxa-2-azaeicosan-25-yl) thioureido) benzyl) -1,4, 7-triazacyclononane-2, 5, 8-triyl) triacetic acid; 2,2',2 "- (3- (4- (3- (1, 2,4, 5-tetrazin-3-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26,29,32,35-undeca-2-aza-tridecan-37-yl) thioureido) benzyl) -1,4, 7-triazacyclononan-2, 5, 8-yl) triacetic acid; 2,2' - (7- (4- (3- (1, 2,4, 5-tetrazin-3-yl) phenyl) -1-oxo-5,8,11,14,17,20,23,26,29,32,35-undeca-2-aza-tridecan-37-yl) thioureido) benzyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid; 2,2',2 "- (3- (4- (3- (1, 2,4, 5-tetrazin-3-yl) phenyl) -3, 7-dioxo-11,14,17,20,23,26,29-heptaoxa-2, 8-diaza-triundec-31-yl) thioureido) benzyl) -1,4, 7-triazacyclononan-2, 5, 8-triyl) triacetic acid; 2,2',2 "- (3- (4- (3- (1, 2,4, 5-tetrazin-3-yl) phenyl) -3, 7-dioxo-11,14,17,20,23,26,29,32,35,38,41-undec-2, 8-diazatetra-tridecan-43-yl) thiourea) benzyl) -1,4, 7-triazacyclononan-2, 5, 8-triyl) triacetic acid; 2,2',2 "- (3- (4- (3- (25, 28-dioxo-28- ((6- (6- (pyridin-2-yl) -1,2,4, 5-tetrazin-3-yl) pyridin-3-yl) amino) -3,6,9,12,15,18, 21-heptaoxa-24-aza-octacosyl) thiourea) benzyl) -1,4, 7-triazacyclononane-2, 5, 8-triyl) triacetic acid; 2,2',2 "- (3- (4- (3- (37, 40-dioxo-40- ((6- (pyridin-2-yl) -1,2,4, 5-tetrazin-3-yl) pyridin-3-yl) amino) -3,6,9,12,15,18,21,24,27,30,33-undeca-36-aza-forty-one) thiourea) benzyl) -1,4, 7-triazacyclononane-2, 5, 8-triyl) triacetic acid; 2,2',2 "- (3- (4- (1- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) phenyl) -3-oxo-6,9,12,15,18,21,24-heptaoxa-2-aza-heptadecane-27-amido) benzyl) -1,4, 7-triazacyclononane-2, 5, 8-triyl) triacetic acid; 2,2',2 "- (2- (4- (1- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) phenoxy) -3,6,9,12,15,18,21,24,27,30,33-undecane-36-amido) benzyl) -1,4, 7-triazacyclononane-1, 4, 7-triyl) triacetic acid; 2,2',2 "- (3- (4- (3- (5-amino-6- ((4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) amino) -6-oxohexyl) thioureido) benzyl) -1,4, 7-triazacyclononane-2, 5, 8-triyl) triacetic acid; 2,2' - (7- (4- (3- (5-amino-6- ((4-6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) amino) -6-oxohexyl) thiourea) benzyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid; 2,2',2 "- (3- (4- (3- (5-amino-6- ((4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) amino) -6-oxohexyl) thiourea) benzyl) -1,4, 7-triazacyclononane-2, 5, 8-triyl) triacetic acid; 2,2',2 "- (3- (4- (3- (5-amino-6- ((4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) amino) -6-oxohexyl) thiourea) benzyl) -1,4, 7-triazacyclononane-2, 5, 8-triyl) triacetic acid.

Bispecific immunocyte cement (BiICE)

In some embodiments, the active agents of the present disclosure may be additional binding moieties (e.g., immune cell binding moieties). The PD-L1 binding peptides of the disclosure (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) can be complexed with one or more additional binding moieties to form a bispecific or multispecific molecule. Bispecific or multispecific molecules may bind to two or more target molecules (e.g., PD-L1 and one or more additional target molecules). An example of a bispecific molecule includes a bispecific immunocyte cement (BiICE) comprising a PD-L1 binding peptide (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) and an immunocyte targeting agent (e.g., an immunocyte targeting antibody or antibody fragment, or immunocyte targeting CDP, peptide, or peptide fragment). The immune cell targeting agent can bind to a molecule on the surface of an immune cell. For example, the immune cell targeting agent can bind to CD3, 4-1BB, CD28, CD137, CD89, CD16, CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX, PD-1, CTLA-4, or STRO-1. Bispecific immune cell cements can function by recruiting immune cells to PD-L1 positive cells (e.g., PD-L1 positive cancer cells, immune cells associated with an autoimmune response, or pancreatic beta cells). In some embodiments, the immune cell binding agent may bind to a T cell, B cell, macrophage, natural killer cell, fibroblast, regulatory T cell, regulatory immune cell, neural stem cell, or mesenchymal stem cell and may recruit a T cell, B cell, macrophage, natural killer cell, fibroblast, regulatory T cell, regulatory immune cell, neural stem cell, or mesenchymal stem cell to a PD-L1 positive cell. For example, an immune cell targeting agent that binds CD3, 4-1BB, CD28, or CD137 can recruit T cells to PD-L1 positive cells. In another example, an immune cell targeting agent that binds CD89 can recruit macrophages to PD-L1 positive cells. In another example, an immune cell targeting agent that binds CD16 can recruit natural killer cells to PD-L1 positive cells. In another example, an immune cell targeting agent that binds CD25 can recruit regulatory T cells to PD-L1 positive cells. In another example, an immune cell targeting agent that binds CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX40, PD-1, CTLA-4, or STRO-1 can recruit mesenchymal stem cells or other immune cells to PD-L1 positive cells. An example of an immune cell targeting agent that binds CD3 may comprise the sequence of SEQ ID NO. 122 or SEQ ID NO. 442-SEQ ID NO. 471. An example of an immune cell targeting agent that binds CD28 may comprise the sequence of SEQ ID NO:472-SEQ ID NO: 481. An example of an immune cell targeting agent that binds CD25 may comprise the sequence of SEQ ID NO:482-SEQ ID NO: 491. Examples of immune cell targeting agents are provided in table 2.

TABLE 2 exemplary immune cell targeting agents

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Immune cell targeting agents (e.g., binding moieties that bind CD3, 4-1BB, CD28, CD137, CD89, CD16, CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX40, PD-1, CTLA-4, or STRO-1) can be complexed with PD-L1 binding peptides via a linker (e.g., a peptide linker). In some embodiments, the immune cell targeting agent and the PD-L1 binding peptide may form a single polypeptide chain. In some embodiments, the immune cell targeting agent and the PD-L1 binding peptide may be complexed by forming a heterodimer via a heterodimerization domain. The immune cell targeting agent can be linked or fused to a first heterodimerization domain and the PD-L1 binding peptide can be linked or fused to a second heterodimerization domain. The first heterodimerization domain can bind to the second heterodimerization domain to form a heterodimeric complex comprising an immune cell targeting agent and a PD-L1 binding peptide. For example, the PD-L1 binding peptide can be linked or fused to an Fc "mortar" peptide (e.g., SEQ ID NO: 124) and the immune cell targeting agent can be linked or fused to an Fc "mortar" peptide (e.g., SEQ ID NO: 125). In another example, the PD-L1 binding peptide can be linked or fused to an Fc "mortar" peptide (e.g., SEQ ID NO: 125) and the immune cell targeting agent can be linked or fused to an Fc "pestle" peptide (e.g., SEQ ID NO: 124). An example of a PD-L1 binding moiety of a heterodimeric T cell cement can comprise the sequence of SEQ ID NO:119 or SEQ ID NO: 120. An example of a CD 3-binding immune cell targeting moiety of a heterodimeric T cell cement can comprise the sequence of SEQ ID No. 123. In some embodiments, a PD-L1 binding peptide (e.g., any of SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO: 554-567) can form a heterodimer with an immune cell binding moiety via a heterodimerization domain provided in Table 3. For example, the PD-L1 binding peptide can be fused to chain 1 of the Fc pair (e.g., SEQ ID NO: 126) and the immune cell binding moiety can be fused to chain 2 of the Fc pair (e.g., SEQ ID NO: 127). In another example, the PD-L1 binding peptide can be fused to chain 2 of the Fc pair (e.g., SEQ ID NO: 129) and the immune cell binding moiety can be fused to chain 1 of the Fc pair (e.g., SEQ ID NO: 128). It will be appreciated that one "chain" of a pair of heterodimerization domains represented by "pair" in table 3 (e.g., chain 1 and chain 2 of a given pair) may be fused to an immune cell binding moiety and the other "chain" fused to a PD-L1 binding peptide, and vice versa, forming a heterodimer.

TABLE 3 exemplary heterodimerization Domain

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In some cases, both the PD-L1 binding peptide and the immune cell targeting agent may be presented as dimers, for example, by placing or locating the PD-L1 binding peptide at the N-or C-terminus of the homodimeric Fc fusion and placing or locating the immune cell targeting agent at the other end (C-or N-terminus) of the homodimeric Fc fusion. Exemplary homodimer Fc and anti-CD 3 scFv of SEQ ID NO. 1 (EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL; SEQ ID NO: 122) are shown in SEQ ID NO. 121. Additional examples of homodimer Fc fusions are provided in SEQ ID NO:438-SEQ ID NO: 441. Examples of components that can homodimerize or heterodimerize to form a bispecific immunocytocement are provided in table 4. For example, an Fc mortar component (e.g., SEQ ID NO:119 or SEQ ID NO: 120) that binds PD-L1 can heterodimerize with an anti-CD 3 Fc pestle component (e.g., SEQ ID NO: 123) to form BiICE that binds PD-L1/CD 3.

TABLE 4 exemplary BiICE Components

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In some embodiments, the immune cell targeting agent can be a single chain variable fragment (scFv), cysteine dense peptide, high affinity multimer, kong Nici domain, affibody, adestin protein, nano phenanthrene Ding Danbai, fenobody, β -hairpin, staple peptide, bicyclic peptide, antibody fragment, protein, peptide fragment, binding domain, small molecule, or nanobody that binds to an immune cell target (e.g., CD3, 4-1BB, CD28, CD137, CD89, CD16, CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX, PD-1, CTLA-4, or STRO-1).

After delivery, immune cells (e.g., T cells, B cells, macrophages, or natural killer cells) can inactivate, inhibit, kill, or protect PD-L1 positive cells. The function of the bispecific immunocyte cement may depend on the type of immunocyte recruited. In some embodiments, immune cells (e.g., regulatory T cells) can inhibit PD-L1 positive cells (e.g., PD-L1 positive T cells). For example, bispecific immunocyte cements comprising CD25 binding agents can recruit regulatory T cells to PD-L1 positive T cells associated with an autoimmune response and inhibit T cells.

The targeted immune cells (e.g., T cells, B cells, macrophages, or natural killer cells) can interact with the PD-L1 expressing cells through the immune cell binding moiety in the biece, creating an energetically favorable interface that brings the immune cell cement into close proximity or sufficiently narrow proximity to the immune synapse of the cell (e.g., the targeted cancer cell). The immune synapse may be sufficiently narrow to enable signal exchange between a targeted cell engaged by the PD-L1 binding peptide and a targeted immune cell engaged by the immune cell binding moiety, thereby enhancing an immune response against a cancer cell. For example, a bispecific PD-L1 binding peptide-CD 3 biece molecule (e.g., a CDP comprising binding PD-L1 complexed with a CD3 binding moiety) can bind to CD3 on immune cells and PD-L1 on target cells, creating an energetically favorable interface. The space between an immune cell and a target cell is called an immune synapse, and in normal T cells it is driven primarily by a complex between a T Cell Receptor (TCR) on the T cell and a peptide-MHC complex on the target cell. In some embodiments, the immune synapse may be used as a kinase or a mobile synapse. The width of the synapse is the distance between the immune cell and the cell membrane of the target cell. In some embodiments, for example in TCR-driven synapses, the width of the synapse may be about 15nm. In some embodiments, the synapses may have a width of about 3nm to about 25nm, about 5nm to about 20nm, or about 10nm to about 15nm. In some embodiments, the synapses may have a width of about 3nm, about 5nm, about 8nm, about 10nm, about 13nm, about 15nm, about 18nm, about 20nm, about 23nm, or about 25nm. In some embodiments, a synapse may have a width of less than 3nm, less than 5nm, less than 8nm, less than 10nm, less than 13nm, less than 15nm, less than 18nm, less than 20nm, less than 23nm, less than 25nm, less than 30nm, less than 35nm, less than 40nm, less than 45nm, less than 50nm, less than 55nm, less than 60nm, less than 65nm, less than 70nm, less than 75nm, less than 80nm, less than 85nm, less than 90nm, less than 95nm, or less than 100nm. The biece molecule may induce immune synapses whose size depends on the size of the biece and the binding site on the respective targets (e.g., immune cells and cancer cells). For example, larger molecules with more space between binding entities will produce larger synapses. The size of the immune synapse may determine the efficacy of T cell responses such as degranulation rate, kinetics of activation and depletion, and T cell mobility in the tumor microenvironment. BiICE molecules containing smaller binding moieties (e.g., CDPs) can form smaller immune synapses than bispecific molecules containing larger binding moieties (e.g., antibodies). In some embodiments, smaller synapses formed between T cells and cancer cells may produce more efficient T cell killing reactions due to the closer proximity of T cells to cancer cells. Interactions between a CDP that binds PD-L1 and PD-L1 (e.g., between SEQ ID NO:1 and PD-L1) may bring the ends of the CDP close to the surface of PD-L1. In contrast, scFv may bind to PD-L1 in an orientation such that the ends of scFv may be furthest 5nm from the PD-L1 surface, resulting in an immune synapse diameter up to 5nm greater than that formed with the CDP binding to PD-L1. An increase in synaptic diameter of 5nm may have significant biochemical or physiological consequences, as a typical immune cell synapse is about 15nm. Narrower synapses, such as those formed by BiICE containing a CDP that binds PD-L1, may trigger an effective T cell killing response. In some embodiments, the immune synapse of the PD-L1 binding peptide complex (e.g., a biece containing a CDP that binds PD-L1) is about 3nm, about 5nm, about 8nm, about 10nm, about 13nm, about 15nm, about 18nm, about 20nm, about 23nm, or about 25nm. In some embodiments, the immune synapse of the PD-L1 binding peptide complex is no greater than 3nm, no greater than 4nm, no greater than 5nm, no greater than 6nm, no greater than 7nm, no greater than 8nm, no greater than 9nm, no greater than 10nm, no greater than 13nm, no greater than 15nm, no greater than 18nm, no greater than 20nm, no greater than 23nm, no greater than 25nm, no greater than 30nm, no greater than 35nm, no greater than 40nm, no greater than 45nm, no greater than 50nm, no greater than 60nm, no greater than 65nm, no greater than 70nm, no greater than 75nm, no greater than 80nm, no greater than 85nm, no greater than 90nm, no greater than 95nm, or no greater than 100nm.

Chimeric antigen receptor

The PD-L1 binding peptides of the disclosure (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) can be complexed with one or more components of a chimeric antigen receptor to form a chimeric antigen receptor that binds PD-L1. In some embodiments, the PD-L1 binding peptide may be linked or fused to a transmembrane domain, an cytoplasmic domain, a heavy chain variable domain, a light chain variable domain, or a combination thereof of a Chimeric Antigen Receptor (CAR). In some embodiments, the PD-L1 binding peptide may replace a single chain variable fragment (scFv) of a chimeric antigen receptor to form a chimeric antigen receptor that binds PD-L1 (CAR that binds PD-L1). For example, a PD-L1-binding CAR can comprise a PD-L1 binding peptide (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567), a linker (e.g., SEQ ID NO:154-SEQ ID NO:241 or SEQ ID NO: 433), a transmembrane domain, and an intracellular co-stimulatory domain. In some embodiments, the nucleotide sequence encoding a chimeric antigen receptor that binds PD-L1 may be expressed in immune cells (e.g., T cells). The chimeric antigen receptor that binds PD-L1 can be expressed on the surface of immune cells. In some embodiments, a chimeric antigen receptor that binds PD-L1 can function by recruiting T cells to PD-L1 positive cells (e.g., PD-L1 positive cancer cells) and killing or inactivating PD-L1 positive cells.

Peptide oligonucleotide complexes

In some embodiments, the active agent may comprise an oligonucleotide. PD-L1 binding peptides (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) can be complexed with oligonucleotides to form peptide oligonucleotide complexes, also known as peptide-nucleotide agent conjugates, peptide oligonucleotide complexes, or peptide target binding agent complexes, which can comprise peptides complexed with nucleotides (e.g., oligonucleotides). The peptide of the peptide oligonucleotide complex may comprise a PD-L1 binding peptide as described herein. In some embodiments, the peptide may be a PD-L1 binding peptide (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567). The PD-L1 binding peptide of the peptide oligonucleotide complex may mediate binding of the peptide oligonucleotide complex to PD-L1, which may facilitate endocytosis or transcytosis of the peptide oligonucleotide complex, across a cell barrier or into a cancer cell or tissue. For example, a peptide oligonucleotide complex comprising a PD-L1 binding peptide may cross a cell membrane, allowing the nucleotides of the peptide oligonucleotide complex to interact with various cytosolic or nuclear components (e.g., genome DNA, ORF, mRNA, pre-mRNA or DNA). In some embodiments, the peptide oligonucleotide complex comprising a PD-L1 binding peptide can cross the cell membrane by being endocytosed into the cell. The PD-L1 binding peptide of the peptide oligonucleotide complex may be a pH-dependent PD-L1 binding peptide engineered to have a higher binding affinity for PD-L1 at extracellular pH (e.g., pH 7.4) and a lower binding affinity at endosomal pH (e.g., pH 5.5 or pH 6.5).

The nucleotides of the peptide oligonucleotide complex may be a target binding agent comprising single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or a combination thereof. As used herein, the term "nucleotide" may refer to an oligonucleotide or polynucleotide molecule or a single nucleotide base. For example, the nucleotides of the peptide complex may comprise DNA or RNA oligonucleotides. In some embodiments, the nucleotide may be a small interfering RNA (siRNA), a microrna (miRNA or miR), an anti-miR, an antisense RNA, an antisense oligonucleotide (ASO), a complementary RNA, a complementary DNA, an interfering RNA, a micronuclear RNA (snRNA), a spliceosome RNA, an inhibitory RNA, a nuclear RNA, an oligonucleotide complementary to a Natural Antisense Transcript (NAT), an aptamer, a vacancy mer, a splice blocking ASO, or a U1 adaptor. For example, the nucleotide of the peptide oligonucleotide complex may comprise any of the sequences SEQ ID NO. 366-396 or a sequence complementary to a portion of any of the sequences provided in the open reading frames set forth in SEQ ID NO. 397-SEQ ID NO. 430 or SEQ ID NO. 549 or Table 17. In some embodiments, the nucleotide may be an siRNA that inhibits translation of the target mRNA by promoting degradation of the target mRNA. In some embodiments, the nucleotide may be an siRNA that inhibits translation of the target mRNA by promoting cleavage or destabilization of the target mRNA. In some embodiments, the nucleotide may be an aptamer that binds to the target protein, thereby inhibiting protein-protein interactions with the target protein, inhibiting enzymatic activity of the target protein, or activating the target protein.

Examples of structures of various peptide oligonucleotide complexes (e.g., CDP-oligonucleotide complexes containing alternative and non-conventional bases) are illustrated in FIG. 23. Examples of oligonucleotides include aptamers, empty-mers, anti-miR, siRNA, splice-blocking ASO, and U1 adaptors. The peptide portion of the peptide oligonucleotide complex (e.g., CDP of the CDP-oligonucleotide complex) can be used to direct a nucleotide sequence (e.g., an oligonucleotide of the CDP-oligonucleotide complex) to a particular tissue, target, or cell.

In some embodiments, the peptide oligonucleotide complex binds PD-L1 with an affinity of no greater than 10nM, 5nM, 1nM, 800pM, 600pM, 500pM, 400pM, 300pM, 250pM, or 200 pM. In some embodiments, the affinity is the same or similar at pH 7.0 as at pH7.4, at pH 6.5 as at pH7.4, at pH 6.0 as at pH7.4, or at pH 5.5 as at pH 7.4. In some embodiments, the affinity is within ±1nM, ±3nM, ±5nM, ±10nM, ±30pM, ±50pM, ±100pM, ±300pM, ±500pM, or ±1000pM when compared at pH 7.0 and pH7.4, pH 6.5 and pH7.4, pH 6.0 and pH7.4, or pH 5.5 and pH 7.4. In some embodiments, the relative difference in affinity is within 1-fold, 2-fold, 3-fold, 5-fold, or 10-fold when compared at pH 7.0 and pH7.4, pH 6.5 and pH7.4, pH 6.0 and pH7.4, or pH 5.5 and pH 7.4. In some aspects, the affinity of the peptide oligonucleotide complex for binding to a PD1 molecule is higher at a higher pH than at a lower pH. In some aspects, the higher pH is pH7.4, pH 7.2, pH 7.0, or pH 6.8. In some aspects, the lower pH is pH 6.5, pH 6.0, pH 5.5, pH 5.0 or pH 4.5. In some aspects, the affinity of the peptide oligonucleotide complex for PD-L1 is higher at pH 7.4 than at pH 6.0. In some aspects, the affinity of the peptide oligonucleotide complex for PD-L1 is higher at pH 7.4 than at pH 5.5. In some aspects, the target binding peptide is capable of having a dissociation constant (K) of no greater than 100nM, no greater than 20nM, no greater than 10nM, no greater than 5nM, no greater than 2nM, no greater than 1nM, no greater than 0.5nM, no greater than 0.2nM, no greater than 1nM, or no greater than 0.1nM at pH 7.4 _D ) Binds to the target molecule. In some aspects, the target binding peptide is capable of binding to a target molecule (K) at a pH of 5.5 with a dissociation constant of no less than 1nM, no less than 2nM, no less than 5nM, no less than 10nM, no less than 20nM, no less than 50nM, no less than 100nM, no less than 200nM, or no less than 500nM _D ). In some aspects, the affinity of the peptide oligonucleotide complex for PD-L1 at pH 7.4 is at least 1.25-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10,000-fold greater than the affinity of the peptide oligonucleotide complex for PD-L1 at pH 5.5.

The peptide oligonucleotide complexes of the present disclosure can include nucleotides and nucleotide variants within the peptide oligonucleotide complexes, wherein the nucleotide moiety targets a particular target molecule for modulation. Modulation of the target molecule may comprise degradation, inhibition of translation, reduction of expression, increased expression, enhancement of binding interactions (e.g., protein-protein interactions), or inhibition of binding interactions (e.g., protein-protein interactions). Nucleotide sequences useful for the nucleotide portion of the peptide oligonucleotide complex are disclosed herein, e.g., targeting nucleotide sequences set forth in SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:549, table 10 or Table 17 (e.g., DNA or RNA molecules) or nucleotide sequences complementary thereto, or targeting nucleotide sequences encoding or complementary to the proteins set forth in SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:549, table 10 or Table 17 (e.g., DNA or RNA molecules), or nucleotide sequences otherwise described herein. Examples of nucleotide sequences that can be used for the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO 366-SEQ ID NO 396 and SEQ ID NO 492-SEQ ID NO 545. As disclosed herein, the nucleic acid sequences, variants, and properties of nucleic acids used in the nucleic acid portion of the peptide oligonucleotide complex may be referred to as the nucleic acids of the present disclosure, the nucleotides of the present disclosure, or similar terms. It is to be understood that such nucleic acids or nucleotides are described in the context of the disclosed peptide oligonucleotide complexes, e.g., comprising within the peptide oligonucleotide complexes a single strand (ssDNA, ssRNA), double strand (dsDNA, dsRNA), or a combination of single and double strands (e.g., having a mismatched sequence, hairpin or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), oligonucleotides complementary to a Native Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, vacancy mer, anti-miR, splice-blocking ASO, or U1 adaptor, describing corresponding changes, functions, and uses.

In some embodiments, the nucleotide sequence (e.g., a target binding agent capable of binding a target molecule) is single-stranded (ssDNA, ssRNA), double-stranded (dsDNA, dsRNA), or a combination of single-and double-stranded (e.g., having a mismatched sequence, hairpin, or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), an oligonucleotide complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, empty mer, anti-miR, splice-blocking ASO, or U1 adaptor. Peptides according to the present disclosure may be conjugated, linked or fused to such nucleotide sequences to prepare peptide oligonucleotide complexes. In addition, other active agents described herein (e.g., small molecules, proteins, or peptide active agents) can be conjugated, linked, complexed, or fused to such nucleotide sequences, peptides, or peptide oligonucleotide complexes to form peptide oligonucleotide complex conjugates.

The nucleotides (e.g., nucleotides of a peptide oligonucleotide complex) may be fully or partially reverse-complementary to all or a portion of a target molecule (e.g., a target DNA or RNA sequence). In some embodiments, the target molecule expresses or encodes a protein (e.g., mRNA encoding a protein associated with a disease). In some embodiments, the nucleotide may be fully or partially reverse-complementary to a portion of the open reading frame encoding a gene or protein of interest. In some embodiments, the nucleotide may be reverse-complementary to any portion of the RNA or open reading frame encoding the transcript or protein of interest. SEQ ID NO. 397-SEQ ID NO. 430 and SEQ ID NO. 549 provide examples of sequences of target molecules that may be used as target binding nucleotides as described herein along any portion of their length. In some embodiments, the target molecule may comprise a fragment of any of the sequences provided in table 17 along any portion of its length. In some embodiments, the target molecule may comprise a fragment of any of the sequences provided in SEQ ID NO 397-SEQ ID NO 430 or SEQ ID NO 549. In some embodiments, the target molecule may comprise a sequence in which one or more T residues are replaced with U or one or more U residues are replaced with T.

A number of techniques are available for generating a therapeutically active nucleotide sequence for use in the peptide oligonucleotide complexes disclosed herein (e.g., SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) comprising a PD-L1 binding peptide. Several examples of molecules with clinical or advanced clinical development and can be used for the nucleotide moiety within the peptide oligonucleotide complexes described herein. The nucleotides of the peptide oligonucleotide complex can bind to a target molecule (e.g., target DNA, RNA, or protein) and modulate the activity of the target molecule. In this way, the nucleotides may act as target binding agents, also referred to as targeting agents. Examples of nucleotides that can be used as target binding agents include nucleotide antisense RNAs, complementary RNAs, inhibitory RNAs, interfering RNAs, nuclear RNAs, antisense oligonucleotides, micrornas, oligonucleotides complementary to natural antisense transcripts, small interfering RNAs, micronuclear RNAs, aptamers, gapmers, anti-miR, splice-blocking antisense oligonucleotides, and U1 adaptors.

Nucleotides (e.g., oligonucleotides targeted to specific sequences to regulate them) can enter a cell by complexing with a PD-L1 binding peptide to form a PD-L1 binding peptide oligonucleotide complex. The PD-L1 binding peptide oligonucleotide complex may then be endocytosed by PD-L1 or may enter the cell by other mechanisms. Oligonucleotides that are complexed or uncomplexed with PD-L1 binding peptides (e.g., after cleavage of the linker) may slowly exit the endosome or lysosome over time, either through an inactive mechanism or through an endosomal escape mechanism or through other mechanisms. The peptide oligonucleotide complex may leave the endosome or lysosome. Fragments or cleavage products of the peptide oligonucleotide complexes may leave the endosome or lysosome. The oligonucleotide, peptide oligonucleotide complex, or any fragment thereof may enter the cytosol and may enter the nucleus.

The possible mechanism of action of the oligonucleotides is shown in FIG. 22. In one embodiment, upon entry into the nucleus, the oligonucleotide may (1) bind directly to the mRNA structure and prevent maturation (e.g., capping or splicing) of the targeting sequence, (2) modulate alternative splicing of the targeting sequence, (3) and recruit RNaseH1 to induce cleavage of the targeting sequence. In another embodiment, oligonucleotides in the cytoplasm can bind directly to the target mRNA and spatially block ribosomal subunits from attaching during translation and/or running along the mRNA transcript, thus resulting in a translational deletion of the targeting sequence. In another embodiment, the oligonucleotides may also be designed to (4) bind directly to microrna (miRNA) sequences or Natural Antisense Transcript (NAT) sequences, such that each prevents miRNA and NAT from inhibiting their own specific RNA targets, which ultimately results in reduced degradation or increased translation of the miRNA or NAT targeted sequence or sequences themselves. In another embodiment, siRNA (which can target a specific sequence and modulate expression of the target sequence) can alternatively be used to bind and modulate the targeting sequence in the cytoplasm (5), engaging an RNA-induced silencing complex (RISC), which is a multiprotein complex comprising a small interfering RNA (siRNA) or microrna (miRNA), using the siRNA or miRNA as a template to recognize complementary mRNA of the targeting sequence. When it finds a complementary strand, its RNase domain cleaves the targeting sequence. In another embodiment, an aptamer (e.g., a nucleotide that modulates a particular protein or other target) may alternatively be used to bind and modulate the target (6). Aptamers (e.g., extracellular or intracellular) can function by directly binding and modulating the activity of a protein target, e.g., by forming aptamer-protein interactions rather than by base pairing or hybridization interactions.

For example, conventional ASOs or antisense oligonucleotides are typically 18-30 nucleotides (nt) in length. Several ASO treatment strategies exist, two of which (differing in terms of the target RNA interference mechanism) are further described. The first ASOs were sometimes referred to as "vacancy mers" because they had a central region with DNA-based sugar nucleotides, typically (but not always) flanked by non-DNA sugar nucleotides that were more resistant to nucleases. The length of the DNA region is at least 4nt, but is typically greater than 6, which results in the ligation of the DNA/RNA hybrid with the RNaseH endonuclease to cleave the target RNA. Among the clinically approved vacancy polymers are fomivirsen (fomivirsen) and Mi Bomei (mipomersen). In some embodiments, the DNA region of the vacancy mer can comprise from about 4 to about 30, from about 4 to about 25, from about 4 to about 20, from about 4 to about 15, from about 4 to about 10, from about 6 to about 30, from about 6 to about 25, from about 6 to about 20, from about 6 to about 15, or from about 6 to about 10 nucleotide residues. In some embodiments, the non-DNA region of the vacancy-polymer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 3' of the DNA region. In some embodiments, the non-DNA region of the vacancy-polymer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 5' of the DNA region. Examples of vacancy polymers are provided in table 5.

TABLE 5 exemplary vacancy mer sequences

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The second conventional ASO is only used to bind to the target transcript, but does not induce RNase degradation, so DNA-based sugars are not used. In contrast, binding is intended to disrupt the processing of mature mRNA. One such activity relies on binding to the mRNA at or near the splice site to drive a specific splice subtype in the target RNA, thereby modulating the target RNA by disrupting mRNA splicing and causing exon skipping. Among other known names, these are commonly referred to as "splice-blockers" or "splice-blockers" ASOs. One example is etoricsen (eteplirsen), which aims at altering the splicing of the DMD (myodystrophin) gene in patients with duchenne muscular dystrophy (Duchenne Muscular Dystrophy), correcting mutations that would otherwise produce a truncated and nonfunctional myodystrophy protein by splicing mutant exons and creating a different truncated (but functional) protein appearance.

Another example is siRNA molecules that interact specifically with classical RNAi pathways (RISC complexes) to drive cleavage or steric blocking of hybridized transcripts; whether the match is perfect (cleavage) or imperfect but still stable (blocking) depends on whether the cleavage or blocking is complete. The length is typically double-stranded RNA, with an overlap region of 19-22, with two additional nucleotides at the 3' end of each strand. Chemistry is primarily RNA-based with some DNA-based sugar at the 3' overhang. Clinical examples include patricia (patisiran) (target TTR) and Ji Foxi blue (target ALAS 1). In some embodiments, the overlapping region of the siRNA can comprise about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 22, about 10 to about 21, about 10 to about 20, about 15 to about 40, about 15 to about 35, about 15 to about 30, about 15 to about 25, about 15 to about 22, about 15 to about 21, about 15 to about 20, about 17 to about 40, about 17 to about 35, about 17 to about 30, about 17 to about 25, about 17 to about 22, about 17 to about 21, about 17 to about 20, about 18 to about 40, about 18 to about 35, about 18 to about 30, about 18 to about 25, about 18 to about 22, about 18 to about 20, about 19 to about 40, about 19 to about 35, about 19 to about 30, about 19 to about 25, about 19 to about 22, or about 19 to about 19. In some embodiments, the overhang region can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues. Examples of sirnas are provided in table 6.

TABLE 6 exemplary siRNA sequences

Another example is an anti-miR. anti-miR can act as a spatial blocker designed for mirnas that will block RISC complexes loaded with specific disease-related mirnas without cleavage by the RNase subunit of the RISC complex. One clinical example is Mi Lawei sen (Mi Lawei sen), a 15 base oligonucleotide with a mixture of DNA and LNA sugars, targeting miR-122 in patients with hepatitis c. The anti-miR nucleotides can be long enough to specifically and stably anneal to the target miR, but the length of the sequence can vary. For example, the length of the anti-miR may be up to about 21nt, corresponding to the maximum size loaded into RISC. In some embodiments, the anti-miR nucleotides can comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues.

Another example is a U1 adapter, which has two portions. One anneals to the U1-snRNA of the U1-snRNP complex and the other binds to the target RNA, bringing U1-snRNP to the polyadenylation site and inhibiting polyadenylation; the lack of a poly A tail can lead to mRNA degradation. The U1 binding region is at least 10nt, but at most 19nt. The target binding region can be about 15nt to about 25nt. LNA and 2' -O-methyl sugar were used in large amounts in chemistry in early studies. In some embodiments, the U1 binding region can comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues. In some embodiments, the target binding region can comprise about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 22, about 10 to about 21, about 10 to about 20, about 15 to about 40, about 15 to about 35, about 15 to about 30, about 15 to about 25, about 15 to about 22, about 15 to about 21, about 15 to about 20, about 17 to about 40, about 17 to about 35, about 17 to about 30, about 17 to about 25, about 17 to about 22, about 17 to about 21, about 17 to about 20, about 18 to about 40, about 18 to about 35, about 18 to about 30, about 18 to about 25, about 18 to about 22, about 18 to about 20, about 19 to about 40, about 19 to about 35, about 19 to about 30, about 19 to about 25, about 19 to about 19, about 19 to about 22, or about 19 to about 20 nucleotide residues.

Another example of a nucleotide of the present disclosure is an aptamer. Aptamers disrupt target activity using a mechanism different from the other nucleotides described herein that form base pairing interactions with the target nucleotide. An aptamer is a nucleic acid that forms a secondary structure (e.g., a single stranded nucleic acid base pairs with itself when folded, creating a loop at a different position). Aptamers can be screened for interactions with target proteins. The aptamers may have different nucleotide chemical properties and may include mixtures of conventional RNA and/or DNA sugars and modified sugars (e.g., 2 '-O-methyl (2' -O-Me) RNA or 2 '-fluoro (2' -F) RNA sugars). For example, a clinically approved aptamer, pegaptanib (an aptamer that binds VEGF), has a mixture of 2 '-O-methyl (2' -O-Me) RNA and 2 '-fluoro (2' -F) RNA sugars with conventional RNA and DNA sugars. The aptamer sequence may be long enough to form a stable secondary structure (e.g., by intramolecular base pairing), but the length may vary. In some embodiments, the aptamer sequence may comprise about 20nt to about 40nt. For example, experiments to identify pipadatinib have used oligonucleotides ranging in length from 20 to 40nt. Shorter nucleotides (e.g., sequences shorter than about 40 nt) may be advantageous because longer oligonucleotides may complicate nucleotide synthesis or participate in the interferon response pathway. In some embodiments, the aptamer may comprise from about 15 to about 60, from about 15 to about 50, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 20 to about 60, from about 20 to about 50, from about 20 to about 40, from about 20 to about 35, from about 20 to about 30, from about 25 to about 60, from about 25 to about 50, from about 25 to about 40, from about 25 to about 35, or from about 25 to about 30 nucleotide residues.

Nucleotides may be designed for use in the peptide nucleotide complexes of the present disclosure. In some embodiments, the nucleotide that modifies processing, translation, or other RNA function (e.g., a vacancy mer, a splice blocker, an siRNA, an anti-miR, or a U1 adapter) has one or more of the following properties: (a) a length of 8-50nt, but preferably a length of 12-30 nt. It should be understood that any length of nucleotide (nt) within the foregoing ranges may be used; (b) Cross species homology (e.g., by targeting highly conserved motifs) is often an ideal feature, but is not essential for activity or clinical development; (c) Avoiding a common SNP in humans unless the SNP involves a disease pathology (e.g., an allele-specific oligonucleotide) is often an ideal feature, but is not necessary for activity or clinical development; (d) Gene specificity (minimal homology to other sequences; e.g., one sequence may have 3 or more mismatches with the other sequence). (e) Avoiding predicted secondary structures in the oligonucleotide and target region (software tools are available to screen on-computer for the formation of such secondary structures); (f) Higher G/C content may be preferred because G/C-rich sequences (e.g., CCAC, TCCC, GCCA) may help to increase affinity of nucleotides for their targets, while a/T-rich sequences (e.g., TAA) or rounds of 4+g (ggggg) may exhibit low or result in formation of a structure (G-quadruplex). The oligonucleotide sequence may be 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary or matched to the target sequence. In some cases, oligonucleotides with 100% complementarity will result in degradation of the target RNA. In some cases, oligonucleotides with complementarity below 100% may not result in degradation of the target RNA, but may prevent translation and production of the encoded protein.

In some embodiments, the vacancy-polymer has one or more of the following characteristics: (a) a length of 12-30 nt. It should be understood that any length of nucleotide (nt) within the foregoing ranges may be used. (b) The target site is located anywhere in the pre-mRNA, including the UTR, exon, or intron. (c) a central DNA region: a minimum of 4 consecutive DNA nucleotides, typically 10 or more are used. Due to RNaseH recognition requirements, manual substitution of the 2' site (e.g., 2' -O-methyl [2' -O-ME ] or 2' -O-methoxyethyl [2' -O-MOE ]) is intolerable. (d) flanking regions: may be a DNA or RNA based sugar. 2' substitutions, such as 2' -O-ME or 2' -O-MOE, are tolerated. Ligation Nucleic Acid (LNA) and morpholino (phosphodiamide morpholino oligonucleotide) chemistry is also acceptable in flanking regions. (e) The backbone may be a natural Phosphodiester (PO) or a non-natural Phosphorothioate (PS) linkage. One clinical example is Fumivirgine, a 21nt vacancy mer in which the entire oligonucleotide is PS-backbone DNA. Another example is miphene, a 20nt vacancy mer in which the entire backbone is PS-linked, the central region uses DNA sugars, flanked by 2' -O-MOE modified RNA. For both examples, all C bases are 5-methyl-C, but this is not a strict requirement for RNase H1 ligation. Similarly, phosphorothioate chemistry may be used.

In some embodiments, the steric blocker has one or more of the following characteristics; (a) Since the molecule does not need to bind to RNase H or any other enzyme, backbone and sugar chemistry can be more diverse; (g) The target site of the nucleotide is complementary to one or more splice sites in the target RNA. One clinical example is eletrogen, a 30nt splice-blocking ASO, in which morpholino (phosphodiamide morpholino oligonucleotide) chemistry is used for the entire oligonucleotide. Another clinical example is sodium norcinacate (nusinesen), an 18nt ASO with a backbone that is fully linked to PS and uses 2' -O-MOE RNA chemistry. All C bases are 5-methyl-C, but this is not a strict requirement for RNase H1 ligation. Similarly, phosphorothioate chemistry may be used.

In some embodiments, the siRNA has one or more of the following properties: (a) The length of each strand may be between 15 and 25nt (between 13 and 23nt overlap, respectively), or up to 25nt (23 nt overlap), but 21nt (19 nt overlap) is common. It should be appreciated that any length of nucleotide (nt) overlap within the foregoing ranges may be used; (b) Complementary to a sequence of typically but not limited to 21nt in length in a target mRNA that typically but not limited to beginning with "AA"; (c) The target site is desirably present in mature spliced mRNA, because the RISC complex for RNA cleavage is primarily cytosolic; (d) Sequences within 100nt of the start site of the mRNA are preferably, but not completely, avoided because transcripts of the start site are more likely to be occupied by RNA polymerase; (e) Successful siRNA constructs will typically have more G/C at the 5 'end of the sense strand and more A/T at the 3' end of the sense strand, with a G/C content of about 30-60%.

In some embodiments, the anti-miR (anti-miRNA) has one or more of the following properties: (a) Perfect match to the target sequence (particularly the 5' end of the miRNA guide strand); (b) The length may vary or may even be greater than the length of the mature guide strand. Screening for effective anti-miR constructs can begin with the shortest sequence that achieves specificity (no off-target homology) and then increase the length therefrom to empirically determine the ideal minimum length for strong miRNA inhibition; (c) 2 '-sugar modifications (2' -O-Me, 2'-O-MOE, 2' -F) and LNA sugars are common. The sugar may be a mixture. One clinical example of an anti-miR is Mi Lawei sen (miravirsen), which uses a mixture of DNA and LNA sugars; PS bonds in the (d) backbone are common. PS bonds may decrease affinity, but sugar modifications may increase affinity.

In some embodiments, the aptamer has one or more of the following properties: (a) The length of the aptamers can vary widely, as there are no biocomposites (e.g., RISC) that interact with them to function. Although composed of nucleic acids, they are functionally more like proteins (e.g., bind to target proteins, etc.). The minimum length can be determined empirically to maintain sufficient stability of the in-chain hybrid folding into a secondary structure, the upper limit of size being limited only by pharmacology, as longer sequences have a higher risk of participating in inflammatory pathways. Aptamer screening typically begins with a library of length 20-40nt (excluding flanking regions required for library amplification during screening); (b) Since they interact by secondary structures rather than base pairing interactions, their base patterns are almost unlimited, as secondary structures are not only ideal but also critical to their function. Each target may be empirically designed; (c) Selection is typically performed by exponential enriched ligand system evolution (SELEX): random or semi-random sequences between flanking regions of the binding primer are exposed to a target of interest on a solid substrate. The pooled oligonucleotide mixture is washed away from the substrate, leaving only the sequence that interacts with the target, and the binding sequence is then eluted and amplified by PCR. (d) Commonly used sugar modifications include 2 '-fluoro (2' -F), 2'-O-MOE, and 2' -O-Me, but other chemicals including, but not limited to, LNA and Unlocking Nucleic Acid (UNA) are also possible; (e) The backbone is typically PO or PS, but other linkages such as methylphosphonate are also possible. One example of a clinical aptamer, pipadatinib, is entirely a PO backbone, but other examples in development use other linkages. (f) The aptamer ends are typically capped with non-natural nucleotides (e.g., 3' reverse thymidine) or biotinylated nucleotides to reduce sensitivity to nucleases; (g) Because the activity is not based on base pairing, the aptamer may be more creative by chemical modification of the base itself; these may include bases intended to induce covalent bonds with the target protein to permanently disable them; (h) Such modifications are tested after selection of active, high affinity aptamers, as unmodified bases are required for nucleic acid amplification during SELEX; (i) If the target protein is extracellular, fewer factors need to be considered than the cell penetrating capacity.

In some embodiments, other general design considerations aimed at enhancing the Pharmacokinetic (PK) properties of a nucleotide, peptide or peptide oligonucleotide complex include one or more of the following properties: (a) Conjugation to a moiety that reduces clearance or increases cellular uptake is constructed, including cholesterol or other lipids, diacylglycerols, galNAc, palmitoyl, PEG, RGD motifs, cell penetrating peptides or moieties (e.g., PD-L1 binding peptides or cell penetrating peptides as described herein). The addition of cholesterol to the peptide oligonucleotide complex can improve the biodistribution of the target tissue, increase cellular uptake by endocytosis, and alter serum pharmacokinetics.

The therapeutic activity and molecular approach of the peptide oligonucleotide complex may depend on the target molecule (e.g., DNA or RNA) to which the nucleic acid is complementary, or in the case of an aptamer, on the target molecule (e.g., protein or other macromolecule) to which it binds. The selection of targets may be categorized into one or more non-mutually exclusive categories, e.g., based on tissue targets or disease selectivity. Targets are known to have known mRNA and genomic sequences that can be used to design a variety of complementary nucleic acids for use in the peptide nucleotide complexes described herein, depending on the activity desired (e.g., gene regulation, protein degradation, reduction of cancer cell activators). Examples of targets are provided in SEQ ID NO 397-SEQ ID NO 430 or SEQ ID NO 549, table 10 and Table 17. For example, tissue targeting may include selecting a target that functions in tissue where the PD-L1 binding peptide portion of the peptide oligonucleotide complex will preferentially enter or accumulate. For example, serum proteins produced in the liver may be targeted, for example, by TTR treatment of transthyretin-related amyloid disease, or by treatment of hypercholesterolemia or cardiovascular disease by various apolipoproteins. In addition, it is possible to target proteins produced in the lung or lung tissue expressing PD-L1, for targeting inflammatory cytokines or cytokine receptors to treat pulmonary diseases (e.g., COPD), or to down-regulate receptors that determine the tropism of an airborne virus (e.g., SARS-CoV-2) (e.g., ACE 2).

Alternatively, targets that function in the region where PD-L1 binding peptide accumulates (e.g., in a cancer cell that expresses PD-L1), or that function in the case where oligonucleotides that do not complex with PD-L1 binding peptide are otherwise excluded, including in the case of a tumor, may be selected based on known expression of PD-L1 in a tissue or target cell type. Tumor targeting can be used with the peptide oligonucleotide complexes of the present disclosure because tumors typically have high levels of PD-L1 and are typically vascularized, sufficient to rapidly perfuse serum-resident PD-L1 binding peptides and their oligonucleotide cargo. Targets for the peptide oligonucleotide complexes may include oncogenes, e.g., genes that lack excess ortholog by designing the nucleic acid portion of the complex to target overexpression or tumors (i.e., normal cells function by using X or Y, tumors do not express Y, so X is targeted). In addition, disease selective targeting can be used to treat conditions where transcripts are selectively found in diseased tissue and accumulate preferentially there, to improve safety and reduce off-target effects. PD-L1 may also be expressed in tissues such as colon or other gastrointestinal tissues, skeletal muscle, adipose tissue, lymphoid tissue, soft tissue, placenta, seminal vesicles, tonsils, resting or activating T cells, B cells, dendritic cells and macrophages. Thus, the PD-L1 binding peptides of the present disclosure can be used to deliver oligonucleotides or other active agents to those tissues.

The target binding agent (e.g., a nucleotide of a peptide oligonucleotide complex) may be capable of binding to a target as set forth in SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:549, table 10 or Table 17, or a nucleotide (e.g., a DNA or RNA molecule) encoding a protein as set forth in SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:549, table 10 or Table 17, or a nucleotide as otherwise described herein. Examples of nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO 366-396 and SEQ ID NO 492-545. It is understood that any oligonucleotide of a molecule complementary to a portion of a target DNA or RNA molecule may be used. Such target binding agents may comprise the following nucleotide sequences: single strand (ssDNA, ssRNA) or double strand (dsDNA, dsRNA) or a combination of single and double strands (e.g., having a mismatched sequence, hairpin or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotides (ASO), micrornas (miRNA), oligonucleotides complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, gapmer, anti-miR, splice-blocking ASO, or U1 adaptor. Such oligonucleotides can be about 5 to 30nt in length, 10 to 25nt in length, 15 to 25nt in length, 19 to 23nt in length, or at least 10nt in length, at least 15nt in length, at least 20nt in length, at least 25nt in length, or at least 30nt in length, at least 50nt in length, at least 100 nucleotides in length, spanning any portion of the target RNA. Examples of sequences to which such oligonucleotides may bind (e.g., complement) include SEQ ID NO:397-SEQ ID NO:430 and SEQ ID NO:546-SEQ ID NO:549, or any of the genomic or ORF sequences recited in Table 17. One of skill in the art can readily design or determine the length of the target binding agent and whether the target binding agent is complementary to a reference target RNA sequence, and thus can use the chemistry of RNA and DNA to determine where such target binding agent will bind to such reference target RNA sequence, spanning any portion of the target RNA to the designed length. Thus, for any RNA target described herein, including any target or target-encoding molecule described in Table 10 and SEQ ID NO:397-SEQ ID NO:430 and SEQ ID NO:546-SEQ ID NO:549, or any genomic or ORF sequence referenced in Table 17, such target binding agents of any nt length are described.

In some embodiments, the nucleotide binds to the target molecule at a melting temperature of no less than 37 ℃ and no more than 99 ℃. In some embodiments, the nucleotide binds to the target molecule at a melting temperature of no less than 40 ℃ and no more than 85 ℃, no less than 40 ℃ and no more than 65 ℃, no less than 40 ℃ and no more than 55 ℃, no less than 50 ℃ and no more than 85 ℃, no less than 60 ℃ and no more than 85 ℃, or no less than 55 ℃ and no more than 65 ℃.

In some embodiments, the nucleotide binds the target molecule with an affinity of no greater than 500nM, no greater than 100nM, no greater than 50nM, no greater than 10nM, no greater than 1nM, no greater than 500pM, no greater than 400pM, no greater than 300pM, no greater than 200pM, or no greater than 100 pM. In some embodiments, the nucleotide binds the target molecule with an affinity of no greater than 500nM and no less than 100pM, no greater than 100nM and no less than 200pM, no greater than 50nM and no less than 300pM, no greater than 10nM and no less than 400pM, or no greater than 1nM and no less than 500 pM.

In some embodiments, the nucleotide comprises at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOS: 366-396. In some embodiments, the nucleotide comprises the sequence of any one of SEQ ID NO 366-SEQ ID NO 396, any one of SEQ ID NO 366-SEQ ID NO 396 wherein U is replaced with T, or any one of SEQ ID NO 366-SEQ ID NO 396 wherein T is replaced with U. In some embodiments, the nucleotide comprises NO more than 1, 2, 3, 4, or 5 base changes relative to the sequence of any of SEQ ID NOS: 366-396.

In some embodiments, the nucleotide is at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% reverse complementary to the target molecule. In some embodiments, the nucleotide is 100% reverse complementary to the target molecule. In some embodiments, the nucleotide comprises no more than 1, 2, 3, 4, or 5 base pair mismatches after binding to the target molecule. In some embodiments, the nucleotide comprises at least 1, 2, 3, 4, or 5 base pair mismatches after binding to the target molecule.

In some embodiments, the nucleotide may modulate the activity of the target molecule. In some embodiments, modulating the activity of the target molecule comprises reducing expression of the target molecule, increasing expression of the target molecule, reducing translation of the target molecule, degrading the target molecule, reducing the level of the target molecule, modifying processing of the target molecule, modifying splicing of the target molecule, inhibiting processing of the target molecule, reducing the level of a protein encoded by the target molecule, or blocking interaction with the target molecule. In some embodiments, expression of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%. In some embodiments, translation of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%. In some embodiments, expression of the target molecule is reduced by at least 2-fold, 4-fold, 8-fold, 10-fold, 15-fold, 16-fold, 20-fold, 32-fold, 50-fold, 64-fold, 100-fold, 128-fold, 200-fold, 256-fold, 500-fold, 512-fold, or 1000-fold. In some embodiments, translation of the target molecule is reduced by at least 2-fold, 4-fold, 8-fold, 10-fold, 15-fold, 16-fold, 20-fold, 32-fold, 50-fold, 64-fold, 100-fold, 128-fold, 200-fold, 256-fold, 500-fold, 512-fold, or 1000-fold. In some embodiments, at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the target molecules are degraded. In some embodiments, the level of protein encoded by the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%. In some embodiments, modification of splicing of a target molecule increases the level of a protein encoded by the target molecule by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.

The peptide oligonucleotide complexes of the present disclosure may comprise a nucleotide complexed with a protein (e.g., a PD-L1 binding peptide). The nucleotides may comprise single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or a combination thereof. In some embodiments, the nucleotides of the peptide oligonucleotide complex may be non-naturally occurring, also referred to as "engineered nucleotides". In some embodiments, the nucleotide may comprise a naturally occurring sequence. The nucleotide may be exogenously expressed, enzymatically synthesized in vitro, or chemically synthesized. For example, the nucleotides may be expressed in bacteria, yeast or mammalian cell lines and purified for use in the peptide oligonucleotide complexes of the present disclosure. In another example, the nucleotide may be enzymatically synthesized in vitro using RNA or DNA polymerase. In another example, the protected nucleotide may be used to chemically synthesize the nucleotide on a solid support.

One example of a chemical synthesis method that can be used to prepare nucleotides for the peptide oligonucleotide complexes of the present disclosure is phosphoramidate synthesis. Briefly, single nucleotide residues can be added sequentially from 3 'to 5' to a growing nucleotide chain by repeating the steps of deblocking (detritylation), coupling, capping, and oxidation. Phosphoramidate synthesis can be performed on a solid support such as Controlled Pore Glass (CPG) or macroporous polystyrene (MPPS). Similarly, phosphorothioates may be used.

The nucleotides of the peptide oligonucleotide complex can bind to a target molecule (e.g., target DNA, target RNA, or target protein). In some embodiments, binding of the oligonucleotide to the target molecule may alter the activity of the target molecule. For example, binding of an oligonucleotide (e.g., siRNA, miRNA, a vacancy mer, or a U1 adaptor) to a target mRNA or pre-mRNA can increase or decrease translation of the target mRNA or pre-mRNA. In another example, binding of a nucleotide to a target DNA may increase or decrease expression of a gene encoded by the target DNA. In another example, binding of a nucleotide to an RNA (e.g., transcript, precursor RNA, non-spliced RNA, nuclear RNA, a sequence complementary to NAT, or mRNA) expressed from a target DNA (e.g., gene or ORF) can increase or decrease expression of the gene encoded by the target DNA. In another example, binding of an oligonucleotide (e.g., an aptamer) to a target protein can increase or decrease the activity (e.g., enzymatic activity or binding activity) of the target protein. In some embodiments, the target molecule may be associated with a disease or disorder, and increasing or decreasing the activity of the target molecule may treat the disease or disorder.

The oligonucleotide sequence of the peptide oligonucleotide complex may be selected for its ability to bind to a target molecule or modulate the activity of a target molecule. In some embodiments, the oligonucleotide may be reverse-complementary to the target DNA or RNA molecule. For example, the siRNA oligonucleotide may be reverse-complementary to the target RNA molecule. In some embodiments, the oligonucleotide may be reverse complementary (e.g., comprise one or more mismatched base pairs) to the target DNA or RNA molecule portion. For example, the siRNA oligonucleotide may comprise a base mismatch relative to the target RNA molecule. In some embodiments, the oligonucleotide sequence may be selected based on the annealing temperature relative to the target DNA or RNA molecule. The preferred annealing temperature may be achieved by selecting the length of the nucleotide, the degree of complementarity of the nucleotide to the target molecule, the chemical nature of the nucleotide, or any combination thereof. Nucleotide sequence parameters (e.g., complementarity, annealing temperature, melting temperature, base mismatches, and binding affinity) can be calculated using any available software, such as ITD OligoAnalyzer, and the like. In some embodiments, the oligonucleotide may employ a secondary structure that binds to a target DNA, RNA, or protein molecule. For example, the aptamer may employ a secondary structure to bind to a target protein. The aptamer sequence may be selected to adopt a secondary structure that binds to the target protein. Any available software can be used to predict nucleotide secondary structure, such as RNAfold and the like. In some embodiments, the nucleotide sequence may be determined experimentally by selecting for the ability to bind a target molecule. For example, a library of nucleotides can be contacted with a target molecule, and sequences that bind to the target molecule can be identified.

In some embodiments, the nucleotide comprises a G/C content of not less than 20% and not more than 80%. In some embodiments, the nucleotide comprises a G/C content of not less than 30% and not more than 65%. In some embodiments, the nucleotide comprises a G/C content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%. In some embodiments, the nucleotide comprises a G/C content of no more than 80%, no more than 75%, no more than 70%, no more than 65%, or no more than 50%. In some embodiments, the nucleotide comprises no less than 20% and no more than 80% of the a/T content or the a/U content. In some embodiments, the nucleotide comprises no less than 30% and no more than 65% of the a/T content or the a/U content. In some embodiments, the nucleotide comprises an a/U (or a/T or a combination of a/U and a/T) content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%. In some embodiments, the nucleotide comprises no more than 80%, no more than 75%, no more than 70%, no more than 65%, or no more than 50% of the a/U content (or a/T or a combination of a/U and a/T). In some embodiments, the nucleotide has a length of no more than 1000nt, 600nt, 200nt, 100nt, 60nt, 56nt, 52nt, 50nt, 48nt, 46nt, 44nt, 22nt, 40nt, 38nt, 36, nt, 34nt, 32nt, 30nt, or 24 nt. In some embodiments, the nucleotides have a length of 24 to 100nt, 24 to 60nt, 24 to 50nt, or 36 to 50 nt. In some embodiments, the nucleotides have a length of about 42 nt.

In some embodiments, the nucleotide has a length of no more than 500nt, 300nt, 100nt, 50nt, 30nt, 28nt, 26nt, 25nt, 24nt, 23nt, 22nt, 21nt, 20nt, 19nt, 18nt, 17nt, 16nt, 15nt, or 12 nt. In some embodiments, the nucleotides have a length of 12 to 50nt, 12 to 30nt, 12 to 25nt, 18 to 25nt, 19 to 23nt, or 20 to 22 nt. In some embodiments, the nucleotides have a length of about 21 nt.

The peptide oligonucleotide complexes of the present disclosure (e.g., peptide oligonucleotide complexes comprising a PD-L1 binding peptide and a nucleotide) can be further conjugated, linked or fused to an active agent other than a nucleotide active agent (e.g., a target binding agent capable of binding a target molecule). Such additional active agents may be complexed, fused, linked or conjugated to one or more of the peptides, nucleotides or linkers within the peptide oligonucleotide complex. In some embodiments, the active agent may be directly or indirectly attached to a peptide of the peptide oligonucleotide complex or a nucleotide of the peptide oligonucleotide complex. Peptide nucleic acid complexes that also contain additional active agents may be referred to as peptide-active agent conjugates or peptide constructs.

The peptide oligonucleotide complexes of the present disclosure may also be used to deliver another active agent. The peptides according to the present disclosure may be conjugated, linked or fused with agents for the treatment of tumors and cancers or other diseases. For example, in certain embodiments, the peptides described herein are fused or conjugated to another molecule, such as an active agent that provides additional functional capabilities. The peptide or nucleotide may be fused to the active agent by expression of a vector containing the sequence of the peptide and the sequence of the active agent. In various embodiments, the sequence of the peptide and the sequence of the active agent may be expressed from the same Open Reading Frame (ORF). In various embodiments, the sequence of the peptide and the sequence of the active agent may comprise a contiguous sequence. The peptide and the active agent may each retain similar functional capabilities in the peptide construct as compared to their functional capabilities when expressed alone. In certain embodiments, examples of active agents may include other peptides.

As another example, in certain embodiments, a peptide or nucleotide described herein is linked to another molecule, such as an active agent that provides functional capability. The active agent can be any of the active agents (e.g., therapeutic agents, detectable agents, or binding moieties) described herein. In some embodiments, the peptide or nucleotide is covalently or non-covalently attached to the active agent, e.g., directly or through a linker. Exemplary linkers suitable for use with embodiments herein are discussed in more detail below.

Modification of peptides

The peptide may be modified in one or more of a variety of ways (e.g., chemically, mutationally, or with a peptide). In some embodiments, the peptide may be mutated to increase function, delete function, or modify in vivo behavior. One or more loops between disulfide bonds of a peptide (e.g., a PD-L1 binding peptide or peptide complex) may be modified or substituted to include active elements from other peptides (e.g., moore and Cochran, methods in Enzymology,503, pages 223-251, 2012). In some embodiments, the peptides of the disclosure (e.g., PD-L1 binding peptides or peptide complexes) can be further functionalized and multimerized by the addition of additional functional domains. For example, the albumin binding domain from the streptococcus uberis albumin binding protein of megafengold (Finegoldia magna) (SEQ ID NO:245, MKLKKKKKVIDEVIVEGQIGAIVGGVVVKVIDEAGAIKKVAPKKVAPQKKKWAKEKVEKKVEKQVEKKKKKVEKKQVEKKKKQVEKKKQVEKKVKKKKQVEKKKQVEKKKKKQWEKKKKKKKQQQKKQWEKQWEIKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKQKQKQKQQQQQQQKQKQKQKQKQKQKQKQKQKQKQKK QKK QK GYGYGK GK GYGK GK GYGYGYGK GK GYK GK K;K; A kind of electronic device A kind of electronic device. For example, the albumin binding domain of SEQ ID NO 243 (LKNAKEDAIAELKKAGITSDFYFNAINKAKTVEEVNALK NEILKA) may be added to the peptides of the disclosure. In some embodiments, the peptides of the present disclosure can be functionalized with albumin binding domains that have been modified to increase albumin affinity, increase stability, decrease immunogenicity, increase manufacturability, or a combination thereof. For example, the peptide may be functionalized with the modified albumin binding domain of SEQ ID NO 244 (LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDE ILKA) having high thermostability and improved serum half-life compared to the albumin binding domain of SEQ ID NO 243. Albumin binding domains comprise a simple triple-helical structure that is unlikely to disrupt the independent folding of other peptide domains (e.g., CDP domains). In some embodiments, a functional domain (e.g., an albumin binding domain) can increase the serum half-life of a peptide or peptide complex of the disclosure. Functional domains (e.g., albumin binding domains) may be included in any orientation relative to the PD-L1 binding peptide. For example, the functional domain may be linked to a PD-L1 binding peptide. The additional functional domains may be linked to one or more peptides (e.g., PD-L1 binding peptides or peptide complexes) by a linker (e.g., a peptide linker of any of SEQ ID NO:154-SEQ ID NO:241 or SEQ ID NO: 433).

The amino acids of the peptide or peptide complex (e.g., PD-L1 binding peptide or peptide complex) may also be mutated, e.g., to increase half-life, improve, add or delete in vivo binding behavior, add new targeting functions, alter surface charge and hydrophobicity, or allow the creation of conjugation sites. N-methylation is one example of methylation that may be present in the peptides of the present disclosure. In some embodiments, the peptide is modified by methylation on a free amine. For example, complete methylation can be achieved by using reductive methylation with formaldehyde and sodium cyanoborohydride.

Peptides may be modified to add functionality, for example to graft loops or sequences from other proteins or peptides onto the peptides of the disclosure. Likewise, domains, loops or sequences from the present disclosure may be grafted onto other peptides or proteins having additional functions, such as antibodies.

In some embodiments, the PD-L1 binding peptide or peptide complex may comprise a tissue targeting domain and may accumulate in a target tissue upon administration to a subject. For example, the PD-L1 binding peptide may be conjugated, linked or fused to a molecule (e.g., a small molecule, peptide or protein) that has a targeting or homing function to a cell of interest or a target protein located on or within the cell surface. In some embodiments, the PD-L1 binding peptide may be conjugated, linked or fused to a molecule that extends the plasma and/or biological half-life of the peptide, or improves the pharmacodynamics (e.g., enhanced binding to a target protein) and/or pharmacokinetic properties (e.g., rate and pattern of clearance) of the peptide, or any combination thereof.

Chemical modifications can, for example, extend the half-life of the peptide, or alter the biodistribution or pharmacokinetic profile. Chemical modifications may include polymers, polyethers, polyethylene glycols, biopolymers, polyamino acids, fatty acids, dendrimers, fc regions, simple saturated carbon chains (e.g. palmitate or myristate) or albumin. The polyamino acids may include, for example, polyamino acid sequences having repeated single amino acids (e.g., poly glycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern (e.g., gly-ala-gly-ala (SEQ ID NO: 550)), or any combination of the foregoing.

The peptides of the present disclosure may be modified such that the modification increases the stability and/or half-life of the peptide. Hydrophobic moieties such as linked to the N-terminal, C-terminal, or internal amino acids can be used to extend the half-life of the peptides of the present disclosure. Peptides may also be modified to increase or decrease intestinal or cellular permeability of the peptide. In some cases, the peptides of the present disclosure exhibit high accumulation in glandular cells of the intestine, thereby demonstrating applicability in the treatment and/or prevention of diseases or disorders of the intestine, such as crohn's disease, or more generally, inflammatory bowel disease. Peptides of the disclosure may include post-translational modifications (e.g., methylation and/or amidation and/or glycosylation) that may affect, for example, serum half-life. In some embodiments, a simple carbon chain (e.g., by myristoylation and/or palmitoylation) may be conjugated to the fusion protein or peptide. A simple carbon chain may render the fusion protein or peptide easily separable from unconjugated material. For example, methods that may be used to separate fusion proteins or peptides from unconjugated materials include, but are not limited to, solvent extraction and reverse phase chromatography. The lipophilic moiety can extend half-life through reversible binding to serum albumin. The conjugated moiety may be, for example, a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety may be cholesterol or a cholesterol derivative, including cholestenes, cholestanes, cholestadienes, and oxidized sterols. In some embodiments, the peptide may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof, linked. In other embodiments, the peptides of the disclosure can be coupled (e.g., conjugated, linked, or fused) to a half-life modulator. Examples of half-life modulators may include, but are not limited to: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly (amino acids), water-soluble polymers of proline, alanine and serine, water-soluble polymers containing glycine, glutamic acid and serine, fc regions, fatty acids, palmitic acid, albumin or molecules bound to albumin. In some embodiments, the half-life modulator may be a serum albumin binding peptide, such as SA21 (SEQ ID NO:242,RLIEDICLPRWGC LWEDD). In some embodiments, the SA21 peptide may be conjugated or fused to a CDP of the disclosure (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567). In addition, conjugation of peptides to near infrared dyes such as cy5.5 or to albumin binders such as album-tags can extend the serum half-life of any peptide as described herein. In some embodiments, immunogenicity is reduced by using minimal non-human protein sequences to extend the serum half-life of the peptide.

In some embodiments, the first two N-terminal amino acids (GS) of SEQ ID NO:60-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO:567 act as spacers or linkers to facilitate conjugation or fusion with another molecule, as well as cleavage of the peptide from such conjugated, linked or fused molecules. In some embodiments, the fusion proteins or peptides of the present disclosure can be conjugated, linked or fused to other moieties that can, for example, improve or effect a change to a property of the peptide.

In some embodiments, the peptides or peptide complexes of the disclosure may also be conjugated, linked or fused to other affinity handles. Other affinity handles may include genetically fused affinity handles. Genetic fusion affinity handles may include 6xHis (HHHHH (SEQ ID NO: 248), immobilized metal affinity column purification possibilities), FLAG (DYKDDDDK (SEQ ID NO: 432), anti-FLAG immunoprecipitation), and "terminal" FLAG (DYKDE (SEQ ID NO: 431). In some embodiments, the peptides or peptide complexes of the present disclosure may also be conjugated, linked or fused to expression tags or sequences to improve protein expression and/or purification.

In addition, more than one peptide sequence (peptide derived from toxin or venom protein) may be present on, conjugated to, linked to or fused to a particular peptide. Peptides can be incorporated into biomolecules by a variety of techniques. Peptides may be incorporated by chemical transformations (e.g., forming covalent bonds, such as amide bonds). Peptides may be incorporated, for example, by solid phase or solution phase peptide synthesis. The peptide may be incorporated by preparing a nucleic acid sequence encoding a biomolecule, wherein the nucleic acid sequence comprises a subsequence encoding the peptide. The subsequence may be in addition to, or may replace, the sequence encoding the biomolecule.

The PD-L1 binding peptides of the present disclosure (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) can be modified with a cell penetrating peptide to form a cell penetrating PD-L1 binding peptide. The cell penetrating peptide may facilitate delivery of the PD-L1 binding peptide or peptide complex into a cell or across a cell layer (e.g., across the blood brain barrier or across the endosome into the cytosol). In some embodiments, the cell penetrating PD-L1 binding peptide may be further complexed with an additional active agent to facilitate the delivery of the additional active agent into the cell or across a cell layer. This may enable delivery of the active agent (e.g., therapeutic agent) to the intracellular target. Examples of cell penetrating peptides that can be used in combination with the PD-L1 binding peptides of the present disclosure are provided in table 7.

TABLE 7 exemplary cell penetrating peptides

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In some embodiments, the tissue targeting domain may comprise a transferrin receptor binding (TfR binding) peptide, e.g., SEQ ID No. 350 (REGCASRCMKYNDELEK CEARMMSMSNTEEDCEQELEDLLYCLDHCHSQ), which may facilitate transcytosis across the blood brain barrier and deliver PD-L1 binding peptides to the central nervous system, including brain tumors. In some embodiments, the TfR binding peptide may be derived from the scaffold of SEQ ID NO:351 (REGCASHCTKYKAELEKCEARVSSRSNTEETCVQELFDFLHCVD HCVSQ) or SEQ ID NO:352 (GSREGCASHCTKYKAELEKCEARVSS RSNTEETCVQELFDFLHCVDHCVSQ). For example, the CNS can be accessed by transcytosis across the BBB by binding a PD-L1 binding peptide complexed with a TfR binding peptide to a transferrin receptor (TfR), and then recycling the complex to the cell surface. Some PD-L1 binding peptide/TfR binding peptide complexes may enter low pH early endosomes. The PD-L1 binding peptide or other active agent complex may be exposed to endosomes following endocytosis of TfR and may remain undegraded due to the stability of the PD-L1 binding peptide. If the PD-L1 binding peptide/PD-L1 binding peptide complex includes additional cell penetration capacity, the peptide may facilitate accelerated escape of the PD-L1 binding peptide or other active agent from the endosomal compartment into the cytosol. Even without increasing the cell penetration capacity, PD-L1 binding peptides or other active agents may slowly leak out of the endosome and into the cytosol.

Nucleotide modification

In some embodiments, the nucleic acid portion of the peptide oligonucleotide complex (e.g., an oligonucleotide of the PD-L1 binding peptide oligonucleotide complex) contains one or more modified bases within the nucleic acid molecule. Such modifications can be made whether the nucleic acid portion is single-stranded (ssDNA, ssRNA) or double-stranded (dsDNA, dsRNA) or a combination of single and double strands (e.g., having a mismatched sequence, hairpin or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), oligonucleotide complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, gapmer, anti-miR, splice-blocking ASO, or U1 adaptor. One or more bases in a given nucleotide sequence may be modified to increase in vivo stability, increase resistance to enzymes such as nucleases, increase protein binding (including binding to serum proteins), increase in vivo half-life, alter tissue biodistribution, or modify the manner of response of the immune system. Phosphonates, ribose or bases may be modified. In some aspects, the modifications include phosphorothioate modifications, phosphodiester modifications, phosphorothioate modifications, methylphosphonate modifications, phosphorodithioate modifications, methoxypropyl phosphonate modifications, 5'- (E) -vinylphosphonate modifications, 5' methylphosphonate modifications, (S) -5 '-C-methylphosphonate modifications, 5' -phosphorothioate modifications, peptide Nucleic Acid (PNA), 2'-O methyl modifications, 2' -O-methoxyethyl (2 '-O-Me) modifications, 2' -fluoro (2 '-F) modifications, 2' -deoxy-2 '-fluoro modifications, 2' -arabino-fluoro modifications, 2 '-O-benzyl modifications, 2' -O-methyl-4-pyridine modifications, locked Nucleic Acid (LNA), amino-LNA, thio-LNA, ENA, amino ENA, carbon-ENA, (S) -cEt-bridging nucleic acid, (S) -MOE, bridging nucleic acid, tricyclic DNA, morpholino nucleic acid (PMO), unlocking nucleic acid (BNA), ethylene Glycol Nucleic Acid (GNA), bridging nucleic acid (S) -pseudoethyl (S) -uridine, uridine (N '-methyl) modifications, 6' -methyl adenosine, 5 '-methyl-6' -methyl-triazole, piperidine, 5 '-methyl-6' -guanosine, 6 '-methyl-6' -triazole modifications, piperidine, and other modifications, 2',4' -difluorotoluene ribonucleoside, 5' nitroindole, 5' methyl, 5' phosphonate, inverted a base, 2' -H (deoxyribose), 2' -OH (ribose), or any combination thereof. The oligonucleotide may consist entirely of a combination of 2'-O-Me and 2' -F modifications. Diastereoisomers or one or both stereoisomers may be used. Any stabilization chemistry or mode disclosed in Hu Signal Transduction and Targeted Therapy 2020,5:101 may be used, including STC, ESC, advanced ESC, AD1-3, AD5. Pyrimidine may be 2' -fluoro modified, which may increase stability to nucleases but may also increase activation of the immune system. The RNA backbone may be replaced with phosphorothioates (where non-bridging oxygen is replaced with sulfur), which may increase resistance to nuclease digestion, as well as alter biodistribution and tissue retention and increase pharmacokinetics, e.g., by increasing protein binding, but may also induce more immune stimulation. Methyl phosphonate modification of RNA can also be used. 2'-O methyl and 2' -F RNA bases can be used that resist base hydrolysis and nucleases and increase the melting temperature of the duplex. Bridging, locking, and other similar forms of bridging nucleic acids (BNA, LNA, cEt), where any chemical bridge, such as an N-O bond between 2 'oxygen and 4' carbon in ribose, can be incorporated to increase resistance to exonucleases and endonucleases and enhance biostability. These include BNA, where an N-O bond occurs between the 2 'and 4' carbons, and any chemical modification of the nitrogen in the bridge (including but not limited to NH, N-CH3, N-benzene) may be added to increase the stability of the RNA backbone, or base modifications may be placed at any position, one, more or all base positions of the RNA sequence. Optionally, phosphorothioate nucleic acid linkages may be used between 2-4 terminal nucleic acids of one or both sequences. Optionally, 2' f modified nucleic acids can be used at least 2-4 positions, at least 5%, at least 10%, at least 25% internal positions, at least 50%, at least 75% or up to 100% internal positions, all internal positions or all positions. Optionally, one or more of a 2'f base, LNA base, BNA base, ENA base, 2' o-MOE base, morpholino base, 2'ome base, 5' -Me base, (S) -cEt base, or a combination thereof may be used at least 2-4 positions, at least 5%, at least 10%, at least 25% internal positions, at least 50%, at least 75% or at most 100% internal positions, all internal positions, or all positions.

The modified bases can be used to increase the in vivo half-life of the oligonucleotide. They can leave the oligonucleotides intact in serum, endosomes, cytosol or nucleus, including days, weeks or months. This may allow for sustained activity, including the case where the oligonucleotide is released slowly from the endosome within a given cell over a period of days, weeks or months (as described in Brown et al, nucleic Acids Research,2020, pages 11827-11844).

In some embodiments, the nucleotide comprises at least one phosphorothioate linkage. In some embodiments, the peptide oligonucleotide complex comprises 1 to 12 phosphorothioate linkages. In some embodiments, the nucleotide comprises at least one phosphorothioate amide linkage. In some embodiments, the nucleotide comprises 1 to 12 phosphorothioate amide linkages. In some embodiments, the nucleotide comprises at least one modified base. In some embodiments, at least the modified base comprises a 2'f base, LNA base, BNA base, ENA base, 2' o-MOE base, 5'-Me base, (S) -cEt base, 2' ome base, morpholino base, or a combination thereof.

Joint

The peptide (e.g., PD-L1 binding peptide or peptide complex) according to the present disclosure can be directly linked to another moiety (e.g., additional active agent) by a linker or in the absence of a linker, such as a small molecule, a second peptide, a second CDP, a protein, a mini-protein, an antibody fragment, an Fc pestle, an Fc mortar, an aptamer, a polypeptide, a polynucleotide, a fluorophore, a radioisotope, a radionuclide chelator, a polymer, a biopolymer, a fatty acid, an acyl adduct, a chemical linker, a binding moiety, or a sugar, or other active or detectable agent described herein. In the absence of a linker, for example, the active agent or detectable agent may be conjugated, linked or fused to the N-terminus or C-terminus of the peptide to produce an active agent or detectable agent fusion peptide. In other embodiments, the linking may be via reductive alkylation by peptide fusion. In some embodiments, cleavable linkers are used for in vivo delivery of the peptide, e.g., linkers that can be cleaved or degraded upon entry into a cell, endosome, or nucleus. In some embodiments, in vivo delivery of the peptide requires a small linker that does not interfere with cell penetration or localization to the nucleus. The linker may also be used to covalently link a peptide as described herein to another moiety or molecule having a separate function, such as targeting, cytotoxicity, therapeutic, homing, imaging or diagnostic functions.

The peptide may be directly linked to another molecule by covalent linkage. For example, a peptide is attached to the end of the amino acid sequence of a larger polypeptide or peptide molecule, or to a side chain such as lysine, serine, threonine, cysteine, tyrosine, aspartic acid, an unnatural amino acid residue, or a glutamic acid residue. The linkage may be via an amide bond, an ester bond, an ether bond, a urethane bond, a carbon-nitrogen bond, a triazole, a macrocyclic, an oxime bond, a hydrazone bond, a carbon-carbon single, double or triple bond, a disulfide bond or a thioether bond. In some embodiments, similar regions of the disclosed peptide(s) themselves (e.g., the ends of amino acid sequences, amino acid side chains (e.g., side chains of lysine, serine, threonine, cysteine, tyrosine, aspartic acid, unnatural amino acid residues, or glutamic acid residues), via amide linkages, ester linkages, ether linkages, carbamate linkages, carbon-nitrogen linkages, triazoles, macrocycles, oxime linkages, hydrazone linkages, carbon-carbon single, double or triple bonds, disulfide or thioether linkages, or linkers as described herein) can be used to attach other molecules.

The attachment via the linker may involve the incorporation of a linker moiety between the other molecule and the peptide. Both the peptide and the other molecule may be covalently linked to a linker. The linker may be cleavable, labile, non-cleavable, stable self-cleaving, hydrophilic or hydrophobic. As used herein, the term "non-cleavable" or "stable" (e.g., used in conjunction with an amide, cyclic, or urethane linker, or as otherwise described herein) is generally used by the skilled artisan to distinguish relatively stable structures from less stable or "cleavable" structures (e.g., used in conjunction with cleavable linkers that can be cleaved or cleaved structurally by enzymes, proteases, self-decomposition, pH, reduction, hydrolysis, certain physiological conditions, or otherwise as described herein). It will be appreciated that in contrast to "cleavable" linkers, "non-cleavable" or "stable" linkers provide stability against cleavage or other cleavage, and that the term is not intended to be construed as absolutely non-cleavable or non-cleavable structures under any condition. Thus, as used herein, a "non-cleavable" linker is also referred to as a "stable" linker. The linker may have at least two functional groups, one bound to the peptide and the other bound to another molecule, and a linking moiety between the two functional groups. Some example linkers are described in Jain, N., pharm Res.32 (11): 3526-40 (2015); doronina, S.O., bioconj chem.19 (10): 1960-3 (2008); pilow, t.h., J Med chem.57 (19): 7890-9 (2014); dorywalksa, m., bioconj chem.26 (4): 650-9 (2015); kellogg, b.a., bioconj chem.22 (4): 717-27 (2011); and Zhao, R.Y., J Med chem.54 (10): 3606-23 (2011).

Non-limiting examples of functional groups for attachment may include functional groups capable of forming amide linkages, ester linkages, ether linkages, carbonate linkages, urethane linkages, or thioether linkages. Non-limiting examples of functional groups capable of forming such bonds may include: an amino group; a carboxyl group; a hydroxyl group; an aldehyde group; an azide group; alkyne and alkene groups; a ketone; a hydrazide; acyl halides such as acyl fluoride, acyl chloride, acyl bromide and acyl iodide; anhydrides, including symmetrical, mixed, and cyclic anhydrides; a carbonate ester; carbonyl functions bonded to a leaving group such as cyano, succinimidyl and N-hydroxysuccinimidyl; a hydroxyl group; a mercapto group; and molecules having, for example, alkyl, alkenyl, alkynyl, allyl, or benzyl leaving groups, such as halides, mesylate, tosylate, triflate, epoxide, phosphate, sulfate, and benzenesulfonate.

Non-limiting examples of linking moieties can include alkylene, alkenylene, alkynylene, polyethers (such as polyethylene glycol (PEG)), hydroxycarboxylic acids, polyesters, polyamides, polyamino acids, polypeptides, cleavable peptides, valine-citrulline, aminobenzyl carbamate, D-amino acids, and polyamines, any of which are unsubstituted or substituted with any number of substituents such as halogen, hydroxy, mercapto, amino, nitro, nitroso, cyano, azido, sulfoxide, sulfone, sulfonamide, carboxyl, formaldehyde, imino, alkyl, halo-alkyl, alkenyl, halo-alkenyl, alkynyl, halo-alkynyl, alkoxy, aryl, aryloxy, aralkyl, arylalkoxy, heterocyclyl, acyl, acyloxy, carbamate, amide, carbamate, epoxide, and ester groups.

In some cases, the linker may comprise a triazole group, e.g., any of those of formula C ₂ H ₃ N ₃ Five-membered rings having two carbon atoms and three nitrogen atoms, optionally hydrogen atoms are bonded to N at any position in the ring, for example:

for example, 1,2, 3-triazole (e.g., 1H1,2,3-triazole, 2H1,2,3-triazole or 1-methyl-4, 5,6,7,8, 9-hexahydro-1H-cycloocta [ d ]][1,2,3]Triazole) or a1,2, 4-triazole (e.g. 1H1,2,4-triazole or 4H1,2,4-triazole).

Additional non-limiting examples of linkers include linear or acyclic linkers, such as:

wherein each n is independently 0 to about 1,000;1 to about 1,000;0 to about 500;1 to about 500;0 to about 250;1 to about 250;0 to about 200;1 to about 200;0 to about 150;1 to about 150;0 to about 100;1 to about 100;0 to about 50;1 to about 50;0 to about 40;1 to about 40;0 to about 30;1 to about 30;0 to about 25;1 to about 25;0 to about 20;1 to about 20;0 to about 15;1 to about 15;0 to about 10;1 to about 10;0 to about 5; or 1 to about 5. In some embodiments, each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. In some embodiments, m is 1 to about 1,000;1 to about 500;1 to about 250;1 to about 200;1 to about 150;1 to about 100;1 to about 50;1 to about 40;1 to about 30;1 to about 25;1 to about 20;1 to about 15;1 to about 10; or 1 to about 5. In some embodiments, m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50, or as in Jain, n., pharm res.32 (11): 3526-40 (2015) or Any of the linkers disclosed in Ducry, l., antibody Drug Conjugates (2013).

In some cases, the linker may comprise a cyclic group, such as an organic non-aromatic or aromatic ring, optionally having 3 to 10 carbons in the ring, optionally constructed of carboxylic acids,such as trans-4- (aminomethyl) cyclohexanecarboxylic acid,

or a substituted analog or stereoisomer thereof. This linker may optionally be used to form urethane linkages. In some cases, urethane linkages may be more resistant to cleavage, for example, by hydrolysis, enzymes (e.g., esterases), or other chemical reactions, than ester linkages.

In some cases, the linker may comprise a cyclic formic acid, such as a cyclic dicarboxylic acid, for example one of the following groups: 1, 4-cyclohexanedicarboxylic acid, 1, 2-cyclohexanedicarboxylic acid or 1, 3-cyclohexanedicarboxylic acid, 1-cyclopentanediacetic acid,

or a substituted analog or stereoisomer thereof. For example, the linker may comprise one of the following groups.

In some cases, linkers may optionally be used to form ester linkages. In some cases, the cyclic ester bond may be more sterically resistant to cleavage, for example, by hydrolysis of water, enzymes (e.g., esterases), or other chemical reactions.

In some cases, the linker may comprise an aromatic dicarboxylic acid, e.g., terephthalic acid, isophthalic acid, phthalic acidOr a substituted analog thereof.

In some cases, the linker may comprise a natural or unnatural amino acid, e.g., cysteineOr a substituted analog or stereoisomer thereof. In some cases, the linker can comprise alanine (A, ala); arginine (R, arg); asparagine (N, asn); aspartic acid (D, asp); glutamic acid (E, glu); glutamine (Q, gln); glycine (G, gly); histidine (H, his); isoleucine (I, ile); leucine (L, leu); lysine (K, lys); methionine (M, met); phenylalanine (F, phe); proline (P, pro); serine (S, ser); threonine (T, thr); tryptophan (W, trp); tyrosine (Y, tyr); valine (V, val); or any multiple or combination thereof. In some embodiments, the unnatural amino acid can comprise one or more functional groups that can be used as a functional handle, e.g., an alkene or alkyne.

In some cases, the linker may comprise one of the following groups:

or a substituted analog or stereoisomer thereof. In some cases, the linker is selected from one of the following groups: / >

Or a substituted analog or stereoisomer thereof.

In some cases, the linker may comprise one of the following groups:

or a substituted analog or stereoisomer thereof. In some cases, the linker is selected from one of the following groups:

or a substituted analog or stereoisomer thereof.

In some cases, a substituted analog or stereoisomer is a structural analog of a compound disclosed herein, one or more hydrogen atoms of which may be substituted with one or more of the following groups: halo (e.g., cl, F, br), alkyl (e.g., methyl, ethyl, propyl), alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or any combination thereof. In some cases, stereoisomers may be enantiomers, diastereomers, cis or trans stereoisomers, E or Z stereoisomers, or R or S stereoisomers.

Non-limiting examples of linear linkers include;

wherein each n1 or n2 or m is independently 0 to about 1,000;1 to about 1,000;0 to about 500;1 to about 500;0 to about 250;1 to about 250;0 to about 200;1 to about 200;0 to about 150;1 to about 150;0 to about 100;1 to about 100;0 to about 50;1 to about 50;0 to about 40;1 to about 40;0 to about 30;1 to about 30;0 to about 25;1 to about 25;0 to about 20;1 to about 20;0 to about 15;1 to about 15;0 to about 10;1 to about 10;0 to about 5; or 1 to about 5. In some embodiments, each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. In some embodiments, m is 1 to about 1,000;1 to about 500;1 to about 250;1 to about 200;1 to about 150; 1 to about 100;1 to about 50;1 to about 40;1 to about 30;1 to about 25;1 to about 20;1 to about 15;1 to about 10; or 1 to about 5. In some embodiments, m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. In some cases, the linker may include a linear dicarboxylic acid, such as one of the following groups: succinic acid, 2, 3-dimethylsuccinic acid, glutaric acid, adipic acid, 2, 5-dimethyladipic acid,

or a substituted analog or stereoisomer thereof. In some cases, linkers may be used to form urethane linkages. In some embodiments, the urethane linkage is more resistant to cleavage, for example, by hydrolysis, enzymes (e.g., esterases), or other chemical reactions, than the ester linkage. In some cases, linkers may be used to form linear ester linkages. In some embodiments, the linear ester linkage may be more susceptible to cleavage, for example, by hydrolysis, enzymes (e.g., esterases), or other chemical reactions, than the cyclic ester or urethane linkage. Side chains such as methyl groups on the linear ester linkages may optionally render the linkages less prone to cleavage than without side chains.

In some cases, the linker may be a succinic acid linker, and a targeting agent (e.g., single-stranded (ssDNA, ssRNA) or double-stranded (dsDNA, dsRNA) or a combination of single-and double-stranded (e.g., having a mismatched sequence, hairpin, or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), oligonucleotide complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, empty mer, anti-miR, splice-blocking ASO, or U1 adaptor), or other active or detectable agent may be linked to the peptide through an ester or amide bond, with two methylene carbons in between. In other cases, the linker may be any linker having both hydroxyl and carboxylic acid, such as hydroxycaproic acid or lactic acid.

In some cases, nucleotides (e.g., single-stranded (ssDNA, ssRNA) or double-stranded (dsDNA, dsRNA) or a combination of single-and double-stranded (e.g., having a mismatched sequence, hairpin, or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), oligonucleotides complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, vacancy-mer, anti-miR, splice-blocking ASO, or U1 adaptor), an active agent, or a detectable agent can be linked to the peptide using any one or more of the linkers shown in table 8 below. In some embodiments, the peptide, additional active agent, or detectable agent may be linked to the nucleotide using any one or more of the linkers shown in table 8 below.

TABLE 8 exemplary linkers for peptide conjugates or complexes

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In some cases, the active agent is attached to the linker, wherein the nucleophilic functional group (e.g., hydroxyl) of the active agent molecule acts as a nucleophile and replaces the leaving group on the linker moiety, thereby attaching it to the linker.

In other cases, the active agent is attached to a linker, wherein a nucleophilic functional group (e.g., thiol group, amine group, etc.) of the linker replaces a leaving group on the active agent, thereby allowing it to attach to the linker. Such a leaving group (or a functional group convertible to a leaving group) may be a primary alcohol to form a thioether bond, thereby allowing it to be attached to the linker. Primary alcohols may be converted to leaving groups such as mesylate, tosylate or p-nitrobenzenesulfonate to accelerate nucleophilic substitution reactions.

The peptide-active agent complexes of the present disclosure (e.g., PD-L1-binding peptide complexes) can comprise an active agent (e.g., a therapeutic agent, a detectable agent, or an immune cell binding moiety), a linker, and/or a peptide of the present disclosure. The general connectivity between these three components may be an active agent-linker-peptide, such that the linker is attached to both the active agent and the peptide. In many cases, the peptide is linked to the linker through an amide bond. Amide linkages may be relatively stable (e.g., in vivo) compared to other linkages described herein, such as esters, carbonates, and the like. Thus, if an active agent seeks to cleave from a peptide-active agent conjugate, after such in vivo cleavage the linker is not attached to the active agent, then the amide bond between the peptide and the linker may provide advantageous properties due to its in vivo stability. Thus, in each case, the active agent is linked to the linker-peptide moiety through a bond such as an ester, carbonate, carbamate, etc., wherein the peptide or active agent is linked to the linker through an amide bond. This may allow selective cleavage of the active agent-linker bond (as opposed to the linker-peptide bond), allowing the active agent to be released, with the non-linker moiety attached to it after cleavage. The use of such different active agent-linker bonds or linkages allows for modulating active agent release in vivo, e.g., to achieve therapeutic function, while minimizing off-target effects (e.g., reducing active agent release during circulation).

The linker may be a cleavable or stable linker. The use of a cleavable linker allows for release of the conjugate moiety (e.g., a nucleotide targeting agent, a therapeutic agent, a detectable agent, or a combination thereof) from the peptide, e.g., after targeting to a target tissue or cell or subcellular compartment. In some cases, the linker is enzymatically cleavable, e.g., a valine-citrulline linker (SEQ ID NO: 217) cleavable by a cathepsin, or an ester linker cleavable by an esterase. In some embodiments, the linker contains a self-degrading moiety. In other embodiments, the linker comprises one or more cleavage sites for a particular protease, such as a cleavage site for a Matrix Metalloproteinase (MMP), thrombin, urokinase-type plasminogen activator, or a cathepsin (e.g., cathepsin K).

Thus, in some cases, the peptide-active agent complexes of the present disclosure may comprise one or more, about two or more, about three or more, about five or more, about ten or more, or about 15 or more amino acids that may form an amino acid sequence that is cleavable by an enzyme. Such enzymes may include proteases. The peptide-active agent complex may comprise an amino acid sequence cleavable by a cathepsin, chymotrypsin, elastase, subtilisin, thrombin I, or urokinase, or any combination thereof.

Alternatively or in combination, the cleavable linker may be cleaved, dissociated or broken by other mechanisms, such as by pH, reduction or hydrolysis. Hydrolysis may occur directly as a result of the water reaction, or may be facilitated by enzymes, or by the presence of other chemicals. Hydrolytically unstable linkers (as well as other cleavable linkers described herein) may be advantageous in terms of releasing the active agent from the peptide. For example, an active agent in conjugated form with a peptide may not be active, but after release from the conjugate after targeting to a target tissue or cell or subcellular compartment, the active agent is active. The cleaved active agent may retain the chemical structure of the active agent prior to cleavage, or may be modified. In some embodiments, the stable linker may optionally not cleave in the buffer over a long period of time (e.g., hours, days, or weeks). In some embodiments, the stabilizing linker may optionally not cleave in bodily fluids such as plasma or synovial fluid over a long period of time (e.g., hours, days, or weeks). In some embodiments, the stabilizing linker optionally can be cleaved, for example, after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that may be present in cells (e.g., macrophages), cellular compartments (e.g., endosomes and lysosomes), inflamed areas of the body (e.g., inflamed joints), or tissues or body compartments. In some embodiments, the stabilizing linker may optionally not cleave in vivo, but instead present an active agent that remains active when conjugated, linked or fused to the peptide.

The rate of hydrolysis of the linker (e.g., of the peptide conjugate) can be adjusted. For example, a linker with an unhindered ester may hydrolyze at a faster rate than a linker with a bulky group adjacent to the ester carbonyl. The bulky group may be a methyl, ethyl, phenyl, ring, or isopropyl group, or any group that provides steric hindrance. In another example, where hydrophilic groups such as alcohols, acids, or ethers are present near the ester carbonyl, the rate of hydrolysis may be faster. In another example, the hydrophobic groups present as side chains or by having longer hydrocarbon linkers can slow down the cleavage of the esters. In some embodiments, cleavage of the urethane group may also be modulated by steric hindrance, hydrophobicity, and the like. In another example, the use of less labile linkers, such as carbamates rather than esters, may slow the cleavage rate of the linker. In some cases, steric hindrance may be provided by the drug itself, for example, ketorolac (ketorolac) when conjugated by a carboxylic acid. The rate of hydrolysis of the linker may be adjusted according to the residence time of the conjugate in the target tissue or cell or subcellular compartment, according to the rapid extent of accumulation of the peptide in the target tissue or cell or subcellular compartment, or according to the time frame required for exposure to the active agent in the target tissue or cell or subcellular compartment. For example, when the peptide is cleared relatively rapidly from the target tissue or cell or subcellular compartment, the linker may be adjusted to hydrolyze rapidly. Conversely, for example, when the peptide has a longer residence time in the target tissue or cell or subcellular compartment, a slower rate of hydrolysis may allow for prolonged delivery of the active agent. This can be important when the peptide is used to deliver a drug to a target tissue or cell or subcellular compartment (e.g., a tumor cell or tumor tissue). An example of improving the hydrolysis rate is provided by "Programmed hydrolysis in designing paclitaxel prodrug for nanocarrier assembly" Sci Rep 2015,5,12023Fu et al. In some embodiments, the rate of cleavage may vary with species, body compartments, and disease states. For example, cleavage by esterases may be faster in rat or mouse plasma than in human plasma, e.g., due to different levels of carboxylesterase. In some embodiments, the linker may be adjusted to achieve different cleavage rates, similar cleavage rates, in different species.

In some cases, the linker may be a succinic linker and the drug may be attached to the peptide via an ester or amide bond with two methylene carbons in between. In other cases, the linker may be any linker having both hydroxyl and carboxylic acid, such as hydroxycaproic acid or lactic acid.

In some embodiments, the linker may release the active agent in an unmodified form. In other embodiments, the active agent may be released upon chemical modification. In other embodiments, catabolism may release an active agent that remains attached to the linker and/or the portion of the peptide.

The linker may be a stable linker or a cleavable linker. In some embodiments, the stabilizing linker may slowly release the conjugate moiety by exchange of the conjugate moiety to a free thiol on serum albumin. In some embodiments, the use of a cleavable linker may allow release of the conjugate moiety (e.g., therapeutic agent) from the peptide, e.g., after administration to a subject in need thereof. In other embodiments, the use of a cleavable linker may allow release of the conjugated therapeutic agent from the peptide. In some cases, the linker is enzymatically cleavable, such as a valine-citrulline (SEQ ID NO: 217) linker. In some embodiments, the linker contains a self-degrading moiety. In other embodiments, the linker comprises one or more cleavage sites for a particular protease, such as a cleavage site for Matrix Metalloproteinase (MMP), thrombin, cathepsin, peptidase, or β -glucuronidase. Alternatively or in combination, the linker may be cleaved by other mechanisms, for example by pH, reduction or hydrolysis.

Depending on the application, the hydrolysis or reduction rate of the linker may be fine tuned or modified. For example, a linker with an unhindered ester may hydrolyze at a faster rate than a linker with a bulky group adjacent to the ester carbonyl. The bulky group may be a methyl, ethyl, phenyl, ring, or isopropyl group, or any group that provides steric hindrance. In some cases, steric hindrance may be provided by the drug itself, for example, by ketorolac when conjugated, linked or fused by a carboxylic acid. The rate of hydrolysis of the linker can be adjusted according to the residence time of the conjugate or fusion in the target site. For example, when the peptide is cleared relatively rapidly from a tumor or brain, the linker may be adjusted to hydrolyze rapidly. When the peptide has a longer residence time in the target site, a slower rate of hydrolysis will allow for prolonged delivery of the active agent.

The rate of hydrolysis of a linker (e.g., of a peptide conjugate) can be measured. Such measurements may include assaying free active agents in the plasma or synovial fluid or other fluid or tissue of a subject in vivo, and/or by incubating the linker or the peptide complex comprising the linker of the disclosure ex vivo with a buffer (e.g., PBS) or plasma from the subject (e.g., rat plasma, human plasma, etc.) or synovial fluid or other fluid or tissue. Methods for measuring the rate of hydrolysis may include taking a sample during incubation or after administration and determining free active agent, free peptide, or any other parameter indicative of hydrolysis, and measuring total peptide, total active agent, or conjugated active agent-peptide. The results of such measurements can then be used to determine the hydrolysis half-life of a given linker or peptide conjugate comprising the linker. The hydrolysis half-life of the linker may vary depending on the plasma or fluid or species or other conditions used to determine this half-life. This may be due to certain enzymes or other compounds present in a certain plasma, e.g. rat plasma. For example, different fluids (e.g., plasma or synovial fluid) may contain different amounts of enzymes, such as esterases, and these levels of these compounds may also vary depending on the species (e.g., rat versus human) and the disease state (e.g., normal versus arthritis).

Conjugates of the present disclosure may be described as having a modular structure comprising various components, wherein each of the components (e.g., peptide, linker, active agent, and/or detectable agent) may be selected in dependence on or independently of any other component. For example, conjugates for treating pain may comprise a PD-L1 binding peptide of the present disclosure (e.g., a peptide having the amino acid sequence of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567), a linker (e.g., any of the linkers described or otherwise described in Table 8 or Table 9, SEQ ID NO:154-SEQ ID NO:241, SEQ ID NO: 433), and an active agent (e.g., a therapeutic agent, a detectable agent, or an immune cell binding moiety). The linker may, for example, be selected and/or modified to achieve a certain active agent release (e.g., a certain release rate) at the target site (e.g., in the brain) by a certain mechanism (e.g., by hydrolysis, e.g., enzyme and/or pH dependent hydrolysis), and/or to minimize systemic active agent exposure. During testing of the conjugates, any one or more of the components of the conjugates may be modified and/or altered to achieve certain in vivo properties of the conjugates, such as pharmacokinetic (e.g., clearance time, bioavailability, uptake and retention in various organs) and/or pharmacodynamic (e.g., target engagement) properties. Thus, conjugates of the present disclosure can be modulated for use in preventing, treating, and/or diagnosing a variety of diseases and conditions, while reducing side effects (e.g., side effects that would occur if such agents were administered alone (i.e., not conjugated to a peptide)).

In some embodiments, the unnatural amino acid can comprise one or more functional groups that can be used as a functional handle, e.g., an alkene or alkyne. For example, multiple bonds of such functional groups may be used to add one or more molecules to the conjugate. One or more molecules may be added using various synthetic strategies, some of which may include addition and/or substitution chemistry. For example, an addition reaction using multiple bonds may include the use of hydrobromic acid, wherein bromine may act as a leaving group, and thus be substituted with various moieties, such as active agents, detectable agents, agents that may improve or alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in the Central Nervous System (CNS) or elsewhere) and/or pharmacodynamic (e.g., hydrolysis rate, e.g., enzymatic hydrolysis rate) properties of the conjugate.

In some embodiments, conjugates as described herein comprise one or more unnatural amino acids and/or one or more linkers. Such one or more unnatural amino acids and/or one or more linkers can comprise one or more functional groups that can be used as functional handles, such as alkenes or alkynes (e.g., non-terminal alkenes and alkynes). For example, multiple bonds of such functional groups may be used to add one or more molecules to the conjugate. One or more molecules may be added using various synthetic strategies, some of which may include addition and/or substitution chemistry, cycloaddition, and the like. For example, an addition reaction using multiple bonds may include the use of hydrogen bromide (e.g., by hydrohalogenation), wherein the bromide substituent, once attached, may act as a leaving group, thus being substituted with various moieties comprising nucleophilic functional groups, e.g., active agents, detectable agents, agents. As another example, multiple bonds may be used as functional handles in a cycloaddition reaction. Cycloaddition reactions may include 1, 3-dipolar cycloaddition, [2+2] -cycloaddition (e.g., photocatalysis), diels-Alder reaction (Diels-Alder reaction), hu Yisi cycloaddition (Huisgen cycloaddition), nitrone-olefin cycloaddition, and the like. Such cycloaddition reactions can be used to attach various functional groups, functional moieties, active agents, detectable agents, and the like to the conjugate. For example, a 1, 3-dipole cycloaddition reaction may be used to attach a molecule to a conjugate, wherein the molecule comprises a 1, 3-dipole that may react with, for example, an alkyne to form a 5-membered ring, thereby attaching the molecule to the conjugate.

The addition of such agents or molecules (e.g., by nucleophilic or electrophilic addition followed by nucleophilic substitution) can have a variety of applications. For example, linking such molecules or agents may improve or alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in the CNS, or biodistribution) and/or pharmacodynamic (e.g., rate of hydrolysis, e.g., rate of enzymatic hydrolysis) properties of the conjugate. Linking such molecules or agents may also alter (e.g., increase) the depot effect of the conjugate, or provide functionality for in vivo tracking, e.g., using fluorescence or other types of detectable agents.

In some embodiments, conjugates of the present disclosure may comprise a linker comprising one or more of the following groups:

or a substituted analogue or stereoisomer thereof, wherein each of n1 and n2 is independently a value of from 1 to 10. Such groups may be used as handles to attach one or more molecules to the conjugate by nucleophilic or electrophilic addition followed by nucleophilic substitution, e.g., to alter the conjugatePharmacokinetic (e.g., plasma half-life, retention and/or uptake in the Central Nervous System (CNS) or elsewhere) and/or pharmacodynamic properties. Functionalization of such groups can be performed using one or more multiple bonds (e.g., double bonds, triple bonds, etc.) of the group. Such functionalization may include addition and/or substitution chemistries. For example, a functional group of the linker, such as a double bond, may be converted to a single bond (e.g., by an addition reaction, such as a nucleophilic addition reaction), wherein one or both of the carbon atoms of the newly formed single bond may have a leaving group (e.g., bromine) attached to them. Such leaving groups can then be used (e.g., via nucleophilic substitution reactions) to attach a particular molecule (e.g., active agent, detectable agent, etc.) to the one or more carbon atoms of the linker.

As another example, multiple bonds may be used as functional handles in a cycloaddition reaction. Cycloaddition reactions may include 1, 3-dipolar cycloaddition, [2+2] -cycloaddition (e.g., photocatalysis), diels-alder reactions, hu Yisi cycloaddition, nitrone-olefin cycloaddition, and the like. Such cycloaddition reactions can be used to attach various functional groups, functional moieties, active agents, detectable agents, and the like to the conjugate. For example, a 1, 3-dipole cycloaddition reaction may be used to attach a molecule to a conjugate, wherein the molecule comprises a 1, 3-dipole that can react with, for example, an alkyne to form a 5-membered ring, thereby attaching the molecule (e.g., active agent, detectable agent, etc.) to the conjugate. In some cases, molecules may be attached to the conjugate to, for example, modulate half-life, increase depot effect, or provide new functionality of the conjugate, such as fluorescence for tracking.

Peptide linker

The peptides of the disclosure (e.g., PD-L1 binding peptides or peptide complexes) can be linked or fused in a variety of ways. For example, the PD-L1 binding peptide may be linked or fused to an active agent (e.g., a therapeutic agent, an immune cell binding moiety, fc, or albumin binding peptide) via a peptide linker to form a PD-L1 binding peptide complex. In some embodiments, the peptide linker does not disrupt the independent folding of the peptide domain (e.g., of the PD-L1 binding peptide). In some embodiments, the peptide linker does not negatively impact the manufacturability (synthesis or recombination) of the peptide complex (e.g., PD-L1 binding peptide complex). In some embodiments, the peptide linker does not impair post-synthesis chemical changes (e.g., conjugation of a fluorophore or albumin binding chemical group) of the peptide complex (e.g., PD-L1 binding peptide complex).

In some embodiments, the peptide linker can connect the C-terminus of a first peptide (e.g., PD-L1 binding peptide) to the N-terminus of a second peptide (e.g., an active agent peptide). In some embodiments, the peptide linker can connect the C-terminus of the second peptide (e.g., the active agent peptide) to the N-terminus of the third peptide (e.g., the PD-L1 binding peptide).

In some embodiments, the linker may comprise tau-hu spider toxin-Hs 1a, also known as DkTx (binode toxin), which is extracted from a natural knottin-knottin dimer from chinese huwenchow (Haplopelma schmidti) (e.g., SEQ ID NO: 166). The linker may lack structural features that would interfere with dimerization of the individual functional proteins or peptides (e.g., PD-L1 binding peptides and immune cell targeting agents). In some embodiments, the linker may comprise a glycine-serine (Gly-Ser or GS) linker (e.g., SEQ ID NO: 154-165 or SEQ ID NO: 194-199). Gly-Ser linkers may have minimal chemical reactivity and may confer flexibility to the linker. Serine can increase the solubility of the linker or peptide complex because the hydroxyl groups on the side chains are hydrophilic. In some embodiments, the linker may be derived from a peptide (e.g., SEQ ID NO: 167) in the heavy chain of human immunoglobulin G that separates the Fc from the Fv domain. In some embodiments, the linker of the peptide from the heavy chain of human IgG may comprise a cysteine to serine mutation relative to the native IgG peptide.

In some embodiments, the peptides of the disclosure can dimerize using immunoglobulin heavy chain Fc domains. These Fc domains may be used to dimerize functional domains (e.g., PD-L1 binding peptides and immune cell targeting agents), based on antibodies or other additional soluble functional domains. In some embodiments, if the Fc sequence is native, dimerization may be homodimeric. In some embodiments, dimerization may be performed by mutating the Fc domains to produce a "knob-to-hole" form, but instead heterodimeric, wherein one Fc CH3 domain contains novel residues (knobs) designed to fit into cavities (holes) on the other Fc CH3 domain. The first peptide domain (e.g., PD-L1 binding peptide) can be coupled to a knob, and the second peptide domain (e.g., immune cell targeting agent) can be coupled to a knob. Pestle+pestle dimers can be highly energetically unfavorable. A purification tag can be added to the "pestle" side to remove the mortar+mortar dimer and select the pestle+mortar dimer.

The peptides of the disclosure (e.g., PD-L1 binding peptides) can be linked at the N-terminus or C-terminus to another peptide (e.g., an active agent peptide). In some embodiments, one or more peptides may be linked or fused by a peptide linker (e.g., a peptide linker comprising the sequence of any one of SEQ ID NO:154-SEQ ID NO:241 or SEQ ID NO: 433). For example, the PD-L1 binding peptide may be fused to the active agent via a peptide linker of any of SEQ ID NO:154-SEQ ID NO:241 or SEQ ID NO: 433. Peptide linkers (e.g., linkers linking the PD-L1 binding peptide and the active agent) can have a length of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid residues. The peptide linker may have a length of about 2 to about 5, about 2 to about 10, about 2 to about 20, about 3 to about 5, about 3 to about 10, about 3 to about 15, about 3 to about 20, about 3 to about 25, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 15 to about 20, about 15 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, about 3 to about 40, about 3 to about 30, about 10 to about 40, about 10 to about 30, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, or about 500 amino acids.

In some embodiments, the peptide may be appended to the N-terminus of any of the peptides of the disclosure, after the N-terminus GS dipeptide and before, for example, the GGGS (SEQ ID NO: 154) spacer. In some embodimentsIn embodiments, peptides (e.g., active agents) may use peptide linkers, such as G _x S _y The (SEQ ID NO: 155) peptide linker is appended to the N-or C-terminus of any of the peptides disclosed herein (e.g., PD-L1 binding peptides), wherein x and y are independently any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16 or 20, and the G and S residues are arranged in any order. In some embodiments, the peptide linker comprises (GS) x (SEQ ID NO: 156), where x can be any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 157), GGGGG (SEQ ID NO: 158), GSGSGSGS (SEQ ID NO: 159), GSGG (SEQ ID NO: 160), GGGGS (SEQ ID NO: 161), GGGS (SEQ ID NO: 154), GGS (SEQ ID NO: 162), GGGSGGGSGGGS (SEQ ID NO: 163), or variants or fragments thereof, or any number of repetitions and combinations thereof. In addition, KKYKPYVPVTTN from DkTx (SEQ ID NO: 166) and from human IgG ₃ EPKSSDKTHT (SEQ ID NO: 167) may be used as peptide linkers or any number of repeats and combinations thereof. In some embodiments, the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 164), or a variant or fragment thereof, or any number and combination thereof.

In some embodiments, the linker of the present disclosure may comprise a cleavable or stable linker moiety. In some embodiments, the cleavable linkers of the present disclosure may include, for example, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linkers), ultrasound sensitive linkers, and X-ray cleavable linkers. In some embodiments, the linker is not a cleavable linker.

The linker may comprise a plurality of amino acids. The linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more amino acids. The linker may comprise any of the linkers in table 9 below (where x=6-azidohexanoic acid and z=citrulline). In some cases, the active agent may be attached to the peptide using any one or more of the linkers shown in table 9 below. In some embodiments, the peptide linker comprises a linker of any one of SEQ ID NO:154-SEQ ID NO:241 or SEQ ID NO: 433. Examples of peptide linkers compatible with the peptide complexes of the present disclosure are provided in table 9. It will be appreciated that any of the foregoing linkers, or variants or fragments thereof, may be used with any number of repetitions or any combination thereof. It is also understood that other peptide linkers of the art, or variants or fragments thereof, may be used with any number of repetitions or any combination thereof.

TABLE 9 exemplary peptide linkers

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The linker may provide a minimum distance between the PD-L1 binding peptide and the active agent such that the active agent does not inhibit or prevent the PD-L1 binding peptide from binding to PD-L1. Similarly, the linker may provide a minimum distance between the PD-L1 binding peptide and the active agent (e.g., additional binding moiety) such that the PD-L1 binding peptide does not inhibit or prevent binding of the active agent to its target (e.g., immune cell target). The linker may be long enough to avoid steric hindrance of the active agent that inhibits binding of the peptide to PD-L1. When bound, the linker may be longer than the shortest distance of the N-terminal amine to PD-L1 in the peptide. The linker may be longer than the salt bridge, which may be 2-4 angstroms long. The length of the linker may be at least 5 angstroms, 10 angstroms, 20 angstroms, 40 angstroms or more. The linker may comprise at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, and at least 25 or more carbon, oxygen, nitrogen, sulfur, and/or phosphorus atoms in the linker backbone between the peptide and the oligonucleotide. The linker may comprise 1, 2, 3, 4, 5, 10, 15, 20 or more amino acids. The linker may comprise 1, 2, 3, 4, 5, 10, 20 or more nucleotide bases.

The peptide or peptide complex according to the present disclosure may be directly linked to another moiety, such as a small molecule, a second peptide, a second CDP, a protein, a mini-protein, a cytokine-receptor chain complex, an antibody fragment, an Fc pestle, an Fc mortar, an aptamer, a polypeptide, a polynucleotide, a fluorophore, a radioisotope, a radionuclide chelator, a polymer, a biopolymer, a fatty acid, an acyl adduct, a binding moiety, a chemical linker or sugar, an immune tumor agent, or other active agent described herein, by a linker or in the absence of a linker.

The peptide or peptide complex may be conjugated to a nucleotide, an active agent or a detectable agent through a linker, which may be described by the formula peptide-a-B-C-active agent, wherein the linker is a-B-C. A may be a stable amide bond with an amine or carboxylic acid on the peptide and linker and may be achieved via a Tetrafluorophenyl (TFP) ester, NHS ester or ATT group (thiazolidine-thione). A may also be a stable urethane linker, for example, formed by reacting an amine on a peptide with an imidazole carbamate reactive intermediate (formed by reacting CDI with a hydroxyl group on the linker). A may be a stable secondary amine bond, for example formed by reductive alkylation of an amine on the peptide with an aldehyde or ketone group on the linker. A may be a stable thioether linker, triazole linker, stable oxime linker or oxacarboline linker formed using maleimide or bromoacetamide in the linker with thiol in the peptide. A may comprise triazole. B may comprise (-CH 2-) _x- Short PEG (-CH) with or without branching ₂ CH ₂ O-) _x (x is 1-20), or short polypeptides such as GGGSGGGS (SEQ ID NO: 433), val-Ala (SEQ ID NO: 200), val-Cit (SEQ ID NO: 217), val-costCit-PABC, gly-Ile (SEQ ID NO: 222), gly-Leu (SEQ ID NO: 223), other spacers or NO spacers. C may be a disulfide bond, an amide bond, a triazole bond, a carbamate, a carbon-carbon single, double or triple bond, or an ester bond with a thiol, amine, hydroxyl, or carboxylic acid on the active agent. C may be a thioether, secondary or tertiary amine, carbamate or other stable bond formed between the maleimide on the linker and the sulfhydryl group on the active agent. In some embodiments, C may refer to a "cleavable" or "stable" portion of the joint. In other embodiments, a and/or B may also be "cleavable" or "stable" moieties. In some embodiments, a may be an amide, carbamate, thioether via maleimide or bromoacetamide, triazole, oxime, or oxacarboline. Any linker chemistry described in "Current ADC Linker Chemistry," Jain et al, pharm Res,2015DOI 10.1007/s11095-015-1657-7 or Bioconjugate Techniques, 3 rd edition, greg Hermanson may be used.

Methods of delivery and treatment using peptides and peptide complexes

In some embodiments, the PD-L1 binding peptides of the present disclosure can induce a biologically relevant response. In some embodiments, the biologically relevant response may be induced following intravenous, subcutaneous, intraperitoneal, intracranial, intrathecal, intratumoral, or intramuscular administration, and in some embodiments, following a single intravenous, subcutaneous, peritoneal, intracranial, or intramuscular administration. In some embodiments, the PD-L1 binding peptide or PD-L1 binding peptide complex may be used alone or in combination with various other classes of therapeutic compounds for treating various diseases or disorders, including cancer or immunological disorders (e.g., autoimmune diseases).

The term "effective amount" as used herein refers to a sufficient amount of an agent or compound that is administered to alleviate one or more symptoms of the disease or condition being treated to some extent. The result may be a alleviation and/or relief of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. Compositions containing such agents or compounds may be administered for achieving prophylactic, therapeutic, enhancing, and/or therapeutic treatment. The appropriate "effective" amount in any individual case can be determined using techniques such as dose escalation studies.

The methods, compositions, and kits of the present disclosure may comprise methods to prevent, treat, arrest, reverse, or ameliorate symptoms of a disorder. Treatment can include treating a subject (e.g., an individual afflicted with a disease or disorder, a domestic animal, a wild animal, or a laboratory animal) with a peptide or peptide complex of the disclosure. The disease may be cancer or a tumor. The disease may be an autoimmune disease. In treating a disease, the peptide may contact a tumor or cancer cell or an immune cell. The subject may be a human. The subject may be a human; non-human primates, such as chimpanzees, as well as other apes and monkey species; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, and the like. The subject may be at any age. The subject may be, for example, an elderly adult, an adolescent, a pre-pubertal adolescent, a child, a toddler (toddler), an infant, and a fetus in utero.

Treatment may be provided to the subject prior to the clinical onset of the disease. Treatment may be provided to the subject following a clinical episode of the disease. Treatment may be provided to the subject 1 day, 1 week, 6 months, 12 months, or 2 years or more after the clinical onset of the disease. Treatment may be provided to the subject after the clinical onset of the disease for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, or more. Treatment may be provided to the subject following a clinical episode of the disease for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years. Treatment may also include treating a human in a clinical trial. Treatment may include administering a pharmaceutical composition to a subject, e.g., one or more of the pharmaceutical compositions described throughout the present disclosure. Treatment may include once daily administration, twice daily administration, every other day administration, every third day administration, weekly administration, every other week administration, monthly administration, every third month administration, or every sixth month administration. Treatment may include delivering a peptide of the present disclosure to a subject intravenously, subcutaneously, intramuscularly, by inhalation, transdermally, locally, by intra-articular injection, orally, sublingually, intrathecally, transdermally, intranasally, by a peritoneal route, intratumorally (e.g., directly into a tumor, e.g., by injection), directly into the brain (e.g., by and intraventricular route), or directly onto a joint (e.g., by local, intra-articular injection route). Treatment may include administering the peptide-active agent complex to the subject intravenously, subcutaneously, intramuscularly, by inhalation, by intra-articular injection, transdermally, topically, orally, intrathecally, transdermally, intranasally, parenterally, orally, by peritoneal route, nasally, sublingually, or directly onto cancerous tissue. Intravenous administration may be in the form of a bolus or by infusion.

PD-L1 and PD-1 inhibition

The PD-L1 binding peptides (e.g., any of SEQ ID NOs: 1-118, 435, 436, or 554-567) or PD-L1 binding peptide complexes of the present disclosure can be administered to a subject (e.g., a human or non-human animal subject) to inhibit PD-L1 activity in the subject. PD-L1 activity may be associated with immunosuppression, T cell depletion, or immune function, and inhibiting PD-L1 may reduce immunosuppression, reduce T cell depletion, restore immune function, or a combination thereof. Inhibition of PD-L1 may be beneficial in diseases such as cancer, where PD-L1 positive cancer cells may evade host immune responses by inhibiting interactions between PD-L1 on cancer cells and PD-1 on host T cells. In some embodiments, inhibiting PD-L1 (e.g., by administering a PD-L1 binding peptide) can enhance the immune response of the host against cancer cells, thereby treating cancer. In some embodiments, inhibiting PD-L1 (e.g., by administering a PD-L1 binding peptide) may enhance a host immune response in the case of chronic infection where T cell depletion may be a problem or in sepsis or other acute infection.

The PD-L1 binding peptides of the present disclosure can inhibit PD-L1 by blocking interactions between PD-L1 and PD-1. For example, SEQ ID NO. 1 binds PD-L1 at the PD-1 binding interface, preventing PD-1 from entering the binding interface. In some embodiments, the PD-L1 binding peptides of the present disclosure can inhibit PD-L1 by binding to PD-L1 and stabilizing PD-L1 in an inactive conformation.

Administration of the PD-L1 binding peptide or peptide complex can be used in a method of treating cancer by binding and inhibiting PD-L1 after administration to a subject. Inhibition of PD-L1 can reduce T cell depletion and enhance the host immune response against cancer. The PD-L1 binding peptides described herein can be used to treat any PD-L1 positive cancer. Examples of cancers that may be treated by administration of the PD-L1 binding peptide or peptide complex include melanoma, non-small cell lung cancer, kidney cancer, esophageal cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, lymphoma, bladder cancer, liver cancer, gastric cancer, breast cancer, pancreatic cancer, prostate cancer, merck cell cancer, mesothelioma, or brain cancer (e.g., glioblastoma, astrocytoma, meningioma, metastatic brain cancer, or primary brain cancer).

Active agent delivery

PD-L1 binding peptide complexes of the present disclosure (e.g., complexes comprising peptides of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO:567 complexed with an active agent) are useful in methods of delivering an active agent to a cell, region, or tissue of interest in a subject. Following administration, the PD-L1 binding peptide complex can target and bind to PD-L1 (e.g., on PD-L1 positive cells) and deliver the active agent to the PD-L1-containing cell, tissue, or region. Targeting the active agent to PD-L1 positive cells, tissues, or regions may increase the therapeutic window of the active agent as compared to administration of the active agent alone, as targeted delivery may cause an increase in the concentration of the active agent at the target cell, tissue, or region as compared to surrounding tissues. This can reduce negative off-target effects, reduce the dosage required to produce a therapeutic effect, or both.

The PD-L1 binding peptide complex can be administered to deliver an active agent to any PD-L1 positive cell, region or tissue. For example, the PD-L1 binding peptide complex can deliver an active agent to cancer cells, immune cells, or pancreatic beta cells. Examples of agents that may be delivered to PD-L1 positive cells may include immune cell targeting agents, immune cells (e.g., T cells, B cells, macrophages, natural killer cells, fibroblasts, regulatory T cells, regulatory immune cells, neural stem cells, or mesenchymal stem cells), anti-cancer agents, chemotherapeutic agents, radiotherapeutic agents, pro-inflammatory cytokines, or oligonucleotides.

Chemotherapeutic or anti-cancer agents may act by killing or inhibiting proliferation of target cancer cells (e.g., PD-L1 positive cancer cells). Examples of chemotherapeutic or anti-cancer agents that can be complexed with the PD-L1 binding peptides of the present disclosure include anti-tumor agents, cytotoxic agents, tyrosine kinase inhibitors, mTOR inhibitors, retinoids, or anti-cancer antibodies. Proinflammatory cytokines can act by stimulating an immune response against a target (e.g., PD-L1 positive cancer cells). Examples of pro-inflammatory cytokines that can be complexed with the PD-L1 binding peptides of the present disclosure include TNFα, IL-2, IL-6, IL-12, IL-15, IL-21, or IFNγ. Anti-inflammatory agents may act by inhibiting inflammatory responses within or around the target (e.g., by inhibiting cyclooxygenase or stimulating glucocorticoid receptors). Examples of anti-inflammatory agents that can be complexed with the PD-L1 binding peptides of the present disclosure include anti-inflammatory cytokines, steroids, glucocorticoids, corticosteroids, or non-steroidal anti-inflammatory drugs (NSAIDs).

Oligonucleotides may function by modulating alternative splicing of a target sequence, indicating the position of a polyadenylation site of a target sequence, inhibiting translation of a target sequence, inhibiting binding of a target sequence to a secondary target sequence, recruiting RISC to a target sequence, recruiting RNaseH1 to a target sequence, inducing cleavage of a target sequence, or modulating a target sequence upon binding of an oligonucleotide to a target sequence. In some embodiments, the oligonucleotide may comprise an oncolytic viral vector, mRNA, miRNA, or siRNA.

In some embodiments, the PD-L1 binding peptide complex may be used to deliver an active agent to treat a disease or disorder associated with PD-L1. For example, the PD-L1 binding peptide complex can be administered to treat cancer (e.g., melanoma, skin cancer, non-small cell lung cancer, non-small cell lung cancer, kidney cancer, esophageal cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, colon cancer, rectal cancer, head and neck cancer, lymphoma, bladder cancer, liver cancer, stomach cancer, breast cancer, pancreatic cancer, prostate cancer, merck cell cancer, mesothelioma or brain cancer (including primary brain cancer or metastatic brain cancer), cancers that express PDL1, primary cancer, metastatic cancer, gastric cancer, squamous cell cancer, urothelial cancer or cervical cancer), autoimmune diseases (e.g., rheumatoid arthritis, atherosclerosis, ischemia reperfusion injury, colitis, psoriasis, lupus, inflammatory bowel disease, crohn's disease, ulcerative colitis, multiple sclerosis, type 1 diabetes or neuroinflammation), hyperglycemia, type 2 diabetes, infection, or neuronal injury. In some embodiments, the treatment of cancer may include delivering an anti-cancer agent or an immunostimulatory agent to the PD-L1 positive cancer cells. In some embodiments, the treatment of an autoimmune disorder may include delivering an anti-inflammatory agent or immunosuppressant to the PD-L1 positive immune cells, thereby reducing an autoimmune response in the subject. In some embodiments, the treatment of hyperglycemia may include delivering a protective agent to pancreatic beta cells, thereby preventing the onset of type 1 diabetes.

Nucleotide and oligonucleotide delivery

Peptides (e.g., PD-L1 binding peptides) can be linked, conjugated, complexed, or fused to nucleotides by various chemistries, thereby producing peptide oligonucleotide complexes that can form cleavable or stable bonds to deliver the oligonucleotides to cells. For example, in some embodiments, the PD-L1 binding peptide may bind to PD-L1 on the cell surface, which may then be taken up into early endosomes by endocytosis. The nucleotides and peptides in the PD-L1 binding peptide oligonucleotide complex may remain together (stable) or be cleaved (cleavable). If the bond is stable, the PD-L1 binding peptide oligonucleotide complex may be recycled back to the cell surface. Some PD-L1 binding peptide oligonucleotide complexes may enter low pH early endosomes. Once the nucleotides within the peptide oligonucleotide complex are exposed to the endosome, they may remain and are not degraded due to stabilization chemistry, for example, due to the oligonucleotide (backbone, sugar, bond, etc.) changes described herein. If the PD-L1 binding peptide includes additional cell penetration capacity, the peptide may facilitate accelerated escape of the oligonucleotide from the endosomal compartment into the cytosol. Even without increasing the cell penetration capacity, the oligonucleotides may slowly leak out of the endosome and into the cytosol.

In order to avoid recirculation to the cell surface or to facilitate endosomal escape of the oligonucleotide, cleavage of the PD-L1 binding peptide from the peptide oligonucleotide complex may be advantageous, in which case a cleavable linker may be used between the oligonucleotide and the peptide. Nucleotides in or cleaved from the peptide oligonucleotide complex may be transported between the cytosol and the nucleus actively or by passive diffusion. Some of the nucleotides in the peptide oligonucleotide complex may function in the nucleus, including vacancy mers, ASO splice blockers and U1 adaptors. Others play a role in the cytosol of cells, including siRNA and anti-miR. Aptamers are unique in that they do not function by hybridization or base pairing interactions with nucleic acid targets. In contrast, aptamers form a secondary structure to bind to proteins or other macromolecules. The aptamer may function wherever the target protein or macromolecule is located. For example, if the target is located on the cell surface, cell penetration through the internal volume may not be required, and it may be advantageous for the linker to be cleavable or non-cleavable, depending on the transport and stability of the PD-L1 binding peptide.

The nucleotide portion of the peptide oligonucleotide complexes described herein can target specific RNAs (e.g., mRNA or pre-mRNA) of genes expressed in cancer and other diseases. For example, the nucleotide sequence in the complex may be complementary to any of the targets provided in SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:549, table 10 or Table 17. The nucleotide sequence in the complex may be complementary to the target RNA or, in the case of an aptamer, may bind to a target protein or other macromolecule. The nucleotide sequence may be single-stranded (ssDNA, ssRNA) or double-stranded (dsDNA, dsRNA) or a combination of single and double strands (e.g., having a mismatched sequence, hairpin or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), oligonucleotide complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, gapmer, anti-miR, splice-blocking ASO, or U1 adaptor.

In some embodiments, the target of the nucleotide in the peptide oligonucleotide complex may be a gastrointestinal target, such as a gene having pro-inflammatory, extracellular matrix-modifying, or immune cell recruiting functions. Peptide oligonucleotide complexes described herein that target a gastrointestinal gene target (e.g., peptide oligonucleotide complexes comprising a PD-L1 binding peptide and nucleotides that bind to a gene target mRNA) can be used to treat various gastrointestinal disorders, including Inflammatory Bowel Disease (IBD), ulcerative colitis, and crohn's disease.

In some embodiments, the target of the nucleotide in the peptide oligonucleotide complex may be a cancer target, such as a gene involved in oncogene signaling, an anti-apoptotic gene, a pro-inflammatory signaling gene, a protein homeostasis gene, a developmental regulation gene, or an adaptor protein gene that initiates downstream cell growth signals. For example, targeting over-expressed growth factors such as HER2 can be challenging, but HER2 and other RTKs (e.g., EGFR, ERBB 3) signaling relies on adapter proteins such as Grb2 to initiate downstream cell growth signaling. The knockdown of Grb2 can stop signaling in a way that is difficult to mutate and compensate because Grb2 loss is episodic with HER2 activity. Cancer cells are often under low levels of protein toxic stress because they grow so fast that their protein folding mechanisms are difficult to keep up, thus targeting protein homeostatic genes such as Heat Shock Proteins (HSPs), hypoxia-sensing proteins (e.g., HIF), and up-regulation factors of the heat shock response can reduce protein toxic stress by helping to fold or stabilize the protein during folding. In some embodiments, the pro-inflammatory cytokine may be delivered through mRNA in the peptide oligonucleotide complex, or an antisense construct targeting the anti-inflammatory signal may be delivered. Delivery of pro-inflammatory signals or reduction of anti-inflammatory signals may aid in recruiting B cells, T cells, macrophages or other immune infiltrates into the tumor microenvironment. The peptide oligonucleotide complexes described herein that target a cancer gene target (e.g., peptide oligonucleotide complexes comprising a PD-L1 binding peptide and nucleotides that bind gene target mRNA) can be used to treat a variety of cancers, including solid tumors. Developmental regulators, such as transcription factors involved in early cell fate and pluripotency, and chromatin remodelling enzymes, may be targeted to specifically harm dedifferentiated cells that may be present in late stage tumors and associated with more mobile and/or mitotic cell states. Peptide oligonucleotide constructs targeting cancer targets can treat or prevent cancer by reducing oncogenic signaling, reducing target overexpression, reducing oncogenic antisense activity (e.g., mirnas targeting tumor suppressors), and/or eliminating the source of oncogenic signaling cascades.

Examples of gene targets (e.g., gastrointestinal tract or cancer gene targets) are provided in table 10.

TABLE 10 examples of disease-specific gene targets

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In some embodiments, the oligonucleotide may target a gene that is down-regulated. For example, the PvRBSA, pvRBP2b, pfEMP1, pfmdr1, pfgch1, GPX4, SLC7a11, alpha-synuclein, PD-L1, NUP98-KDM5A, NTRK1, JAK2, K-/N-RAS, JAK-STAT pathway, hedgehog pathway, PI3K/AKT pathway, RAF/MEK/ERK pathway, mTOR pathway, HDAC, MDM2, LSD1, CALR, PKC, NF-kappa B, HSP90, HIV Tat, TNF-alpha, CCR2, CCR5, TAR (Tat), RRE (rev), vpr, U5 leader, nef, gag, vif, env, IL1b, IL6, TNFa, IFNg, LRRK2, or myostatin may be targeted for down-regulation.

An example of An Da (antagomir) that can be complexed with a PD-L1 binding peptide to target a gene includes cobomassen (cobomassen). One example of an aptamer that can complex with a PD-L1 binding peptide to target a gene includes pipadatinib. Examples of vacancy mers that can be complexed with PD-L1 binding peptides to target genes include Fumivir, mi Bomei, inodorsen, velanesersen (volanessen), tolfensen (tofersen), tolmiphene (tominesen), perracainesen (pelalarsen), A Li Kafu (aliafsen), apathosen (apatosen), and Qu Beide (trabedersen). Examples of siRNAs that can be complexed with PD-L1 binding peptides to target genes include Parkibroccoli, vi Qu Xilan (vutrisiran), rafubroccoli (revusiran), cetuximab (fitusiran), lu Maxi blue (lumasiran), ji Foxi blue He Yingke broccoli (inclisiiran). Examples of splice blockers that can be complexed with the PD-L1 binding peptide to target a gene include sodium norcinacate, eptifibatide, golodirsen (golodirsen), viltolasen (viltolarsen), casimepirn (casimersen), and nipagin (sepofarsen). An example of a translational blocker that can be complexed with a PD-L1 binding peptide to target a gene includes prazibuxine (prexgebesen).

Any target of the nucleic acid portion of the peptide oligonucleotide complexes described herein can be used in combination with a U1 adapter to degrade the targeted mRNA. The target recognition (or complementary nucleic acid of the target mRNA) moiety directs the peptide oligonucleotide complex to the target mRNA selected for degradation, while the U1 moiety prevents addition of polyadenylic acid to the mRNA, resulting in degradation of the target mRNA. The U1 adapter may comprise any nucleotide sequence that is complementary to the ssRNA component of U1 ribonucleoprotein (U1 snRNP). In some embodiments, the U1 adapter sequence engages the U1 snRNP near its polyadenylation site. In some embodiments, the length of the U1 adapter is 15 to 25nt long or about 20nt long. In some embodiments, the G/C content of the U1 adapter is greater than 40%. Exemplary U1 adaptors are shown in table 11, along with a target nucleic acid "target recognition" portion comprising the nucleotide sequences: single-stranded (ssDNA, ssRNA) or double-stranded (dsDNA, dsRNA) or a combination of single-and double-stranded (e.g., having a mismatched sequence, hairpin, or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotides (ASO), micrornas (miRNA), oligonucleotides complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, gapmer, anti-miR, or splice-blocking ASO.10-19nt U1 adaptors are shown in italics.

TABLE 11 examples of target recognition constructs with U1 adaptors

Exemplary U1 adaptors include: UCCCCUGCCAGGUAAGUAU (SEQ ID NO: 366); CCCUGCCAGGUAAGUAU (SEQ ID NO: 367); CUGCCAGGUAAGUAU (SEQ ID NO: 368); UGCCAGGUAAGUAU (SEQ ID NO: 369); GCCAGGUAAGUAU (SEQ ID NO: 370); CCAGGUAAGUAU (SEQ ID NO: 371); CAGGUAAGUAU (SEQ ID NO: 372); and CAGGUAAGUA (SEQ ID NO: 373).

Detectable label and imaging

PD-L1 binding peptide complexes of the present disclosure (e.g., complexes comprising a peptide of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO:567 complexed with a detectable agent) are useful in methods of labeling a cell, region, or tissue of interest in a subject. Following administration, the PD-L1 binding peptide complex can target and bind to PD-L1 (e.g., on PD-L1 positive cells) and deliver a detectable agent to a cell, tissue, or region containing PD-L1. The detectable agent of the peptide complex can generate a detectable signal and can be used to label a cell or tissue (e.g., a PD-L1 positive cell or tissue). In some embodiments, generating a detectable signal may include emitting fluorescence (e.g., visible light, ultraviolet light, or infrared light), emitting electromagnetic radiation, absorbing electromagnetic radiation (e.g., light or X-rays), generating a contrast signal, generating an electron spin signal, emitting radiation, generating a magnetic signal, or a combination thereof. Examples of detectable agents that can be complexed with the PD-L1 binding peptides of the present disclosure include fluorophores, near infrared dyes, contrast agents, nanoparticles, metal-containing nanoparticles, metal chelates, X-ray contrast agents, PET agents, radionuclides, or radionuclide chelators.

Delivery of a detectable agent to a PD-L1 positive region can be used in a method of diagnosing a disease or disorder in a subject, such as a disorder associated with PD-L1. For example, a PD-L1 binding peptide complex comprising a detectable agent may be administered to a subject having or suspected of having cancer. The peptide complex can target and bind to PD-L1 positive cancer cells, thereby labeling PD-L1 positive cancer cells. The presence and location of the detectable agent can be imaged to diagnose cancer.

Immune cell recruitment

PD-L1 binding peptide complexes of the present disclosure (e.g., complexes comprising peptides of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO:567 complexed with immune cell targeting agents) are useful in methods of recruiting immune cells to PD-L1 positive cells, tissues, or regions. The PD-L1 binding peptide complex may comprise a bispecific immunocyte cement. After administration, the bispecific immune cell cement can bind to PD-L1 positive cells via PD-L1 binding peptides and to immune cells (e.g., T cells, B cells, macrophages, natural killer cells, fibroblasts, regulatory T cells, regulatory immune cells, neural stem cells, or mesenchymal stem cells) via immune cell targeting agents. Bispecific immunocyte cement can recruit immune cells to PD-L1 positive cells. In some embodiments, recruitment of immune cells may stimulate an immune response against a target cell. For example, recruitment of T cells, NK cells, macrophages, fibroblasts, regulatory immune cells, neural stem cells, or mesenchymal stem cells to PD-L1 positive cancer cells may induce an immune response in the host against the cancer cells or otherwise modulate the microenvironment surrounding the PD-L1 positive tissue. In some embodiments, recruitment of immune cells may inhibit an immune response against a target cell. For example, regulatory T cells (T _reg ) Recruitment to pancreatic beta cells may protect pancreatic beta cells and prevent the onset of type 1 diabetes. Type 1 diabetes occurs when T cells bind to insulin-producing pancreatic beta cells and attack them as autoimmune disorders. By PD-L1+ beta cells and T _reg Double-engagement of cells will T _reg Cell recruitment to islets may reduce this autoimmune destruction of beta cells. In another example, recruitment of regulatory T cells or natural killer cells to T cells involved in an autoimmune response can inhibit the autoimmune response and treat autoimmune disorders. Regulatory T cells recruited to inflamed tissue may produce anti-inflammatory signaling, thereby reducing inflammation at the inflamed tissue site. The development of anti-inflammatory signaling at the site of PD-L1 positive islet cells invaded by activated T cells may delay, slow or reverse type 1 diabetes. In some embodiments, biICE comprising a PD-L1 binding peptide (e.g., a peptide of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) may be used in a method of recruiting a regulatory T cell or natural killer cell to a PD-L1 positive cell, tissue or region complexed with a regulatory T cell or natural killer cell binding moiety. The recruitment of neural stem cells may treat sites or neuronal damage. Anti-inflammatory, regulatory immune cell (e.g., treg) recruitment may be helpful in chronic or sepsis or acute infections. Bispecific immunocyte cements using PD-L1 binding peptides can also be administered to treat cancers (e.g., melanoma, skin cancer, non-small cell lung cancer, kidney cancer, esophageal cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, colon cancer, rectal cancer, head and neck cancer, lymphoma, bladder cancer, liver cancer, stomach cancer, breast cancer, pancreatic cancer, prostate cancer, merck cell cancer, mesothelioma, or brain cancer, including primary brain cancer or metastatic brain cancer, cancers that express PDL1, primary cancer, metastatic cancer), autoimmune or inflammatory diseases (e.g., rheumatoid arthritis, atherosclerosis, ischemia reperfusion injury, colitis, psoriasis, lupus, inflammatory bowel disease, crohn's disease, ulcerative colitis, multiple sclerosis, type 1 diabetes or neuro-inflammation), hyperglycemia, type 2 diabetes, infection, or neuronal injury.

CAR T cell therapy

The PD-L1 binding peptide complexes of the disclosure (e.g., chimeric antigen receptors comprising a PD-L1 binding peptide of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567) can be used in methods of recruiting T cells to PD-L1 positive cells, tissues, or regions. For example, a Chimeric Antigen Receptor (CAR) that binds PD-L1 may be used in CAR T. The chimeric antigen receptor that binds PD-L1 can be expressed in T cells (e.g., T cells collected from a subject), and T cells expressing the CAR can be administered back to the subject. The PD-L1 binding peptide of the CAR can deliver T cells to PD-L1 positive cells (e.g., PD-L1 positive cancer cells) of the subject. CAR T cells can stimulate an immune response against PD-L1 positive cells. In some embodiments, the PD-L1-binding CAR can be administered to treat cancer (e.g., melanoma, skin cancer, non-small cell lung cancer, kidney cancer, esophageal cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, colon cancer, rectal cancer, head and neck cancer, lymphoma, bladder cancer, liver cancer, stomach cancer, breast cancer, pancreatic cancer, prostate cancer, mercker cell cancer, mesothelioma, or brain cancer, including primary brain cancer or metastatic brain cancer, PDL 1-expressing cancer, primary cancer, metastatic cancer).

Peptide stability

The peptides of the disclosure may be stable under a variety of biological or physiological conditions, such as physiological extracellular pH, endosomal or lysosomal pH, or a reducing environment inside a cell, in the cytosol, in the nucleus or endosome or tumor. For example, any peptide or peptide complex comprising any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO:567 may exhibit resistance to reducing agents, proteases, oxidizing conditions, or acidic conditions.

In some cases, biomolecules (e.g., peptides and proteins) may provide therapeutic functions, but such therapeutic functions are reduced or blocked due to instability caused by the in vivo environment. (Moroz et al Adv Drug Deliv Rev 101:108-21 (2016); mitragotri et al Nat Rev Drug Discov (9): 655-72 (2014); bruno et al Ther Deliv (11): 1443-67 (2013); sinha et al Crit Rev Ther Drug Carrier Syst.24 (1): 63-92 (2007); hamman et al Biodrugs 19 (3): 165-77 (2005)). For example, the GI tract may contain a low pH (e.g., about pH 1) region, a reducing environment, or a protease-rich environment that can degrade peptides and proteins. Proteolytic activity in other areas of the body, such as the mouth, eyes, lungs, nasal cavity, joints, skin, vagina, mucous membranes and serum can also be an obstacle to the delivery of functionally active peptides and polypeptides. In addition, the half-life of a peptide in serum may be extremely short due in part to proteases, such that when administered in a reasonable dosing regimen, the peptide may be degraded too rapidly to have a sustained therapeutic effect. Likewise, proteolytic activity in cellular compartments such as lysosomes, as well as reductive activity in lysosomes and cytosol, can degrade peptides and proteins so that they cannot provide therapeutic function to intracellular targets. Thus, peptides resistant to reducing agents, proteases and low pH can provide enhanced therapeutic effects, or enhance therapeutic efficacy in vivo of co-formulated or conjugated, linked or fused active agents.

In addition, oral delivery of drugs may be desirable to target certain areas of the body (e.g., diseases in the GI tract such as colon cancer, irritable bowel disorder, infection, metabolic disorder, and constipation), although such methods of administration present a barrier to the delivery of functionally active peptides and polypeptides. For example, oral delivery of a drug may increase compliance by providing a dosage form that is more convenient for a patient to take than parenteral delivery. Oral delivery can be used in treatment regimens with large therapeutic windows. Thus, peptides that are resistant to reducing agents, proteases, and low pH may allow oral delivery of peptides without compromising their therapeutic function.

The PD-L1 binding peptides or peptide complexes of the present disclosure may contain one or more cysteines that may participate in disulfide bridges that may be necessary to maintain the folded state of the peptide. Exposure of the peptide to a biological environment with a reducing agent can result in the deployment of the peptide, as well as loss of functionality and biological activity. For example, glutathione (GSH) is a reducing agent that may be present in many areas of the body, in the blood, and inside cells, and may reduce disulfide bonds. As another example, following oral administration, the peptide may become reduced during migration of the peptide across the gastrointestinal epithelium. The peptide may become reduced upon exposure to various portions of the GI tract. The GI tract may be a reducing environment that may inhibit the ability of therapeutic molecules with disulfide bonds to have optimal therapeutic efficacy due to the reduction of disulfide bonds. Peptides may also be reduced after entry into cells, for example, by endosomal or lysosomal internalization, or into the cytosol or other cellular compartments. Reduction of disulfide bonds and expansion of peptides can lead to loss of functionality or affect key pharmacokinetic parameters such as bioavailability, peak plasma concentration, bioactivity, and half-life. Reduction of disulfide bonds can also result in loss of functionality due to increased susceptibility of the peptide to subsequent degradation by proteases, which leads to rapid loss of the intact peptide after administration. In some embodiments, peptides that are resistant to reduction may remain intact in various compartments of the body and in the cells for a longer period of time than peptides that are more susceptible to reduction, and may confer functional activity.

In certain embodiments, the peptides of the present disclosure can be analyzed for their resistance characteristics to reducing agents to identify stable peptides. In some embodiments, the peptides of the present disclosure may remain intact after exposure to different molar concentrations of reducing agent, e.g., 0.00001M-0.0001M, 0.0001M-0.001M, 0.001M-0.01M, 0.01M-0.05M, 0.05M-0.1M, or 0.1M to 0.2M, for 15 minutes or more. In some embodiments, the reducing agent used to determine the stability of the peptide may be Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 2-mercaptoethanol, (reduced) Glutathione (GSH), or any combination thereof. In some embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90%, or at least 90% -100% of the peptide remains intact after exposure to the reducing agent. In some embodiments, the peptide is fully resistant to GSH reducing conditions and is partially resistant to degradation under DTT reducing conditions. In some embodiments, the peptides described herein can withstand, or be resistant to, degradation under physiological reducing conditions.

The stability of the peptides of the present disclosure can be determined by resistance to degradation by proteases. Proteases, also known as peptidases or proteases, are enzymes that degrade peptides and proteins by breaking bonds between adjacent amino acids. Families of proteases specific for targeting a particular amino acid may include serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and asparagine proteases. In addition, metalloproteases, matrix metalloproteases, elastase, carboxypeptidase, cytochrome P450 enzymes, and cathepsins can digest peptides and proteins. Proteases may be present in high concentrations in the blood, in mucous membranes, lungs, skin, GI tract, mouth, nose, eyes, and in cellular compartments. Deregulation of proteases may also occur in a variety of diseases, such as rheumatoid arthritis and other immune disorders. Degradation by proteases can reduce the bioavailability, biodistribution, half-life and bioactivity of therapeutic molecules so that they cannot perform their therapeutic function. In some embodiments, protease resistant peptides may better provide therapeutic activity in vivo at reasonably tolerated concentrations.

In some embodiments, the peptides of the disclosure are resistant to degradation by any class of proteases. In certain embodiments, the peptides of the present disclosure are resistant to degradation by pepsin (as may be found in the stomach), trypsin (as may be found in the duodenum), serum protease, or any combination thereof. In some embodiments, the protease used to determine peptide stability may be pepsin, trypsin, chymotrypsin, or any combination thereof. In certain embodiments, the peptides of the present disclosure are resistant to degradation by pulmonary proteases (e.g., serine, cysteinyl, and aspartyl proteases, metalloproteases, neutrophil elastase, alpha-1 antitrypsin, secreted leukocyte protease inhibitors, and elastase inhibitors), or any combination thereof. In some embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% of the peptide remains intact after exposure to the protease.

The peptides of the present disclosure can be administered in an acidic biological environment. For example, following oral administration, peptides may be subjected to acidic environmental conditions in the gastric juice and Gastrointestinal (GI) tract of the stomach. The pH of the stomach may be in the range of about 1-4 and decrease from the upper GI tract to the colon, with the pH of the GI tract being in the range of acidic to normal physiological pH. In addition, the vagina, late endosomes and lysosomes can also have acidic pH values, e.g., less than pH 7. These acidic conditions can cause the peptides and proteins to denature into an expanded state. The unfolding of peptides and proteins can lead to increased susceptibility to subsequent digestion by other enzymes, as well as loss of biological activity of the peptide. In certain embodiments, the peptides of the present disclosure are resistant to denaturation and degradation under acidic conditions as well as in buffers that mimic acidic conditions. In certain embodiments, the peptides of the present disclosure are resistant to denaturation or degradation in buffers having a pH of less than 1, a pH of less than 2, a pH of less than 3, a pH of less than 4, a pH of less than 5, a pH of less than 6, a pH of less than 7, or a pH of less than 8. In some embodiments, the peptides of the disclosure remain intact at a pH of 1-3. In certain embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% of the peptide remains intact after exposure to a buffer having a pH of less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7 or less than 8. In other embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% of the peptides remain intact after exposure to a buffer having a pH of 1-3. In other embodiments, the peptides of the present disclosure may be resistant to denaturation or degradation in simulated gastric fluid (pH 1-2). In some embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% of the peptide remains intact after exposure to simulated gastric fluid. In some embodiments, low pH solutions, such as simulated gastric fluid, may be used to determine peptide stability.

In some embodiments, the peptides described herein are resistant to degradation in vivo, in the serum of a subject, or inside a cell. In some embodiments, the peptide is stable at physiological pH ranges, for example, between about pH7, about pH 7.5, between about pH 5 and 7.5, between about 6.5 and 7.5, between about pH 5 and 8, or between about pH 5 and 7. In some embodiments, the peptides described herein are stable under acidic conditions, e.g., less than or equal to about pH 5, less than or equal to about pH 3, or in the range of about 3 to about 5. In some embodiments, the peptide is stable under endosomal or lysosomal conditions or within the nucleus.

The peptides of the present disclosure can be administered in a biological environment having a high temperature. For example, following oral administration, peptides may be subjected to elevated temperatures in the body. The body temperature may be in the range of 36 ℃ to 40 ℃. The high temperature can cause the peptides and proteins to denature into an expanded state. The unfolding of peptides and proteins can lead to increased susceptibility to subsequent digestion by other enzymes, as well as loss of biological activity of the peptide. In some embodiments, the peptides of the present disclosure may remain intact at a temperature of 25 ℃ to 100 ℃. High temperatures can lead to faster degradation of peptides. Stability at higher temperatures may allow for storage of peptides in tropical environments or in areas where access to refrigeration is limited. In certain embodiments, 5% -100% of the peptide may remain intact after exposure to 25 ℃ for 6 months to 5 years. After exposure to 70 ℃ for 15 minutes to 1 hour, 5% -100% of the peptide may remain intact. After exposure to 100 ℃ for 15 minutes to 1 hour, 5% -100% of the peptide may remain intact. In other embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% of the peptides remain intact after exposure to 25 ℃ for at least 6 months to 5 years. In other embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% of the peptides remain intact after exposure to 70 ℃ for 15 minutes to 1 hour. In other embodiments, at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% of the peptide remains intact after exposure to 100 ℃ for 15 minutes to 1 hour.

Method of manufacture

Various expression vector/host systems may be used to recombinantly express the peptides described herein. Non-limiting examples of such systems include microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing nucleic acid sequences encoding the peptides, peptide complexes, or peptide fusion proteins/chimeric proteins described herein, yeasts transformed with recombinant yeast expression vectors containing the above-mentioned nucleic acid sequences, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the above-mentioned nucleic acid sequences, plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., ti plasmid) containing the above-mentioned nucleic acid sequences, or animal cell systems infected with recombinant viral expression vectors (e.g., adenovirus, vaccinia virus, lentivirus), including cell lines engineered to contain multiple copies of the above-mentioned nucleic acid sequences that are stably amplified (e.g., CHO/dhfr, CHO/glutamine synthetase), or that are not stably amplified in double microcolonies (e.g., murine cell lines). Disulfide bond formation and folding of the peptide may occur during expression, or after expression, or both during and after expression.

The host cell may be suitable for expressing one or more peptides described herein. The host cell may be a prokaryotic, eukaryotic, or insect cell. In some cases, the host cell is capable of modulating the expression of the inserted sequence, or modifying and processing the gene or protein product in a particular manner as desired. For example, expression from certain promoters may be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for metallothionein promoters). In some cases, modification (e.g., phosphorylation) and processing (e.g., cleavage) of the peptide product may be important for the function of the peptide. Host cells may have features and specific mechanisms for post-translational processing and modification of peptides. In some cases, the host cell used to express the peptide secretes a minimal amount of proteolytic enzyme.

The PD-L1 binding peptides or peptide complexes of the present disclosure can advantageously be prepared by a single recombinant expression system without the need for chemical synthesis or modification. For example, the PD-L1 binding peptide or peptide complex may be expressed in CHO cells, yeast, pichia pastoris, e.coli, or other organisms.

In the case of a cell or virus-based sample, the organism may be treated to retain and/or release the target polypeptide prior to purification. In some embodiments, the cells are fixed using a fixative. In some embodiments, the cells are lysed. The cellular material may be treated in a manner that does not disrupt a significant proportion of the cells, but removes proteins from the surface of the cellular material and/or from interstices between the cells. For example, the cellular material may be immersed in a liquid buffer, or in the case of plant material, may be subjected to a vacuum to remove proteins located in the cell gap and/or in the plant cell wall. If the cellular material is a microorganism, the protein may be extracted from the microorganism culture medium. Alternatively, the peptide may be packaged in inclusion bodies. The inclusion bodies may be further separated from cellular components in the medium. In some embodiments, the cells are not destroyed. The cellular or viral peptides presented by the cells or viruses may be used for attachment and/or purification of whole cells or viral particles. In addition to recombinant systems, peptides can be synthesized in cell-free systems using a variety of known techniques employed in protein and peptide synthesis, followed by extraction.

In some cases, the host cell produces a peptide having a point of attachment for a cargo molecule (e.g., a therapeutic agent). The point of attachment may comprise a lysine residue, an N-terminal, a cysteine residue, a cysteine disulfide, a glutamic acid or aspartic acid residue, a C-terminal or unnatural amino acid. Peptides may also be produced synthetically, for example by solid phase peptide synthesis or solution phase peptide synthesis. Peptide synthesis can be performed by fluorenylmethoxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. The peptide may be folded (disulfide bond formed) during synthesis, or after synthesis, or both during and after synthesis. Peptide fragments may be produced synthetically or recombinantly. The peptide fragments may then be joined together enzymatically or synthetically.

In other aspects, the peptides of the present disclosure can be prepared by conventional solid phase chemical synthesis techniques, for example, according to the Fmoc solid phase peptide synthesis method ("Fmoc solid phase peptide synthesis, apractical approach," w.c.chan and p.d.white edit, oxford University Press, 2000).

Nucleic acids, including RNA and DNA polynucleotides, used in the peptide nucleotide complexes described therein can also be produced using the methods described in U.S. patent No. 9,279,149, which is incorporated herein by reference. In some embodiments, the RNA or DNA polynucleotide is synthesized by an enzymatic/PCR method. For example, an RNA polynucleotide may be synthesized using an enzyme such as a nucleotide transferase (e.g., escherichia coli poly (adenylate) polymerase or escherichia coli poly (uridylate) polymerase), which may add an RNA nucleotide to the 3' end. Alternatively, E.coli poly (uridylic acid) polymerase may be used. A 3' unblocked reversible terminator ribotriphosphate (rtp) can be used during polynucleotide synthesis. Alternatively, a 3', 2', or 2'-3' blocked rNTP may be used with any of the enzymes described above. RNA or DNA polynucleotides can also be synthesized using standard solid phase synthesis techniques and phosphoramidate-based or phosphorothioate-based methods. The RNA or DNA polynucleotides of the present disclosure can be prepared by conventional solid phase oligonucleotide synthesis. For example, any solid phase synthesis method may be employed, including, but not limited to, the methods described below: as shown in https:// www.atdbio.com/content/17/Solid-Phase-oligoside-Synthesis, and Albericci (Solid-Phase Synthesis: A practical guide, CRC Press, 2000); lambert et al (Oligonucleotide Synthesis: solid-Phase Synthesis, DNA, DNA Sequencing, RNA, small Interfering RNA, nucleic Acid, phosphoamidite, sense, betascript Publishing, 2010) and Guzaev, A.P. et al (Current Protocols in Nucleic Acid chemistry.2013; 53:3.1:3.1.1-3.1.60), each of which is incorporated herein by reference. Solid supports such as CPG or polystyrene may be used. Protected 2' -deoxynucleosides (dA, dC, dG and T), ribonucleosides (A, C, G and U) or chemically modified nucleosides, such as LNA or BNA, can be used. Phosphoramidate chemistry can be recycled by the steps of: detritylation, activation and coupling, capping and oxidation of the carrier-bound 3' -nucleoside. At the end of the synthesis, the protected nucleotide may be cleaved from the vector and then deprotected. The product may be purified by HPLC. Protecting groups for solid phase synthesis of RNA polynucleotides may include t-butyldimethylsilyl (TBDMS) or Triisopropylsiloxymethyl (TOM). RNA or DNA polynucleotides may have modified backbones to enhance stability. Furthermore, non-natural or modified bases can be used as unique functional handles for subsequent chemical conjugation. In some embodiments, the 5 'and/or 3' ends of the RNA or DNA may be modified to produce a desired functional group, stability, or activity. In some embodiments, the functional handle comprises modified bases, including one or more modified uridine, modified guanosine, modified cytidine, or modified adenosine bases of the RNA. An example of such a modified base is uridine with extended amines. Nucleic acids can be made using such methods, including single-stranded (ssDNA, ssRNA) or double-stranded (dsDNA, dsRNA) or a combination of single-and double-stranded (e.g., having a mismatched sequence, hairpin, or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microrna (miRNA), oligonucleotides complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, aptamer, gapmer, anti-miR, splice-blocking ASO, or U1 adaptor. It may be advantageous to manufacture the oligonucleotides and peptides by synthetic methods and then conjugate them together, thereby increasing purity, safety and commodity costs. Oligonucleotides, including modified oligonucleotides, can be made by any of the methods disclosed in: glazier et al Chemical synthesis and biological application of modified oligonucleotides, bioconjugation Chemistry,2020,31,1213-1233.

In some embodiments, the peptides of the disclosure may be more stable during manufacture. For example, the peptides of the present disclosure may be more stable during recombinant expression and purification, such that the rate of degradation by proteases present in the manufacturing process is lower, the purity of the peptides is higher, the yield of peptides is higher, or any combination thereof. In some embodiments, the peptides may also be more stable to degradation at high and low temperatures during manufacture, storage, and sale. For example, in some embodiments, the peptides of the present disclosure may be stable at 25 ℃. In other embodiments, the peptides of the disclosure may be stable at 70 ℃ or above 70 ℃. In some embodiments, the peptides of the disclosure may be stable at 100 ℃ or above 100 ℃.

Pharmaceutical composition

The pharmaceutical compositions of the present disclosure may be a combination of any peptide as described herein with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, antioxidants, solubilizers, buffers, osmotic agents (e.g., sugars, disaccharides, and sugar alcohols), salts, surfactants, amino acids, encapsulating agents, bulking agents, cryoprotectants, and/or excipients. The pharmaceutical compositions facilitate administration of the peptides described herein to an organism. In some cases, the pharmaceutical composition comprises a factor that increases the half-life of the peptide and/or aids in the penetration of the peptide into the target cell. In some embodiments, the pharmaceutical composition comprises a cell modified to express and secrete a PD-L1 binding peptide or peptide complex of the present disclosure.

The pharmaceutical compositions may be administered in therapeutically effective amounts in the form of pharmaceutical compositions by a variety of forms and routes including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, ophthalmic, nasal, oral, sublingual, inhalation, dermal, intrathecal, intratumoral, intranasal, and topical administration. The pharmaceutical composition may be administered locally or systemically, e.g., by injection of the peptides described herein directly into the organ, optionally in a depot.

Parenteral injection may be formulated for bolus injection, infusion or continuous infusion. The pharmaceutical compositions may be in a form suitable for parenteral injection, in sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the peptides described herein in water-soluble form. Suspensions of the peptide-antibody complexes described herein may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. The aqueous injection suspension may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain suitable stabilizers or agents that increase the solubility of such peptide-antibody complexes described herein and/or reduce aggregation of the complexes to allow for the preparation of highly concentrated solutions.

Alternatively, the peptides described herein may be lyophilized, or in powder form for reconstitution with a suitable vehicle, such as sterile pyrogen-free water, prior to use. In some embodiments, the purified peptide is administered intravenously. The peptides described herein can be administered to a subject to home to, target to, migrate to, or target to CNS cells, brain cells, cancer cells, or tumors. In some embodiments, the peptide may be conjugated, linked or fused to another peptide that provides a targeting function for a particular target cell type in the central nervous system or across the blood brain barrier. Exemplary target cells include CNS cells, erythrocytes, erythrocyte precursor cells, immune cells, stem cells, muscle cells, brain cells, thyroid cells, parathyroid cells, adrenal cells, bone marrow cells, appendiceal cells, lymph node cells, tonsil cells, spleen cells, muscle cells, liver cells, gall bladder cells, pancreas cells, gastrointestinal tract cells, glandular cells, kidney cells, bladder cells, endothelial cells, epithelial cells, choroid plexus epithelial cells, neurons, glial cells, astrocytes, or cells associated with the nervous system.

The peptides of the present disclosure may be applied directly to an organ or organ tissue or cell, such as the brain or brain tissue or cell, during a surgical procedure. The recombinant peptides described herein can be topically administered and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, sticks, balms, creams and ointments. Such pharmaceutical compositions may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a peptide described herein can be administered in the form of a pharmaceutical composition to a subject suffering from a disorder, e.g., a disorder affecting the immune system. In some embodiments, the subject is a mammal, e.g., a human or primate. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors.

In some embodiments, the peptide is cloned into a viral or non-viral expression vector. Such expression vectors may be packaged in viral particles, virosomes or non-viral vectors or delivery mechanisms for administration to a patient in the form of gene therapy. In other embodiments, patient cells are extracted and modified to express a peptide capable of binding PD-L1 ex vivo, and the modified cells are then returned to the patient in the form of a cell-based therapy, such that the modified cells will express the peptide once transplanted back into the patient.

Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Depending on the route of administration selected, the formulation may be modified. Pharmaceutical compositions comprising the peptides described herein can be manufactured, for example, by: expressing the peptide in a recombinant system, purifying the peptide, buffer exchanging the peptide, lyophilizing the peptide, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, or compacting the peptide. The pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier, diluent or excipient, a compound described herein in free base or pharmaceutically acceptable salt form.

Methods for preparing the peptides described herein comprising the compounds described herein include formulating the peptides described herein with one or more inert pharmaceutically acceptable excipients or carriers to form solid, semi-solid, or liquid compositions. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. These compositions may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.

Non-limiting examples of pharmaceutically acceptable excipients can be found, for example, in the following: remington, the Science and Practice of Pharmacy, 19 th edition (Easton, pa.: mack Publishing Company, 1995); hoover, john e., remington' sPharmaceutical Sciences, mack Publishing co., easton, pennsylvania1975; liberman, h.a. and Lachman, l. Edit, pharmaceutical Dosage Forms, marcel Decker, new York, n.y.,1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, seventh edition (Lippincott Williams & Wilkins 1999), each of which is incorporated herein by reference in its entirety.

The pharmaceutical composition may also include a permeation or absorption enhancer (Aungst et al AAPS J.14 (1): 10-8. (2012) and Moroz et al Adv Drug Deliv Rev 101:108-21. (2016)). Permeation enhancers may aid in the uptake of molecules from the GI tract into the systemic circulation. Penetration enhancers may include salts of medium chain fatty acids, sodium caprate, sodium caprylate, N- (8- [ 2-hydroxybenzoyl ] amino) caprylic acid (SNAC), N- (5-chlorosalicyl) -8-amino caprylic acid (5-CNAC), hydrophilic aromatic alcohols (e.g., phenoxyethanol), benzyl alcohol and phenyl alcohol, chitosan, alkyl glycosides, dodecyl-2-N, N-dimethylaminopropionate (DDAIPP), chelators of divalent cations (including EDTA, EGTA and citric acid), sodium alkyl sulfate, sodium salicylate, lecithin-based agents or bile salt derivatizing agents, such as deoxycholate.

The composition may further comprise protease inhibitors including soybean trypsin inhibitor, aprotinin, sodium glycocholate, camostat mesylate (camostat mesilate), bacitracin or cyclopentadecanolide.

Medicine box

In one aspect, the peptides described herein can be provided in a kit form. In another embodiment, the peptide complexes described herein may be provided in a kit form. In another embodiment, the kit comprises amino acids encoding the peptides described herein, vectors, host organisms and instruction manual. In some embodiments, the kit includes written instructions for use or administration of the peptide.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments and its several details are capable of modification in various respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

As used herein, the terms "about" and "approximately" with respect to a number are used herein to include numbers that fall within the range of 10%, 5%, or 1% of the number in either direction (greater or less), unless otherwise indicated or otherwise apparent from the context (except for such numbers that would exceed 100% of the possible values).

Examples

The invention is further illustrated by the following non-limiting examples.

Example 1

Peptide production

This example describes the manufacture of peptides and peptide complexes described herein (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO: 567). Peptides derived from proteins are produced in mammalian cell culture using published methods. (A.D.Bandaranayke, C.Correnti, B.Y.Ryu, M.Brault, R.K.Strong, D.Rawlings.2011.Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids research. (39) 21, e 143).

Peptide sequences were reverse translated into DNA, synthesized, and cloned in-frame with ferritin using standard molecular biology techniques (m.r.green, joseph sambrook. Molecular cloning.2012cold Spring Harbor Press). The resulting construct was packaged into lentiviruses, transduced into HEK-293 cells, amplified, isolated by Immobilized Metal Affinity Chromatography (IMAC), cleaved with Tobacco Etch Virus (TEV) protease, and purified by reverse phase chromatography to obtain homogeneity. After purification, each peptide was lyophilized and stored frozen.

Example 2

Construction of cystine compact peptide screening library

This example describes the construction of a cystine dense peptide screening library using a surface folding model and protease resistance data. Surface display can be used as a highly efficient method for screening peptides for target engagement, and mammalian surface display may be particularly useful for using Cystine Dense Peptides (CDPs) that are capable of folding the secretory pathway of cysteine-rich surface proteins. A mammalian surface display system for screening an improved cystine dense peptide library is illustrated in FIG. 1A. Under conditions where CDP is unlabeled but cells are stained with an affinity-labeled (e.g., biotinylated) target protein and labeled with a fluorescent co-stain (e.g., streptavidin), cells expressing the target binding CDP are sorted and grown for several rounds to enrich for the sequence of interest (left, "vector SDGF"). Under conditions where the CDP itself is labeled (e.g., 6XHis (SEQ ID NO: 248)), a fluorescent co-stain (e.g., anti-6 XHis) is used to detect the complete CDP on the cell surface (right panel, "vector SDPR").

CDP libraries were scaled down to a subset of CDP scaffolds (n=953), which were particularly high in score in quantitative surface folding assays. The composite surface folding score, which combines surface expression and protease resistance, was used to evaluate the surface folding of large CDP library members, as shown in fig. 1B. The highest scoring CDP represents a cystine scaffold predicted to have high surface expression, protease resistance, or both. A database of 96,000 CDP sequences collected from the public sequence database (Swiss-Prot and TrEMBL) was searched to identify peptides having structural or sequence homology to the 953 CDP scaffolds identified. Additional 7,940 CDPs were identified and combined with 953 identified CDP scaffolds to create an optimized library containing 8,893 CDP members, as shown in fig. 1C.

To determine if the best performing CDP homolog improved library performance, the optimized library (optimized CDP library) and the original library (diverse CDP library) were cloned into an SDPR surface display lentiviral vector with a C-terminal 6XHis tag (SEQ ID NO: 248) and tested as a pool by low multiplicity of infection (MOI of 1) transduction, as shown in FIG. 1D. Cells were treated with 5 μg/mL trypsin, 20 μg/mL trypsin or PBS (no trypsin) for 5 min, reduced with 10mM DTT for 5 min, and stained with anti-6 XHis antibody. In cells treated with PBS (no trypsin), anti-His staining represents total surface protein levels. In trypsin-treated cells, anti-His staining represents trypsin resistance, as CDP cleaved by trypsin will release its His tag after reduction with DTT and will not confer staining to the cells. PBS treated samples stained 2.14 times more in the optimized library than in the variegated library, indicating better surface expression of the optimized pool. Trypsin treatment reveals similar improvements in optimizing libraries over diverse libraries. Since only 11% of the optimized library represented the highest scoring diversified sequences, the higher staining of the optimized library, whether or not trypsin treated, indicated homology to the highest scoring CDP as an efficient method of identifying untested sequences that could be expected to exhibit high surface expression and protease resistance similar to the highest scoring CDP.

Example 3

Optimizing structural analysis of cystine compact peptide library

This example describes computational modeling of structural analysis performed on an optimized Cystine Dense Peptide (CDP) library. The structure of the optimized library members was modeled and compared to known crystal structures to facilitate hypothesis driven docking simulations and mutant structure-activity relationships (mutational structure-activity relationship, SAR). Protein Structure modeling tools I-TASSER and Rosetta in combination were used to model the structure of CDP library members. I-TASSER has previously been used in small stents with complex folds with high success. Their one program, "ForceDisulfides," selected Rosetta protein modeling software, which can convert very close cysteine pairs with beta carbon in the model to disulfides. As shown in FIG. 2A, the structural modeling pipeline uses modeling software version I-TASSER 5.1 to create structural models from CDP sequences. This model was used to determine the most likely disulfide pairing by minimizing the average pairing distance between the bonded cysteine sulfur atoms. Using the forcedisclufides program, rosetta relaxes the disulfide structure through the full atom refinement and reassembly algorithm to minimize spatial conflicts.

The structurally modeled pipeline was applied to the previously crystallized CDP and the model was compared to the experimentally obtained crystal structure. PyMol protein visualization software was used to align the crystal structure and backbone atoms of the computational model. An alignment of the CDP structure is provided in fig. 2B. The structures were aligned with an average Root Mean Square Deviation (RMSD) of 0.924 as shown in fig. 2C. When Asymmetric Units (AU) of the crystals are aligned with each other, the RMSD is greater thanAverage RMSD-> About +.f. of the average RMSD of (A) and what is generally considered to be proof of successful modeling of the de novo design of mini-proteins>RMSD values perform well. The tubing was then applied to optimize the remaining members of the library. Of the 8893 members of the modeling, 4298 structures passed a set confidence threshold (I-TASSER C score>0 and Rosetta energy fraction<80)。

Example 4

Pre-screening target compatible scaffolds using low resolution docking

This example describes low resolution docking using a cystine dense peptide structural model for computer pre-screening of target compatible scaffolds. The CDP structural library generated as described in example 3 was used to predict favorable binding to a target of clinical interest. Since high resolution docking simulations are computationally expensive on a large scale (thousands of candidate binders have no prior interface knowledge), low resolution RosettaDock scripts are used. By converting the side chains into a single large pseudo atom or centroid, the low resolution simulation runs faster than the high resolution simulation by eliminating rotamer stacking to simplify the energy calculation. This transformation was used to predict favorable docking regions for high-molecular docking aggregation (using DBSCAN) and rank scaffolds for docking compatibility with targets of interest, allowing generation of focused sub-libraries from high-diversity optimization libraries, as identified in example 2. To generate the model, the target protein of interest and CDP are input into RosettaDock in low resolution mode and run at least 2000 times. For each docking point, the centroid (CoM) of the CDP is identified and the docking interactions are scored. The centroids of the first 100-200 docking points were analyzed using DBSCAN to identify highly docked clusters, and the center of each cluster was defined as the likely peptide docking site.

To test the computer pre-screening of the optimized CDP library, an optimized model library containing 4298 members was docked with the domains seen in the high resolution eutectic structure of PD-L1 and PD-1 (PDB ID No:4 ZQK). After docking all 4298 CDP scaffolds with targets, a common target region for CDP docking at the assembled candidate docking site was identified using DBSCAN aggregation. As shown in FIG. 3, four clusters were identified, one of which was located at the PD-L1:PD-1 interface and may represent a cluster of binding agents from which PD-L1 inhibitor candidates could be identified.

Such full-text library docking is used to select libraries enriched in scaffolds with surface shapes/energetics that favor target binding to PD-L1 at the PD-1 binding interface. A sub-library of high-molecular PD-L1 scaffolds predicted to dock at the PD-1 interface is shown in fig. 4A and 4B. Fig. 4A shows Rosetta docking energy for a high-fraction PD-L1 scaffold plotted against Solvent Accessible Surface Area (SASA). The stent is color coded according to the main structural elements in the stent. The lighter shaded areas represent scaffolds for docking-rich Met/Tyr scan (DEMYS) library generation, described in more detail in example 5. Even though there are fewer than 300 members, the scaffold library still has structural and taxonomic diversity, as seen in the phylogenetic tree shown in fig. 4B.

Example 5

Identification of PD-L1-conjugated cystine compact peptides

This example describes the identification of cystine-dense peptides that bind to PD-L1 using a dock-rich methionine (M) -tyrosine (Y) scan (DEMYS). The surface display assay and library generation techniques described in example 2-example 4 were implemented as a high throughput screening platform to identify CDPs that bind to PD-L1. The finite library of potential PD-L1 binding scaffolds allows for further diversification using tyrosine and methionine scanning to create hydrophobic patches that can seed novel proteins-protein interactions. Tyrosine (Tyr) and methionine (Met) are chosen because they are aromatic and aliphatic residues containing polar atoms, respectively, avoiding the extreme hydrophobic nature of similarly sized phenylalanine (Phe) and leucine (Leu) residues that can affect solubility. The rich docking MY scan (DEMYS) strategy for combining sub-library selection of dockable scaffolds with tyrosine and methionine scans is shown in fig. 5A. Scaffolds with a high fraction in low resolution target docking were selected from the optimized model library and further diversified by Met/Tyr scanning of hydrophilic surface residues. Sample scaffolds were color coded as hydrophobic such that lighter shading indicated carbon atoms did not contact polar atoms, middle shading indicated acidic atoms, darker shading indicated basic atoms, and the remaining atoms were shown as white, these scaffolds being shown in their parent (WT) form and using three example Met or Tyr mutations (D18M, R26Y and R36M). The sample holder was used to illustrate Met/Tyr scanning as a way to seed or amplify hydrophobic patches for novel proteins, protein interactions.

DEMYS was performed to identify PD-L1 binding peptides that bound at the PD-1 binding interface. A library of DEMYS was created for the PD-L1: PD-1 interface. The generated optimized library and PD-L1: PD-1 DEYS library as described in example 4 were both screened by SDGF mammalian display, as described in example 2, to identify CDPs that bind PD-L1. The optimized mammalian display screen shown in FIG. 5B produced a validated binding scaffold corresponding to SEQ ID NO. 4. The verification of the binding of SEQ ID NO 4 to PD-L1 is shown in FIG. 5E. The DEMYS library mammalian display screen shown in fig. 5C resulted in hits representing four different parental CDP scaffolds, corresponding to SEQ ID No. 3, SEQ ID No. 57, SEQ ID No. 58, and SEQ ID No. 59, derived from SEQ ID No. 353 (EEDCKVHCVKEWAAYKACAERIKSDTTGQAHCSGQYFDFWKC VDHCAAP, corresponding to SEQ ID No. 354 (GSEEDCKVHCVKEWAAY KACAERIKSDTTGQAHCSGQYFDFWKCVDHCAAP), NO N-terminal GS), SEQ ID No. 355 (EESCKPQCVKAWLEYQACAERVEKDESGEAH CTGQYFDYWHCVDKCAAK), SEQ ID No. 356 (ARTCESQSHRFK GPCVSDTNCASVCRTERFSGGHCRGFRRRCLCTKHC), and SEQ ID No. 357 (EERCKPQCVKSLYEYEKCVKRVENDDTGHKHCTGQY FDYWSCIDKCVAS), respectively. The highest staining of the four DEMYS hits corresponds to SEQ ID No. 3, a variant of SEQ ID No. 4, containing the same cystine scaffold. The remaining three hits, corresponding to SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59, represent three different cystine scaffolds. The verification of the binding of SEQ ID NO:2, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59 to PD-L1 is shown in FIG. 5F. Three of the four DEMYS hits were highly helical in nature and had high Solvent Accessible Surface Area (SASA). While the optimized library as a whole contains high SASA CDPs (coil-rich, fold-rich, or helix-rich) of various structures, the scanning of M-Y focuses on helix-rich structures based on the results of the docking enrichment. M-Y scanning of helix-rich CDPs from the second generation library resulted in multiple PD-L1 binding hits. The I-TASSER/Rosetta modeled scaffold of SEQ ID NO. 4 is shown in FIG. 5D, which scaffold was identified as PD-L1 binding hit in both screens.

As predicted by libraries derived from the highest fractional scaffolds at the PD-L1 interface, high concentrations of PD-1-Fc were observed to disrupt the binding of PD-L1 to cell surface expressed SEQ ID NO: 4. Furthermore, clusters of high-split docking points predicted to disrupt PD-1 binding were found on the surface of PD-L1, with high homology to cynomolgus monkeys, but poor homology to murine species, as shown in fig. 6A. Computer docking was used to predict the binding of the identified CDP that binds PD-L1 to PD-L1. The top 200 predicted PD-L1 binders (cyan grid) of the optimized library were docked on the PD-1 interface and shown superimposed on the surface rendition of PD-L1 (PDB ID No:4 ZQK), color coded for cynomolgus monkey (left)) and mouse (right) homology. PD-1, which binds to PD-L1, is shown as a band structure. The ability of SEQ ID NO. 4 to disrupt PD-1 interactions with PD-L1 has been demonstrated using competition assays, as shown in FIG. 6B. Reduced staining was confirmed by HEK 293F cells expressing SEQ ID NO:4 on the surface of SDGF when PD-1 competitive Fc fusion was added at a concentration of 50nM, 150nM, 500nM, 1.5. Mu.M, 5. Mu.M or 15. Mu.M, but there was NO difference in staining when control Fc fusion protein competitors were used at comparable concentrations. In the flow staining assay shown in FIG. 6C, cells surface-expressing SEQ ID NO. 4 bound human and cynomolgus PD-L1, but NO interaction with murine PD-L1 was observed. These results further verify the modeling interface PD-L1-CDP binding interface. These results also demonstrate that SEQ ID NO. 4 binds at a site that competes with PD-1, and also demonstrate that SEQ ID NO. 4 binds to human and cynomolgus monkey PD-1.

Site-directed saturation mutagenesis (SSM) was used to develop high affinity variants of SEQ ID NO. 4. Cells displaying CDP variants of SEQ ID NO. 4 were stained with fluorescent PD-L1 and flow sorted based on binding to PD-L1 as shown in FIG. 7A. The sorted cells re-grow and confluent as shown in fig. 7B. Flow-sorting of sorted cells stained with co-stain only (no PD-L1) is shown in fig. 7C.

After two rounds of staining, flow sorting and regrowth, the resulting pool of variants was sequenced to identify enriched and depleted variants, as shown in fig. 8. Enriched variants represent amino acid substitutions that may improve target binding (either directly at the interface, or indirectly through improved folding and stability), while depletion represents amino acid variants that disrupt binding capacity. As shown in FIG. 8, "beneficial in SEQ ID NO: 3" means those point mutations that are combined and included in SEQ ID NO:3 to increase the affinity of SEQ ID NO:3 for PD-L1 relative to SEQ ID NO: 4. "omitted from SEQ ID NO: 3" means a point mutation that appears to be beneficial alone, but when combined with other beneficial point mutations, disrupts binding. This point mutation is omitted in SEQ ID NO. 3. "destructive reversion" means point mutations with reduced binding when reverted to the parent amino acid, indicating that those point mutations in SEQ ID NO. 4 favor PD-L1 binding. "neutral reversion" means that reversion to the parent amino acid present in SEQ ID NO:353 does not affect binding to PD-L1, indicating that these mutations were obtained during library generation, but does not contribute to the binding of SEQ ID NO:4 to PD-L1. This neutral reversion is also known as a passenger break.

The enrichment result table format shown in fig. 8 is provided in table 12. The relative enrichment of each amino acid variant (in columns) is shown relative to the amino acid (in rows) at the corresponding position in SEQ ID NO: 4. Positive values indicate amino acid substitutions that may improve target binding, while negative values indicate amino acid variants that disrupt binding capacity.

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The heat map shown in fig. 8 was recoloured in fig. 21 to identify each amino acid that promoted PD-L1 binding. As seen in FIG. 21, amino acid substitutions determined to have a neutral or beneficial effect relative to SEQ ID NO. 4 are colored red, with residues of the original SEQ ID NO. 4 sequence being colored black. Dark grey or black shaded amino acids are believed to contribute to binding to PD-L1. For example, V6 of SEQ ID NO. 4 has five amino acid substitutions, E, Q, S, M or L, which are neutral or beneficial for PD-L1 binding. Thus, either of E, Q, S, M, L or V located at the sixth site of CDP facilitates binding to PD-L1. The consensus sequence SEQ ID NO 358-SEQ ID NO 363 was identified based on this enrichment data.

Six highly enriched amino acid substitutions (E11W, A13M, Y15G, I22N, Y36K and W40F) were combined (SEQ ID NO: 8) and evaluated for SEQ ID NO:4 and each single revertant (SEQ ID NO:9-SEQ ID NO:14, corresponding to revertant W11E, M13A, G15Y, N22I, K Y and F40W, respectively) as shown in FIG. 9 to see if all mutations had synergistic effects. Increased PD-L1 staining indicates higher affinity for PD-L1. The highest stained variant contained all mutations except the E11W mutation, so SEQ ID NO:3 corresponding to SEQ ID NO:4 with amino acid substitutions A13M, Y15G, I22N, Y K and W40F was selected as affinity matured variant. SSM was also used to evaluate mutations found in SEQ ID No. 4 relative to the parental scaffold (SEQ ID No. 353). SEQ ID NO. 4 differs from SEQ ID NO. 353 by five amino acid substitutions (D25Y, T27I, Q R, F V and V43L). SSM enrichment/depletion data showed that two of these substitutions (F39V and V43L) favour PD-L1 binding, as the corresponding back mutations were depleted. The other three replies (D25Y, T27I and Q29R) were not depleted, indicating that the Q29R, F V and V43L substitutions found in SEQ ID NO. 4 may be benign passenger mutations.

Example 6

Stability and binding affinity of high affinity cystine dense peptides that bind PD-L1

This example describes characterization of stability, purity, folding and binding affinity of cystine dense peptides that bind PD-L1 with high affinity. CDP is produced as a soluble molecule as described in example 1. PD-L1 binding of three recombinant CDPs was evaluated: SEQ ID NO. 4; SEQ ID NO. 3; and SEQ ID NO. 1, which contains a single N22Q amino acid substitution relative to SEQ ID NO. 3. The N22Q substitution present in SEQ ID NO. 1 removes the classical N-linked glycosylation site obtained during affinity maturation. All three CDPs were produced in high yield and high homogeneity as assessed by reverse phase high performance liquid chromatography (RP-HPLC, top) and SDS-PAGE (bottom) in fig. 10A. Although SEQ ID NO. 3 contains classical N-linked glycosylation sites, there is little evidence of glycosylation, which may be seen as a small peak, indicated by the arrow in the top middle panel. No tailing consistent with extensive glycosylation was seen in the SDS-PAGE gel (bottom). Exposure of CDP to high concentration of reducing agent (10 mM DTT, measured by RP-HPLC in FIG. 10A) showed that the immature hit SEQ ID NO:4 showed significant mobility change in RP-HPLC, consistent with loss of the cystine-stabilized tertiary structure. However, affinity matured variants SEQ ID NO. 3 and SEQ ID NO. 1 show only subtle mobility changes in RP-HPLC. From MS data (FIG. 10B) it was confirmed that DTT-treated samples were about 6Da heavier than PBS-treated samples, indicating that SEQ ID NO:3 and SEQ ID NO:1 variants were indeed reduced by DTT but retained most of their tertiary structure. This suggests that some or all of the mutations obtained during affinity maturation contribute to the stabilization of the non-cystine dependent tertiary structure. The mobility change observed upon boiling in LDS sample buffer with 10mM DTT (bottom, fig. 10A) shows CDP folding mediated by disulfide. The full gel is provided in fig. 18. The mass spectral data shown in fig. 10B further demonstrates CDP folding, where the m/z values of all three CDPs are consistent with oxidized cysteines.

The binding of the three recombinant CDPs to PD-L1 was assessed by Surface Plasmon Resonance (SPR), as shown in fig. 10C. SPR analysis demonstrated improved affinity after maturation, as seen by the higher affinity of SEQ ID NO:3 and SEQ ID NO:1 compared to SEQ ID NO: 4. Specifically, SEQ ID NO. 4 shows an equilibrium dissociation constant (K) of 39.6.+ -. 0.3nM _D ) PD-L1 is bound, and SEQ ID NO. 3 and SEQ ID NO. 1 are represented by K, respectively _D =160.+ -.1 pM and 202.+ -.2 pM bound PD-L1, confirming that SEQ ID NO 4K after affinity maturation _D The improvement is about 200 times. Further SPR analysis was performed to measure competition of SEQ ID NO 1 with the PD-L1 binding domain of PD-1 for PD-L1, which PD-L1 binding domain is shown in bold and underlined in the full-length sequence of PD-1

As shown in fig. 10D. This data demonstrates that SEQ ID NO 1 competes with PD-1 and that this competition is not an artifact of cell surface staining.

N22Q variant CDP (SEQ ID NO: 1) was co-crystallized with PD-L1 to confirm the CDP binding site and to visualize the surface interactions with PD-L1 as shown in FIG. 20A. SEQ ID NO. 1 (a variant that eliminates the classical N-linked glycosylation site obtained during affinity maturation) was produced as a soluble molecule as described in example 1 and co-crystallized with PD-L1. Part of CDP (from A19 to Q35) is in The structure is not resolved. Enrichment analysis was performed to determine the effect of amino acid substitutions of resolved residues in the crystal structure relative to non-resolved residues in the crystal structure. The average SSM enrichment score for the unresolved residues was less extreme (less deviation from 0) than seen in the case of resolved residues, as shown in figure 20B, indicating that the specific side chain identity of the unresolved residues is less important for high affinity binding. The resolved part matches the model from E1 to C18 and from D38 to A48. K36 and F37 resolved but were not part of the D38-A48 helix.

The resolved part has, as assessed by PISA (PDBe PISA v 1.52)This is in contrast to the interfacial surface area of PD-L1 with PD-1 observed (+.>PDB 4 ZQK) are similar. The location of the CDP on PD-L1 is entirely within the PD-1 footprint, as shown in fig. 20C, showing the interface for computer low resolution docking enrichment to predict such hits. Looking at the interfaces shown in FIGS. 20D and 20G, it is revealed that CDP uses many of the same interaction sites as PD-1. Both K5 of SEQ ID NO. 1 and K78 of PD-1 form a salt bridge with the A121 backbone oxygen of PD-L1, while both D44 of SEQ ID NO. 1 and E136 of PD-1 similarly form a salt bridge with Y123 of PD-L1. F40 of SEQ ID NO. 1 is located in the pocket formed by Y56, R113, M115 and Y123 of PD-L1, thereby forming a hydrophobic contact (M115), a herringbone loop stacking interaction (two Y) and a cation-pi interaction (R113). This pocket is also occupied by I134 of PD-1. In addition, V9, W12 and L43 of SEQ ID NO. 1 share the hydrophobic interaction sites used by L128, A132 and I126, respectively, of PD-1. The interfacial adjacent mutation that distinguishes SEQ ID NO. 1 from its parent scaffold is expected to disrupt binding upon reversion to the parent side chain, as shown in FIG. 20E. Hydrophobic interactions of both M13 and L43 with the PD-L1 surface will be lost in parents a13 and V43; the pocket occupied by F40 will not be Have to be deformed to accommodate the parent W40, thereby altering the interface elsewhere; and parent F39 does not conform as well to the surface as V39. Finally, analysis of human/mouse and human/cynomolgus monkey (cyno) homology on the surface of PD-L1 revealed that the interaction site contained several non-homologous side chains between human and mouse, as shown in figure 20F. Human/cynomolgus monkey homology is perfect at this interface, matching cross-reactivity data. SEQ ID NO. 4 does not cross-react with murine PD-L1, but will bind cynomolgus monkey PD-L1.

Example 7

Incorporation of PD-L1 binding peptides into bispecific immunocytocement

This example describes the incorporation of a high affinity binding to the cystine dense peptide of PD-L1 into bispecific immunocytocement (biece) to produce a highly potent anti-tumor agent. CDPs that disrupt the PD-L1-1 interface that bind PD-L1 can be used as immune tumor drugs, inhibiting checkpoint signals that are normally transmitted by tumor cells to infiltrating T cells. The use of such a PD-L1-binding CDP in a bispecific immune cell cement (biece) format intended to bind CD3 on T cells and PD-L1 on cancer cells simultaneously can further convert PD-L1 from a signal on tumor cells that protects tumor cells from immune attack to a signal on tumor cells that promotes the immune system's attack by encouraging activation of T cells to bind and kill cells that overexpress PD-L1. To test this, SEQ ID NO:2, corresponding to SEQ ID NO:4 with an N22D substitution, was cloned into a heterodimeric Fc fusion construct containing a set of mutations that promote pestle-mortar heterodimerization. This CDP Fc fusion construct that binds PD-L1 was paired with a fusion of Fc and CD 3-binding scFv using knob-to-socket heterodimerization to form a CDP/scFv bispecific immunocytocement (CS-BiICE) that binds PD-L1. CDP-based BiICE ("CS-BiICE") is formed from a first fusion protein (METDTLLLWVLLLWVPGSTGDYKDEGGSEEDCKVHCVKEWMAGKACAERDKSYTIGRAHCSGQKFDVFKCLDHCAAPGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID NO: 342) containing a PD-L1-binding CDP (SEQ ID NO: 2) fused via a linker to an Fc "mortar" sequence with an N-terminal signal peptide (METDTLLLWVLLLWVPGSTG; SEQ ID NO:247; SP ") and a FLAG tag (DYKEGGS; SEQ ID NO: 246) and a second fusion protein (METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSHHHHHH; SEQ ID NO: 347) containing an anti-CD 3 single chain variable fragment (scFv) fused to an Fc" pest "sequence with an N-terminal signal peptide and a C-terminal 6XHis tag (HHHHH; SEQ ID NO: 248). The "mortar" sequence heterodimerizes with the "pestle" sequence.

A comparison molecule was also constructed. This comparison molecule is called SS-BiICE, which contains an anti-PD-L1 scFv instead of a CDP that binds PD-L1, but is otherwise identical to the CS-BiICE molecule. scFv-based bice ("SS-bice") is formed from a first fusion protein (METDTLLLWVLLLWVPGSTGDYKDEGGSDIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAATYFCQQSRRVPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQAWGYPWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID NO: 346) containing an anti-PD-L1 scFc comprising SEQ ID NO:343 (EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQAWGYPWGQGTLVTVSS) and SEQ ID NO:344 (DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAATYFCQQSRRVPYTFGQGTKLEIK) with an N-terminal signal peptide and a FLAG tag fused to an Fc "mortar" sequence and a second fusion protein (SEQ ID NO: 347) containing an anti-CD 3 single chain variable fragment (scFv) fused to an Fc "mortar" sequence comprising an N-terminal signal peptide and a C-terminal 6xHis tag (SEQ ID NO: 248). A schematic of a CDP-containing CS-BiICE and a comparative SS-BiICE is shown in FIG. 11.

As with SEQ ID NO. 4, SEQ ID NO. 3 contains a single D22N amino acid substitution relative to SEQ ID NO. 2, and the anti-PD-L1 scFv also lacks murine cross-reactivity, as shown in FIG. 12. Cross-reactivity of SEQ ID NO 3, anti-PD-L1 scFv (DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAATYFCQQSRRVPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQAWGYPWGQGTLVTVSS; SEQ ID NO 345) and scFv derived from alemtuzumab (EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK; SEQ ID NO 348) to human, cynomolgus monkey ("Cyno") or mouse PD-L1 was determined by measuring staining cells expressing the PD-L1 binding moiety with a fluorescently labeled PD-L1 ortholog. SEQ ID NO. 3 demonstrates binding to human and cynomolgus PD-L1, but not to murine PD-L1. anti-PD-L1 scFv (SEQ ID NO: 345) demonstrated binding only to human PD-L1. The scFv derived from alemtuzumab (SEQ ID NO: 348) demonstrated binding to all three PD-L1 orthologs, with highest binding to humans and lowest binding to mice. Both scFv domains are VHVL oriented, [ GGGGS ] ] ₃ The (SEQ ID NO: 165) linker connects the VH and VL domains. The observed cross-reactivity of the scFv derived from alemtuzumab with murine PD-L1 made it a poor in vivo comparison of SEQ ID No. 3 and related variants as a component of biece in murine studies, as target off-target tumor T cell engagement may occur. Unlike scFv derived from alemtuzumab, anti-PD-L1 scFv did not show cross-reactivity with murine PD-L1. For this purpose, anti-PD-L1 scFv was chosen as a comparison of SEQ ID NO. 3 and related variantsTo eliminate off-target tumor toxicity as a variable.

CS-BiICE and SS-BiICE molecules were produced as recombinant proteins as described in example 1 and purified by Immobilized Metal Affinity Chromatography (IMAC). SDS-PAGE gels of purified CS-BiICE and SS-BiICE molecules are shown in FIGS. 13A and 13C, respectively. The heterodimer (H) species (representing a complex of anti-CD 3-Fc pestle (SEQ ID NO: 347) and PD-L1-binding-Fc mortar (SEQ ID NO:342 in CS-BiICE and SEQ ID NO:346 in SS-BiICE)) and anti-CD 3-scFv-Fc monomer (M) were both visible under non-reducing (NR) conditions of CS-BiICE and SS-BiICE. The anti-CD 3 monomer bands were identical to those observed when the anti-CD 3-Fc pestle was expressed alone. The presence of the monomer band is due to the fact that SEQ ID NO. 347 is more efficient than SEQ ID NO. 346 or SEQ ID NO. 342. The heterodimer band is consistent with the molecular weight of the CS-BiICE and SS-BiICE heterodimers under non-reducing conditions, where the disulfide links the two Fc halves. In the reduction lanes ("DTT") of fig. 13A and 13C, the "S" band was consistent with the molecular weight of the scFv-Fc fusion, while the "C" band was consistent with the molecular weight of the CDP-Fc fusion, and was observed only in CS-biece samples. The presence of anti-CD 3-scFv-Fc monomers did not affect T cell killing properties. Under reducing conditions (DTT), separate CDP-Fc and scFv-Fc species of CS-BiICE can be seen due to the different sizes, whereas scFv-Fc species are shown as a single band in SS-BiICE due to the similarity in molecular size of both. The binding affinity of CS-BiICE and SS-BiICE to PD-L1 in vitro was measured using SPR as shown in FIG. 13B and FIG. 13D, respectively. Both CS-BiICE and SS-BiICE demonstrated binding to PD-L1 in vitro. SPR of CS-BiICE (FIG. 13B) showed a decrease in overall PD-L1 affinity (K of CS-BiICE) following incorporation into the BiICE scaffold relative to CDP binding PD-L1 alone _D =11.2 nM, and SEQ ID No. 1 is 202 pM). This decrease in affinity may be due to a decrease in association constant, since the dissociation rate of CS-BiICE is actually slower than that of CDP binding PD-L1 alone (k of CS-BiICE) _d ＝6.06×10 ^-3 s ^-1 And SEQ ID NO. 1 is 1.96×10 ^-2 s ^-1 ) While the association constant of CS-BiICE is much lower (k of CS-BiICE _a ＝4.07×10 ⁵ M ^-1 s ^-1 And SEQ ID NO. 1 is 9.73X10 ⁷ M ^-1 s ¹ ). In the case of immune synapses, local receptor concentrations are expected to be high, and thus reduced association rates may not adversely affect performance. SPR of SS-BiICE (FIG. 13D) showed that the binding of the PD-L1 scFv of SS-BiICE to K _D =65 nM promotes binding to PD-L1. Rate of association (k) of SS-BiICE _a ＝1.71×10 ⁵ M ^-1 s ^-1 ) Dissociation rate (k) _d ＝1.10×10 ^-2 s ^-1 ) Are inferior to CS-biece. This demonstrates that SEQ ID NO 2 containing a single Q22D substitution relative to SEQ ID NO 1 can assemble into an Fc fusion form with a CD3 binding moiety, and that the assembled molecule binds PD-L1 and has a higher affinity than a comparison molecule using an scFv that binds PD-L1.

Example 8

Induction of T cell killing using bispecific immunocyte cement that binds PD-L1

This example describes the induction of T cell killing using bispecific immune cell cement (biece) molecules containing CDP that binds PD-L1. The biece molecules prepared and validated as described in example 7 demonstrated binding to primary T cells purified from peripheral blood mononuclear cells (PBMCs, fig. 14A) derived from human patients. In vitro T Cell Killing (TCK) assays using cancer cells incubated with Activated T Cells (ATC) showed that BiICE, CS-BiICE (SEQ ID NO:342 heterodimerized with SEQ ID NO: 347) and SS-BiICE (SEQ ID NO:346 heterodimerized with SEQ ID NO: 347) binding to PD-L1 were effective in inducing T cell killing against three human cancer cell lines: prostate cancer PC3 (FIG. 14B), triple negative breast cancer MDA-MB-231 (FIG. 14C) and patient-derived pediatric brain tumor PBT-05 (FIG. 14D). The EC50 on PC3 cells was 28pM and SS-BiICE was 97pM for CS-BiICE, 142pM and SS-BiICE were 333pM for MDA-MB-231 cells, and 2.4pM and 7.7pM for CS-BiICE, PBT-05 cells. In each case, CS-BiICE is more efficient than SS-BiICE. An exemplary tumor killing mechanism using biece is shown in fig. 14E.

These assays also demonstrate that PD-L1 engagement is necessary for maximum activity, as pooled PD-L1 knockdown PC3 cells show significantly less T cell killing after BiICE incubation (fig. 14B); the small amount of activity observed may be related to the remaining PD-L1 positive cells in the knockout pool. Since both the CS-BiICE and SS-BiICE formulations were observed to contain impurities representing monomeric scFv-Fc that binds CD3, T cell killing assays were performed using the additional purified BiICE formulations to further verify that T cell killing activity was dependent on PD-L1. The PD-L1 engagement arm of each molecule also contains a short FLAG tag (DYKDE, SEQ ID NO: 431) and thus bispecific BiICE anti-FLAG-M1 affinity purification was performed away from the anti-CD 3 scFv-Fc monomer. SDS-PAGE gels of additional purified BiICE preparations of IMAC and IMAC plus FLAG alone are provided in FIG. 15A. In a TCK assay using PBT-05 cells, the additional purified BiICE preparation purified by IMAG and FLAG was compared to the IMAC purified preparation, as shown in FIG. 15B. The presence of scFv-Fc monomers that bind CD3 did not affect performance, indicating that the activity of the molecule was dependent on bispecific binding of the two targets, and that impurities found in the formulation did not confer TCK activity. This data demonstrates that bispecific T cell cements constructed using the CDP and CD3 binding moieties of the present disclosure that bind to PD-L1 can cause T cell-mediated killing of human cancer cells in vitro, and that this is more effective than bispecific T cell cements constructed using CDP that bind to PD-L1.

Example 9

Treatment of cancer in vivo using bispecific immunocytocement binding to PD-L1

This example describes the treatment of cancer in vivo using bispecific immunocytocement (biece) molecules containing CDP that binds PD-L1. As a preclinical proof of concept, the biece molecules prepared and validated as described in example 7 were tested in mice bearing a flank tumor. Tumor mass of nude mice carrying flank tumor is 100-200mm when entering group ³ It was treated with activated human T cells (ATC, 7.5X10 per dose ⁶ Individual) and 1nmol dose of CS-biece or SS-biece (1 nmol = 100 μg SS-biece, 80 μg CS-biece per dose). T cells are activated using a T cell activation kit containing microspheres capable of simultaneously binding and cross-linking CD3 and CD 28. Two weeks of treatment included four BiICE injections (day 1, day 4, day 8, and day 11) and two ATC infusionsNotes (day 2 and day 7) were given as shown in the experimental schedule provided in fig. 16A. Tumor size was measured over time. When the tumor reaches 1500mm ³ At this time, if the tumor forms an open ulcer on its surface, or if body weight drops to 80% of the initial value, the mice are removed from the study and euthanized. PC3 and MDA-MB-231 flank tumors were tested. PC 3-bearing mice treated with ATC and SS-biece had a lifespan that was approximately 2.5 times longer (median survival of 61.5 days after group entry) than those mice treated with ATC alone (median survival of 25 days), which was itself longer than those mice treated with vehicle alone (median survival of 20 days), as shown in fig. 16B. However, of 10 PC 3-bearing mice receiving ATC and SS-biece treatments, 9 eventually needed to be euthanized according to tumor regrowth. Only 1 of 10 mice with PC3 tumors need to be euthanized by ATC and CS-biece treatment; since the tumor was significantly completely eliminated, the other 9 survived until the end of the 95 th day experiment at the time of selection. There were no obvious signs of toxicity (weight loss or observed behavioral changes) in either of the biece-treated groups. The trend of body weight in the study over the course of the mice treatment group is shown in fig. 17. Tumor volumes of PC3 tumors were measured during the study as shown in fig. 16C. These results indicate that 8 out of 10 SS-biece mice responded to treatment, but 7 out of 8 responders eventually seen tumor regrowth. In contrast, of the 9 out of 10 CS-BiICE responders, all 9 exhibited complete tumor clearance. As shown in fig. 16D and 16E, the second group of PC3 flank tumor-loaded mice demonstrated elimination of CS-biece, while also demonstrating that the anti-PD-L1 antibody, divaline You Shan, was not effective in this context, suggesting that biece effect was not due to inhibition of PD-L1 alone, and that a 10-fold reduction in CS-biece dose produced similar tumor elimination. These results indicate that CS-BiICE is at least 10 times more potent than SS-BiICE because a 1/10 dose (0.1 nmol) of CS-BiICE eliminates PC3 tumors, whereas a full SS-BiICE dose (1 nmol) is not.

In MDA-MB-231 tumors where biece exhibited lower efficacy in vitro, both bieces significantly prolonged life as shown in fig. 16F; the median survival of only ATC mice was prolonged to 49 days and 52 days for atc+ss-biece and atc+cs-biece, respectively, although no mice exhibited complete MDA-MB-231 tumor clearance. Comparing BiICE in MDA-MB-231 flank tumors, CS-BiICE treatment did not significantly extend survival relative to SS-BiICE treatment, but CS-BiICE treatment did produce slower tumor growth kinetics than SS-BiICE (FIG. 16H), as measured by the time taken for tumor volume to increase three times after entry into the group (FIG. 16G). In both tumor models and both modes of PD-L1 conjugation, PD-L1/CD3 biece treatment exhibited significant tumor mass reduction and survival enhancement, whereas CS-biece was either mild (MDA-MB-231 tumor) or significantly (PC 3 tumor) superior to SS-biece in performance.

Example 10

Treatment of cancer using bispecific immunocytocement binding to PD-L1

This example describes the treatment of cancer using bispecific immunocyte cement that binds to PD-L1. The PD-L1-binding cystine compact peptide of any one of SEQ ID nos. 1-118, 435, 436, 554-567 is complexed with a T cell targeting agent to form a bispecific immunocyte cement. The T cell targeting agent binds to CD3, 4-1BB, CD137 or CD28. The bispecific immunocytocement is administered to a human subject suffering from cancer. After administration, the bispecific immune cell cement recruits T cells to PD-L1 positive cancer cells by binding to PD-L1 positive cancer cells via CDP that binds to PD-L1 and to T cells via T cell targeting agents. The recruited T cells target and kill PD-L1 positive cancer cells, thereby treating cancer.

Example 11

Treatment of autoimmune disorders using bispecific immunocytocement that binds to PD-L1

This example describes the treatment of autoimmune disorders using bispecific immunobinders that bind to PD-L1. The PD-L1 binding cysteine dense peptide of any of SEQ ID NOs 1-118, 435, 436, or 554-567 is complexed with an immune cell targeting agent that binds regulatory T cells or mesenchymal stem cells to form a bispecific immune cell cement. The immune cell targeting agent binds to CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX40, PD-1, CTLA-4 or STRO-1. The bispecific immunocytocement is administered to a human subject suffering from an autoimmune disorder. After administration, the bispecific immune cell cement recruits regulatory T cells or mesenchymal stem cells to PD-L1 positive cells, such as pancreatic β cells, by binding to PD-L1 positive cells via CDP that binds to PD-L1 and to regulatory T cells or mesenchymal stem cells via immune cell targeting agents. Recruited regulatory T cells or mesenchymal stem cells target PD-L1 enriched cells, such as islets, to avoid their killing by autoimmune T cells, thereby treating autoimmune disorders, such as type 1 diabetes.

Example 12

Treatment of cancer using PD-L1 binding peptides

This example describes the treatment of cancer using PD-L1 binding peptides. The PD-L1-binding cysteine dense peptide of any of SEQ ID nos. 1-118, 435, 436, or 554-567 is administered to a subject having cancer. Upon administration to a subject, the PD-L1 binding peptide targets and binds to PD-L1 on PD-L1 positive cancer cells. The PD-L1 binding peptide binds to a site on PD-L1 that overlaps the PD-1 binding interface, prevents PD-L1 binding, and inhibits PD-L1. Binding and inhibition of PD-L1 reduces immunosuppression, reduces T cell depletion, and restores immune function within the cancer cell microenvironment, thereby treating cancer.

Example 13

Treatment of cancer using PD-L1 binding peptides complexed with anticancer agents

This example describes the treatment of cancer using PD-L1 binding peptides complexed with anticancer agents. The PD-L1-binding cystine compact peptide of any one of SEQ ID NO. 1-SEQ ID NO. 118, SEQ ID NO. 435, SEQ ID NO. 436 or SEQ ID NO. 554-SEQ ID NO. 567 is complexed with an anticancer agent. The PD-L1 binding peptide anticancer agent complex is administered to a subject suffering from cancer. Upon administration to a subject, the PD-L1 binding peptide targets and binds to PD-L1 on PD-L1 positive cancer cells and delivers the anti-cancer agent to the cancer cells. Anticancer agents kill cancer cells, thereby treating cancer.

Example 14

Imaging cancer using PD-L1 binding peptides complexed with a detectable agent

This example describes the imaging of cancer using PD-L1 binding peptides complexed with a detectable agent. The PD-L1-binding cystine compact peptide of any one of SEQ ID NO. 1-SEQ ID NO. 118, SEQ ID NO. 435, SEQ ID NO. 436 or SEQ ID NO. 554-SEQ ID NO. 567 is complexed with a detectable agent. The PD-L1 binding peptide detectable agent complex is administered to a subject having cancer or suspected of having cancer. Upon administration to a subject, the PD-L1 binding peptide targets and binds to PD-L1 on PD-L1 positive cancer cells and delivers a detectable agent to the cancer cells. The detectable agent marks cancer cells and the presence or absence of the detectable agent in an area of the subject suspected of having the cancer is detected, thereby imaging the cancer.

Example 15

Treatment of cancer using chimeric antigen receptor that binds PD-L1

This example describes the treatment of cancer using chimeric antigen receptors that bind PD-L1. A chimeric antigen receptor comprising any of SEQ ID No. 1-SEQ ID No. 118, SEQ ID No. 435, SEQ ID No. 436 or SEQ ID No. 554-SEQ ID No. 567, in which the dense peptide of cystine that binds PD-L1 replaces a single chain variable fragment (scFv), a transmembrane domain and an intracellular domain, is expressed in T cells collected from a subject having cancer. T cells expressing the chimeric antigen receptor are administered to a subject. Following administration, the PD-L1 binding peptide of the chimeric antigen receptor targets and binds to PDL-L1 positive cancer cells and delivers T cells to the cancer cells. T cells kill cancer cells, thereby treating cancer.

Example 16

PD-L1 binding peptide engineering for pH dependent binding

This example describes the development and in vitro testing of PD-L1 binding peptides or peptide complexes that are capable of dissociating from PD-L1 in a pH-dependent manner, e.g., at endosomal pH (e.g., pH 5.5).

The pH-dependent binding to the target binding domain (CDP or otherwise) can be conferred in a variety of ways, an example of which is provided herein. Here, libraries of variants containing histidine substitutions were designed. Histidine residues were introduced because of the fact that, among all natural amino acids, his is the only one whose side chain charge varies significantly between neutral (e.g. pH 7.4) and acidic (e.g. pH < 6) endosomal conditions. With a change in pH, for example, with endosomal acidification changing from a physiological extracellular environment to an endosomal environment, such charge changes can either directly alter binding (introduction of a positive charge at low pH can result in charge repulsion of nearby cationic groups) or indirectly alter binding (change in charge imparts a subtle change in binding agent structure, thereby disrupting the protein-protein interface). In its simplest form, this can be performed by generating a double His-doped library, where for CDP each non-Cys non-His residue can be His-substituted one or two at a time. FIG. 19 shows the CDP sequence (SEQ ID NO:1,EEDCKVHCVKEWMAGKACAERQKSYTIGRAH CSGQKFDVFKCLDHCAAP) for high affinity binding to PD-L1 above and to one side of the histidine substitution matrix. Each black box represents a first and a second site in which His may be substituted. Those positions from top left to bottom right all along the diagonal represent a single His substitution. Each black box represents variants with one or two natural to His substitutions, representing 821 peptide variants to be screened. A library of variants containing the parent sequence and variants with one or two natural to His substitutions was generated and tested.

The PD-L1 binding of the resulting histidine-rich PD-L1 binding peptide was evaluated in a comparative binding experiment at various pH levels or ranges. A library of variants expressing PD-L1 binding peptides, each variant containing zero, one or two His substitutions, was displayed by mammalian surface. These variants were tested for maintenance of binding at extracellular pH (e.g., pH 7.4) and reduction of binding at endosomal pH (e.g., pH 5.5). Sequential screening was performed as shown in fig. 26. The input library was initially screened for PD-L1 binding at pH 7.4 and strong binding agents (shaded areas) were selected. The second and third rounds of screening (respectively "sort 1" and "sort 2") were performed at pH5.5 to simulate endosomal pH and collect weak binders (shaded areas). The last round of screening ("sort 3") was performed at pH 7.4 and a strong binding agent was selected. Differential binding at pH 7.4 and pH5.5 was observed after screening ("sort 4").

Variants of SEQ ID NO. 1 containing histidine substitutions at one, two or three of the E2H, M13H and K16H amino acid positions were identified in the pooling screen as pH dependent binders to PD-L1. The pH dependent binding was verified by measuring the binding of PD-L1 to the surface of cells expressing the single variants at pH 7.4 and pH5.5, as shown in figure 27. Peptides containing substitutions at E2H (EHDCKVHCVKEWMAGKACAERQKSYTIGRAHCSGQKFDVFKCLDHCAAP, SEQ ID NO: 555), M13H (EEDCKVHCVKEWHAGKACAERQKSYTIGRAHCSGQKFDVFKCLDHCAAP, SEQ ID NO: 556), K16H (EEDCKVHCVKEWMAGHACAERQKSYTIGRAHCSGQKFDVFKCLDHCAAP, SEQ ID NO: 557), E2H and M13H (EHDCKVHCVKEWHAGKACAERQKSYTIGRAHCSGQKFDVFKCLDHCAAP, SEQ ID NO: 558), E2H and K16H (EHDCKVHCVKEWMAGHACAERQKSYTIGRAHCSGQKFDVFKCLDHCAAP, SEQ ID NO: 554), M13H and K16H (EEDCKVHCVKEWHAGHACAERQKSYTIGRAHCSGQKFDVFKCLDHCAAP, SEQ ID NO: 559) or E2H, M13H and K16H (EHDCKVHCVKEWHAGHACAERQKSYTIGRAHCSGQKFDVFKCLDHCAAP, SEQ ID NO: 560) were compared to SEQ ID NO: 1. Variants containing substitutions at E2H and K16H corresponding to SEQ ID NO:554 showed strong binding to PD-L1 at pH 7.4 and significant loss of binding at pH5.5 (black arrow). Other variants and parent peptides showed different levels of PD-L1 binding at pH 7.4 and pH5.5, and had different degrees of pH dependence on binding.

Example 17

Use of serum albumin binding peptide complexes to extend peptide plasma half-life

This example demonstrates a method of extending the serum or plasma half-life of a peptide using a serum albumin binding peptide complex as disclosed herein. A peptide or peptide complex having the sequence of any one of SEQ ID NO. 1-118, SEQ ID NO. 435, SEQ ID NO. 436 or SEQ ID NO. 554-567 or SEQ ID NO. 119-153 is modified to increase its plasma half-life. The peptide and serum half-life extending moiety are recombinantly fused, chemically synthesized as a single fusion, recombinantly expressed and conjugated separately, or chemically synthesized and conjugated separately. Fusion of the peptide to serum albumin binding peptide extends the serum half-life of the peptide complex. The peptide or peptide complex is conjugated to a serum albumin binding peptide, such as SA21 (SEQ ID NO: 242). Optionally, a peptide fused to SA21Has the sequence of any one of SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 342. Optionally, the peptide fused to SA21 is linked to SA21 by a peptide linker. A linker connects the two separate functional CDPs to incorporate a serum half-life extending function into the peptide or peptide complex. The linker enables cyclization of SA21 without steric hindrance from either member of the peptide complex. Alternatively, the peptide is conjugated to albumin, an albumin binding agent (e.g., an album tag), or a fatty acid (e.g., C ₁₄ -C ₁₈ Fatty acid or palmitic acid) is used to extend plasma half-life. Plasma half-life is also optionally prolonged due to reduced immunogenicity through the use of minimal non-human protein sequences.

Example 18

Treatment of CNS cancers using PD-L1 binding peptides

This example describes the treatment of cancer using PD-L1 binding peptides. The PD-L1 binding cysteine compact peptide of any of SEQ ID NO. 1-SEQ ID NO. 118, SEQ ID NO. 435, SEQ ID NO. 436 or SEQ ID NO. 554-567 is fused to a TfR binding peptide such as SEQ ID NO. 350, optionally using a linker, e.g. SEQ ID NO. 154-SEQ ID NO. 241 or SEQ ID NO. 433. The CDP-TfR binding peptide complex that binds to PD-L1 is administered to a subject having cancer. After administration to a subject, the CDP-TfR binding peptide complex that binds to PD-L1 crosses the blood brain barrier and binds to PD-L1 on PD-L1 positive primary or metastatic cancer cells in the brain. The CDP-TfR binding peptide complex that binds PD-L1 binds to a site on PD-L1 that overlaps the PD-1 binding interface, preventing PD-L1 binding, and inhibiting PD-L1. Binding and inhibition of PD-L1 reduces immunosuppression, reduces T cell depletion, and restores immune function within the cancer cell microenvironment, thereby treating cancer.

Example 19

Synthesis of peptide oligonucleotide complexes for antisense therapy

Genes that are silenced for treatment of disease are identified and the desired single stranded antisense oligonucleotide sequences are designed and synthesized based on the target coding or complementary sequences. The antisense oligonucleotide is conjugated to any of the PD-L1 binding peptides disclosed herein, including the peptides of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO:567, by any of the methods disclosed herein, e.g., according to example 26-example 31, e.g., using a cleavable or stable linker. Optionally, the nucleotides (including the backbone) are modified, for example, to increase in vivo stability, increase resistance to enzymes such as nucleases, increase protein binding, including binding to serum proteins, increase in vivo half-life, alter tissue biodistribution, or reduce activation of the immune system.

Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 20

Synthesis of peptide-oligonucleotide conjugates for RNAi therapy

Genes that are silenced for treatment of disease are identified and the desired double stranded RNAi sequences are designed and synthesized based on the target coding or complementary sequences. The sense or antisense oligonucleotide of RNAi is conjugated to any of the PD-L1 binding peptides disclosed herein, including the peptide of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO:567, by any of the methods disclosed herein, e.g., according to example 26-example 31, e.g., using a cleavable or stable linker. Optionally, the peptide is conjugated to the sense (passenger) strand of the oligonucleotide. Optionally, the nucleotides (including the backbone) are modified, for example, to increase in vivo stability, increase resistance to enzymes such as nucleases, increase protein binding, including binding to serum proteins, increase in vivo half-life, alter tissue biodistribution, or reduce activation of the immune system. The sense strand and the antisense strand hybridize together either before or after conjugation.

Example 21

Synthesis of peptide-oligonucleotide conjugates for U1 adapter therapy

Genes that are silenced for treatment of disease are identified and the desired oligonucleotide sequences for U1 adapter therapy are designed and synthesized based on the target coding or complementary sequences. The oligonucleotides are conjugated to any of the PD-L1 binding peptides disclosed herein, including any of the peptides of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO:567, by any of the methods disclosed herein, e.g., according to example 26-example 31, e.g., using cleavable or stable linkers. Optionally, the nucleotides (including the backbone) are modified, for example, to increase in vivo stability, increase resistance to enzymes such as nucleases, increase protein binding, including binding to serum proteins, increase in vivo half-life, alter tissue biodistribution, or reduce activation of the immune system.

Example 22

Synthesis of peptide-oligonucleotide conjugates for aptamer therapy

The aptamer sequences that interact with the target molecule are selected to treat the disease, identified and synthesized against the target. The aptamer oligonucleotide is conjugated to any of the peptides disclosed herein, including any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436, or SEQ ID NO:554-SEQ ID NO:567, by any of the methods disclosed herein, e.g., according to example 26-example 31, e.g., using a cleavable or stable linker. Optionally, the nucleotides (including the backbone) are modified, for example, to increase in vivo stability, increase resistance to enzymes such as nucleases, increase protein binding, including binding to serum proteins, increase in vivo half-life, alter tissue biodistribution, or reduce activation of the immune system.

Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO: 545) linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 23

Conjugation of oligonucleotides and peptides using click chemistry

An alkyne or azide is installed in an oligonucleotide, for example, by adding a hexynyl group at the 5' or 3' end of the oligonucleotide, installing a 5-octanediynyl dU, installing a DIBO at the 5' end for use (optionally using a DIBO phosphoramidate installation), or installing an azide by attaching an azide to a dT base using an NHS ester reaction. Azido or alkynyl groups are attached to peptides, for example, by incorporation of an N-terminal 6-azidohexanoic acid, azido homoalanine residue or homopropargylglycine residue. Optionally, the alkynyl group comprises a tension ring, such as tension ring Xin Guihuan, e.g., DIBO. The oligonucleotide is conjugated to any of the PD-L1 binding peptides disclosed herein, including any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567. Oligonucleotides and peptides are conjugated together by combining an azide group on one with an alkyne group on the other to form a triazole bond using copper-catalyzed azide-alkyne cycloaddition or tension-promoted azide-alkyne cycloaddition.

Any of the peptide oligonucleotide complexes of the present disclosure can be so modified and described with alkyne or azide groups. Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 24

Conjugation of RNAi sequences and peptides using click chemistry

The alkynyl group within the strained cyclooctyne is attached to an oligonucleotide, optionally attached to the 5 'or 3' end of the sense or antisense strand. Optionally, the strained cyclooctyne is DIBO, which is optionally mounted at the 5' end using a DIBO phosphoramidate. Azido groups are attached to peptides. Optionally, a 6-azidohexanoyl group is added to the N-terminus of any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO:567, optionally with a linker between the 6-azidohexanoyl group and the peptide. Optionally, the peptide is prepared as a TFA salt. The alkyne-containing oligonucleotide and the azide-containing peptide are contacted together, for example in a buffer, solution or solvent. Azide and alkyne react to form a triazole linkage that links the oligonucleotide and peptide. The sense and antisense strands of the RNAi hybridize together before or after the conjugation reaction.

Any of the peptide oligonucleotide complexes of the present disclosure can be modified to include an alkynyl group within a strained cyclooctyne and described. Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 25

Conjugation of U1 adaptor sequences and peptides using click chemistry

The alkynyl group within the tension loop octyne is mounted on an oligonucleotide, optionally attached to the 5 'or 3' end of the sequence, designed for U1 adapter therapy. Optionally, the strained cyclooctyne is DIBO, which is optionally mounted at the 5' end using a DIBO phosphoramidate. Azido groups are attached to peptides. Optionally, the peptide is prepared as a TFA salt. The alkyne-containing oligonucleotide and the azide-containing peptide are contacted together, for example in a buffer, solution or solvent. Azide and alkyne react to form a triazole linkage that links the oligonucleotide and peptide.

Any peptide oligonucleotide complex of the present disclosure may be described as modified with an alkynyl group within a strained cyclooctyne. Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 26

Mounting thiol, amine or aldehyde groups in oligonucleotides

This example describes the incorporation of thiol, amine or aldehyde groups in RNA or DNA or any oligonucleotide. FIGS. 24A-24E illustrate the incorporation or addition of these groups on RNA or DNA. Thiol groups were added to the oligonucleotides using EDC and imidazole to activate the 5' phosphate group to phosphorylimidazole, followed by reaction of the resulting product with cystamine. And then reduced with Dithiothreitol (DTT) to form phosphoramidate linkages with free thiol groups. Alternatively, thiol groups are added to oligonucleotides by incorporating thiol-containing phosphoramidates at the 5 'or 3' end of the oligonucleotide during solid phase phosphoramidate oligonucleotide synthesis, as shown in FIGS. 24A-24E. Phosphoramidates used during synthesis may have protecting groups on thiols during synthesis, which protecting groups are removed during cleavage, purification and work-up. FIG. 24A illustrates the structure of oligonucleotides containing 5 '-Thiol (cyclohexyl; C6) modifications (left) and 3' -Thiol (C3) modifications (right), as shown in https:// www.atdbio.com/content/50/thio-modified-oligonucleotides.

Amine groups are added to RNA or DNA by incorporating phosphoramidates during synthesis, which contain a protected amine group, which are then deprotected. FIG. 24B illustrates MMT-hexyl amino linker phosphoramidate. FIG. 24C illustrates a TFA-amyl amino-linker phosphoramidate as shown in https:// www.sigmaaldrich.com/category/product/sigma/m 01023 hhlang=en & region=US.

Alternatively, thiol-or amine-containing oligonucleotide residues are included in the sequence at any selected position in the RNA or DNA, as described by Jin et al (J Org chem.2005, 27, 70 (11): 4284-99). FIG. 24D illustrates RNA residues incorporating amine or thiol residues as described by Jin et al (J Org chem.2005, 5, 27; 70 (11): 4284-99). In addition, oligonucleotide residues containing phosphorothioate groups in the phosphodiester backbone (wherein sulfur atoms replace non-bridging oxygens in the phosphobackbone of the oligonucleotide) provide reactive groups similarly useful for conjugation to thiol groups. The use of phosphorothioate-containing residues may also render the RNA more resistant to nuclease degradation.

Aldehyde functionality can be incorporated at the 3' end of the RNA by converting the diol to two aldehyde groups using periodate oxidation.

Other methods of incorporating or modifying functional groups are performed using the techniques set forth in Bioconjugate Techniques, greg Hermanson, 3 rd edition.

Any of the peptide oligonucleotide complexes of the present disclosure can be modified with thiol, amine, or aldehyde groups and described. Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 27

Generation of cleavable linkers between oligonucleotides and peptides

This example describes the generation of a cleavable linker between an oligonucleotide and any one of the peptides of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567. Disulfide linkers are generated by combining thiol-containing oligonucleotides with peptides comprising free thiol groups. Thiols are incorporated onto peptides using the special reagent (Traut's reagent), SATA, SPDP, or other suitable reactive amine reagents (e.g., heterobifunctional SPDP and NHS ester linkers with N-terminal or lysine residues) or by incorporating free cysteine residues into the peptides, as shown in fig. 25. Disulfide bond cleavage occurs in the reducing environment of the cytoplasm or in the endosomal/lysosomal pathway.

The ester linkage is generated by combining the free hydroxyl group (e.g., at the 3' end of the oligonucleotide) with a carboxylic acid group on the peptide (e.g., from a C-terminal, aspartic acid, glutamic acid residue, or via a linker introduction linked to a lysine residue or the N-terminal), e.g., via fischer-tropsch esterification (Fisher esterification) or via the use of an acid chloride. The ester linker is cleaved by hydrolysis, which is accelerated by lower pH or enzymatic esterase cleavage of the endosomes and lysosomes.

Oxime or hydrazone linkages are formed by combining aldehyde groups on the oligonucleotide with peptides that have been functionalized with aminooxy groups (forming oxime linkages) or hydrazide groups (forming hydrazone linkages). The stability or instability of the oxime or hydrazone bond is tailored by adjacent groups (Kalia et al Angew Chem Int Ed Engl.2008;47 (39): 7523-6.) hydrolytic cleavage is accelerated in acidic compartments, such as endosomes/lysosomes.

The hydrazide group is incorporated onto the peptide by reacting adipic acid dihydrazide or carbohydrazide with carboxylic acid groups in the C-terminal or aspartic acid or glutamic acid residues. Aminooxy groups are incorporated onto peptides by reacting the N-terminal or lysine residue with a heterobifunctional molecule containing a NHS ester at one end and a phthalimido oxy group at the other end, followed by cleavage with hydrazine. The reaction is optionally catalyzed by the addition of aniline.

For example, the cleavage rate of any linker can be adjusted by changing the electron density near the cleavable bond or by spatially affecting access to the cleavage site (e.g., by adding bulky groups such as methyl, ethyl, or cyclic groups).

Alternatively, cleavable linkers were generated using the method set forth in Bioconjugate Techniques, greg Hermanson, 3 rd edition.

As described in example 26 above, thiol, amine or aldehyde groups are installed in RNA or DNA as functional handles.

Peptide oligonucleotide complexes of the present disclosure may contain cleavable linkers and are described. Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 28

Generation of stable linkers between oligonucleotides and peptides

This example describes the generation of a stable linker between RNA, DNA or any oligonucleotide and any of the peptides SEQ ID NO. 1-SEQ ID NO. 118, SEQ ID NO. 435, SEQ ID NO. 436 or SEQ ID NO. 554-SEQ ID NO. 567. Stable linkers through secondary amines are formed by reductive amination by combining an aldehyde-containing oligonucleotide with an amine in the N-terminus or lysine residue of the peptide, followed by reduction with sodium cyanoborohydride.

Stable amide linkages are formed by combining the amine groups on the oligonucleotide with the carboxylic acid esters in the peptide C-terminal or aspartic acid or glutamic acid residues.

Stable urethane linkages can be formed by activating the hydroxyl groups in an oligonucleotide with Carbonyl Diimidazole (CDI) or N, N' -disuccinimidyl carbonate (DSC) and then reacting with the N-terminal or lysine residues of the peptide.

Maleimide linkers are produced by combining thiol-containing oligonucleotides with maleimide-functionalized peptides. Peptides are functionalized on reactive amines in the peptide using NHS-X-maleimide heterobifunctional agents, where X is any linker. Maleimide linkers serve as stabilizing linkers or as slowly cleavable linkers that cleave by exchange with other thiol-containing molecules in biological fluids. Maleimide linkers are also stabilized by hydrolysis of the succinimide moiety of the linker using various substituents, including those described in Fontaine et al Bioconjugate chem.,2015,26 (1), pages 145-152.

Other methods of incorporating, adding or modifying functional groups in polynucleotides are performed, for example, using the techniques set forth in Bioconjugate Techniques, greg Hermanson, 3 rd edition.

As described in example 26 above, thiol, amine or aldehyde groups are installed in the oligonucleotides as functional handles.

Any of the peptide oligonucleotide complexes of the present disclosure may contain a stable linker and are described. Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 29

Generation of enzymatically cleavable bonds between an oligonucleotide and a peptide

This example describes the generation of an enzymatically cleavable bond between RNA, DNA or any oligonucleotide and any one of the peptides of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567. An enzymatically cleavable bond is generated between the oligonucleotide and the peptide. Conjugates with cleavable linkages are administered in vitro or in vivo and cleaved by enzymes in the cell or in vivo to release the oligonucleotide. The enzyme is present in endosomes/lysosomes, cytosol, cell surface, or up-regulated in tumor microenvironment or tissue microenvironment. These enzymes include, but are not limited to, cathepsins (e.g., kramer et al, trends Pharmacol Sci.2017, month 10; 38 (10): 873-898, all of those cathepsins), such as cathepsin B; glucuronidase, including beta-glucuronidase; hyaluronidase; and matrix metalloproteinases such as MMP-1, MMP-2, MMP-7, MMP-9, MMP-13 or MMP-14 (Kessenblock et al, cell.2010, month 2; 141 (1): 52-67). The amino acid sequences of either SEQ ID NO:200, SEQ ID NO:204 or SEQ ID NO:216-SEQ ID NO:241 or SEQ ID NO:433 are cleaved by cathepsins or MMPs, as shown in Table 14 below (see also Nagase, hideaki. "Substrate specificity of MMPs." Matrix Metalloproteinase Inhibitors in Cancer treatment. Humana Press,2001.39-66; dal Corso et al, bioconjugate chem.,2017,28 (7), pages 1826-1833; dal Corso et al, chemistry-A European Journal 21.18.18 (2015): 6921-6929; doronina et al, bioconjug chem.2008, month 10; 19 (10): 1960-3.). Glucuronidase linkers include any of those described in Jeffrey et al Bioconjugate chem, 2006,17 (3), pages 831-840.

TABLE 14 enzyme cleavable linkers

Such as Jain et al, pharm Res.2015, month 11; 32 (11) the Val-Cit-PABC enzyme cleavable linker described in 3526-40 was prepared by conjugating the end of PABC with an amine group on an oligonucleotide. The valine terminus is further linked to the peptide, for example by amide bond formation with the C-terminus of the peptide. A spacer on either side of the molecule is optionally incorporated to facilitate spatial proximity of the enzyme to the Val-Cit bond (SEQ ID NO: 217). Alternatively, the linkage to the peptide is generated by: the N-terminus of the peptide is activated with SATA and generates a thiol group which is then reacted with a maleimidocaproyl group attached to the N-terminus of the Val-Cit pair (SEQ ID NO: 217). After cleavage by cathepsin B, the self-cleaving PABC group spontaneously eliminates, releasing the amine-containing oligonucleotide without further chemical modification. Other amino acid pairs include Glu-Glu, glu-Gly and Gly-Phe-Leu-Gly (SEQ ID NO: 551).

Any of the peptide oligonucleotide complexes of the present disclosure may contain an enzymatically cleavable linker and are described. Any peptide oligonucleotide complex of the disclosure is described (e.g., including an oligonucleotide of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO:492-SEQ ID NO:545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17.

Example 30

Conjugation of oligonucleotides and peptides

This example describes the conjugation of an oligonucleotide to a peptide of the present disclosure. The peptide is SEQ ID NO. 1. The N-terminus of SEQ ID NO. 1 was conjugated to 4-formyl-PBA by reductive amination. The PBA-containing peptide complexes with the 3' diol group of the oligonucleotide to form a boronate ester.

Alternatively, the oligonucleotide has thiol-or phosphorothioate-containing nucleotide residues included in the sequence during synthesis. The N-terminus of SEQ ID NO. 1 is modified with SATA (followed by deprotection using hydroxylamine) to form a thiol group.

Alternatively, the N-terminus of SEQ ID NO. 1 is SPDP-PEG ₄ -NHS ester modification to form a protected thiol group with a flexible hydrophilic PEG spacer. The modified oligonucleotide and the two thiol groups of SEQ ID NO. 1 combine to form a cleavable disulfide bond. Alternatively, the N-terminus of SEQ ID NO. 1 is treated with bromoacetamido-PEG ₄ TFP esters are modified to form amide linkages and then react with thiol groups within the oligonucleotide to form stable thioether linkages.

Alternatively, the oligonucleotide has amine-containing nucleotides included in the sequence during synthesis. The N-terminus of SEQ ID NO. 1 is modified with SATA to form a thiol group. The maleimidocaproyl-Val-Cit-PABC linker was conjugated to the amine in the oligonucleotide and the thiol in SEQ ID NO. 1.

Alternatively, the oligonucleotide is conjugated to the N-terminus of SEQ ID NO. 1 by reductive amination after oxidation of the 3' diol to form a secondary amine conjugate.

Alternatively, the 3' end of the oligonucleotide is oxidized to the aldehyde by periodate oxidation. The aldehyde then reacts with the peptide of SEQ ID NO. 1, which is functionalized at the N-terminus with an aminooxy group to form a cleavable oxime bond.

Alternatively, dsRNA is used. The 3' end of the sense strand was synthesized with thiol modification as shown in FIGS. 24A-24E. The N-terminal of SEQ ID NO. 1 is treated with bromoacetamido-PEG ₄ TFP esters are modified to form amide linkages and then react with thiol groups in the dsRNA to form stable thioether linkages. Alternatively, the 5' end of the sense strand or amino-terminated nucleotide is used as a modification site.

Alternatively, the peptide is any one of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567.

Alternatively, instead of using the N-terminus of the peptide, a lysine residue in the peptide is used. Optionally, one or more or all lysine residues are mutated to arginine residues, so that only one or no lysine residues are available for amine-based reactions.

Optionally, the oligonucleotides are synthesized using any one or more modified bases to alter the stability, tissue biodistribution, immunoreactivity, or any other property of the oligonucleotides.

Example 31

Surface Plasmon Resonance (SPR) analysis of peptide binding interactions

This example illustrates Surface Plasmon Resonance (SPR) analysis of the binding interactions of peptide-oligonucleotide conjugates with PD-L1.

A peptide-oligonucleotide conjugate (also referred to herein as a peptide oligonucleotide complex) is constructed by combining any PD-L1 binding peptide of the present disclosure with an oligonucleotide. Optionally, the oligonucleotide is designed for RNase H junction antisense, splice blocking antisense, siRNA, anti-miR, U1 adapter or aptamer therapy. Optionally, a stable or cleavable linker is used between the peptide and the oligonucleotide.

SPR (surface plasmon resonance) analysis was performed on the peptide-oligonucleotide conjugates. The affinity of the peptide-oligonucleotide for PD-L1 was measured by SPR using the PD-L1 moiety or the peptide-oligonucleotide conjugate as an analyte. Optionally, PD-L1 is human, murine, rat, canine, or non-human primate (e.g., cynomolgus monkey or rhesus monkey). Measurement of k of peptide-oligonucleotide conjugate to PD-L1 _{Association with} 、k _{Dissociation of} And/or K _D . Optionally, k of individual peptides (not conjugated to oligonucleotides) to PD-L1 was also measured _{Association with} 、k _{Dissociation of} And K _D . Discovery of peptide-oligonucleotidesK of acid conjugate with PD-L1 _D Sufficient to bind to the desired target cell and drive endocytic uptake or transcytosis of the peptide-oligonucleotide conjugate. Optionally, K of the peptide-oligonucleotide conjugate to PD-L1 is found _D Less than 1 μM, less than 100nM, less than 10nM, less than 1nM, or less than 0.5nM. Optionally, the peptide-oligonucleotide conjugate is found to be K with PD-L1 _D K with peptide alone _D Similarly, for example, within 100-fold, 10-fold, 5-fold, or 2-fold of each other. Optionally, k of the peptide-oligonucleotide conjugate is found _{Dissociation of} Sufficient for uptake of the peptide-oligonucleotide conjugate into endosomes and release from PD-L1 prior to circulation or release from PD-L1 following transcytosis. In some cases, increased PD-L1 binding affinity may correspond to reduced transcytosis function, wherein in some cases, increased PD-L1 binding affinity does not correspond to a change in transcytosis function as compared to the reference peptide. Let k be _{Association with} /k _{Dissociation of} The ratio of (2) may affect the transcytosis function of the peptide and thus regulate k _{Association with} And/or k _{Dissociation of} Can be used to generate PD-L1 binding peptides with optimal PD-L1 binding affinity and transcytosis function. Optionally, the linker between the peptide and the oligonucleotide and/or the oligonucleotide or peptide sequence is altered such that the modified peptide-oligonucleotide conjugate pair PD-L1 k _{Association with} 、k _{Dissociation of} And/or K _D Closer to the desired value. Optionally, comparing the different peptide-oligonucleotide conjugates and selecting the one with the most desirable k _{Association with} 、k _{Dissociation of} And/or K _D For further use.

Example 32

Extension of plasma half-life of peptide oligonucleotide complexes

This example demonstrates a method of extending the serum or plasma half-life of a peptide as disclosed herein. The peptide oligonucleotide complex having the peptide sequence of any one of SEQ ID NO. 1-SEQ ID NO. 118, SEQ ID NO. 435, SEQ ID NO. 436 or SEQ ID NO. 554-SEQ ID NO. 567 is modified (e.g. at the peptide, oligonucleotide, linker or at either end) to increase its plasma half-life. Conjugation of peptide oligonucleotide complexes with near infrared dyes such as Cy5.5 for elongating peptidesSerum half-life of the construct. Alternatively, the peptide oligonucleotide complex is conjugated to an albumin binding agent such as an album-tag or C ₁₄ -C ₁₈ Conjugation of fatty acids is used to extend plasma half-life. Optionally, plasma half-life is also optionally prolonged due to reduced immunogenicity through the use of minimal non-human protein sequences.

Example 33

Cell penetrating peptide oligonucleotide complex fusions

This example describes a fusion of a peptide with an additional cell penetrating peptide. The PD-L1 binding peptide oligonucleotide complexes of the present disclosure are chemically conjugated to or recombinantly expressed as fusions with additional cell penetrating peptide moieties. Any peptide oligonucleotide complex of the disclosure is described (e.g., comprising an oligonucleotide sequence of one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO: 492-545 linked or conjugated to SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17. The additional cell penetrating peptide moiety is one or more Arg residues conjugated, linked or fused at the N-or C-terminus of any PD-L1 binding peptide of the disclosure, such as the RRRRRRRRRRR (SEQ ID NO: 251) sequence, or a Tat peptide having the sequence YGRKRRRRRRRRR (SEQ ID NO: 276) conjugated, linked or fused to the N-or C-terminus of any PD-L1 binding peptide of the disclosure. Alternatively, the additional cell penetrating peptide moiety is selected from the group consisting of mokaline, imperatorin, cholecalciferol, hemi-calcitonin, modulin-1, modulin-2, midkine (62-104), MCoTI-II, or chlorotoxin, fused to the N-terminus or C-terminus of any PD-L1 binding peptide of the disclosure. Alternatively, the additional cell penetrating peptide moiety is selected from TAT, such as CysTAT (SEQ ID NO: 249), S19-TAT (SEQ ID NO: 250), R8 (SEQ ID NO: 251), pantp (SEQ ID NO: 252), pas-TAT (SEQ ID NO: 253), pas-R8 (SEQ ID NO: 254), pas-FHV (SEQ ID NO: 255), pas-pAntP (SEQ ID NO: 256), F2R4 (SEQ ID NO: 257), B55 (SEQ ID NO: 258), azurin (azurin) (SEQ ID NO: 259), IMT-P8 (SEQ ID NO: 260), BR2 (SEQ ID NO: 261), OMOTAG1 (SEQ ID NO: 262), OMOTAG2 (SEQ ID NO: 263), pVEC (SEQ ID NO: 264), synB3 (SEQ ID NO: 265), DPV1047 (SEQ ID NO: 266), C105Y (SEQ ID NO: 268), transporter (SEQ ID NO: 267), BBR 2 (SEQ ID NO: 267), or a fusion of any of these with the end of the present disclosure. Alternatively, the additional cell penetrating peptide moiety is fused to the N-terminus or C-terminus of any PD-L1 binding peptide of the present disclosure via a linker. The linker is selected from GGGSGGGSGGGS (SEQ ID NO: 163), KKYKPYVPVTTN (SEQ ID NO: 166) (linker from DkTx) or EPKSSDKTHT (SEQ ID NO: 167) (linker from human IgG 3) or any other linker. Alternatively, the PD-L1 binding peptide, additional cell penetrating peptide moiety, and optionally the linker are joined by other means. For example, other means include, but are not limited to, chemical conjugation at any location, fusion of additional cell penetrating peptide moieties and/or linkers to the C-terminus of the PD-L1 binding peptide, coformulation with liposomes, or other methods.

The cell penetrating peptide fusion or conjugate is administered to a subject in need thereof. The subject is a human or animal and has a disease, such as brain cancer or other brain disorder. After administration, additional cells penetrate the peptide to facilitate crossing the cell membrane to enter intracellular compartments. Alternatively, following administration, the PD-L1 binding peptide facilitates endocytosis to cells expressing PD-L1, and the PD-L1 binding peptide and/or additional cell penetrating peptide facilitate release of the oligonucleotide into the cytoplasm or other subcellular compartment.

Example 34

Peptide oligonucleotide complexes that promote nuclear localization

This example describes peptide complexes to facilitate nuclear localization. The peptides and oligonucleotides of the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are expressed recombinantly or chemically synthesized and then conjugated to a linker. The peptide of the peptide oligonucleotide complex is selected from any of SEQ ID NO. 1-SEQ ID NO. 118, SEQ ID NO. 435, SEQ ID NO. 436 or SEQ ID NO. 554-SEQ ID NO. 567. Any peptide oligonucleotide complex of the disclosure is described (e.g., comprising an oligonucleotide sequence of any one of SEQ ID NO:366-SEQ ID NO:396 or SEQ ID NO: 492-545 linked or conjugated to SEQ ID NO: 1-118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO: 554-567). Any peptide oligonucleotide complex of the present disclosure may have an oligonucleotide complementary to any target in Table 10 or to any of SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO:549 or to any genomic or ORF sequence provided in Table 17. The peptide oligonucleotide complex is conjugated, linked or fused to a nuclear localization signal, such as the four residue sequence of K-K/R-X-K/R (SEQ ID NO: 434) or a variant thereof, wherein X may be any amino acid (Lange et al, J Biol chem.2007, 2, 23; 282 (8): 5101-5). The complex is administered to a subject in need thereof. The subject is a human or animal and has a disease, such as cancer. Following administration, the PD-L1 binding peptide facilitates uptake by cells expressing PD-L1, and the nuclear localization signal facilitates transport to the nucleus, and/or the PD-L1 binding peptide facilitates endocytosis to cells expressing PD-L1.

Example 35

PD-L1 binding peptide oligonucleotide complexes for pH dependent endosomal delivery

This example describes the development and in vitro testing of PD-L1 binding peptide oligonucleotide complexes capable of dissociating from PD-L1, e.g., at endosomal pH (e.g., pH 5.5) in a pH-dependent manner.

One or more additional histidine residues are introduced into the sequence of the PD-L1 binding peptide (e.g., any of SEQ ID NO:1-SEQ ID NO:118, SEQ ID NO:435, SEQ ID NO:436 or SEQ ID NO:554-SEQ ID NO: 567) within the peptide oligonucleotide complex with the oligonucleotide. The resulting histidine-enriched PD-L1 binding peptide oligonucleotide complexes were evaluated for PD-L1 binding at various pH levels or ranges in a comparative binding experiment. Peptides with high PD-L1 binding affinity at physiological pH but significantly reduced binding affinity at lower pH levels, e.g., endosomal pH of 5.4, were selected for cell binding, uptake, and endosomal or intravesicular release experiments.

Identification and characterization of PD-L1 binding peptide oligonucleotide complexes with high endosomal delivery capacity. These results demonstrate that the PD-L1 binding peptide oligonucleotide complexes of the present disclosure can exhibit or can be modified to exhibit pH-dependent PD-L1 binding kinetics, allowing for intravesicular release of the PD-L1 binding peptide oligonucleotide complexes and PD-L1 binding peptide oligonucleotide complexes comprising one or more active agents to achieve endosomal and/or intracellular delivery. Because of dissociation from PD-L1 before PD-L1 circulates back to the cell surface, higher levels of peptide oligonucleotide complexes may be delivered to or accumulated in endosomes.

To improve intracellular delivery function, the PD-L1 binding peptide oligonucleotide complexes as described herein are optionally modified to include motifs that facilitate low pH endosomal release or escape of the peptide oligonucleotide complex or are constructed with cleavable linkers.

Cell uptake and release experiments demonstrated that PD-L1 binding peptide oligonucleotide complexes comprising motifs for low pH endosomal escape appear to be present in cytosol at higher concentrations than peptides that do not comprise motifs for low pH endosomal escape. This data demonstrates that the PD-L1-binding oligonucleotide complexes of the present disclosure can be successfully modified to achieve enhanced intra-vesicle and intracellular delivery, including delivery to subcellular compartments, while retaining their PD-L1 binding capacity. These peptide oligonucleotide complexes may be combined with various therapeutic agents and/or compounds for the treatment and/or diagnosis of diseases and conditions.

Example 36

Dose toxicity comparison of PD-L1 binding peptide oligonucleotide Complex to PD-L1 binding antibody oligonucleotide Complex

This example describes a comparison of dose toxicity of the PD-L1 binding peptide oligonucleotide complexes of the present disclosure, when administered to murine subjects, with anti-PD-L1 antibody oligonucleotide complexes. Optionally, the oligonucleotide targeting agent targets a gene encoding BACE. The anti-PD-L1 antibody oligonucleotide complex is administered to a subject at a dose of 5mg/kg, 25mg/kg or 50mg/kg corresponding to a molar dose of about 0.84nmol, 4.2nmol and 8.4nmol/25g mouse mass, respectively, as described in Couch et al, 2013 (Couch et al, sci Transl Med.2013, 5 month 1; 5 (183): 183ra 57). The PD-L1 binding peptide oligonucleotide complexes of the present disclosure are administered to a subject at a dose of about 31mg/kg at a molar concentration corresponding to about 100nmol/25g mouse mass. Alternatively, the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are administered to a subject at doses of 0.84nmol, 4.2nmol, and 8.4nmol or 100 nmol. Subjects receiving a mass of PD-L1 binding peptide oligonucleotide complex of 31mg/kg or about 100nmol/25g mice showed effective pharmacodynamic and pharmacokinetic properties over the course of at least 24 hours, with no signs of pain or toxicity. Meanwhile, subjects receiving 5mg/kg or about 0.84nmol/25g mouse mass of the anti-PD-L1 antibody oligonucleotide complex show reduced therapeutic efficacy, e.g., reduced pharmacodynamic amyloid β inhibition, compared to subjects receiving 25 or 50mg/kg or 4.2 or 0.4nmol/25kg mouse mass. A dose of 25 or 50mg/kg or 4.2 or 0.4nmol/25kg mouse mass of anti-PD-L1 antibody induces sleepiness, pain and hemolysis, or reduced reticulocyte count or other toxicity, for at least 30 minutes after administration. The results demonstrate that the therapeutic window (higher than the dose at which it would observe a therapeutic pharmacodynamic response, but lower than the dose at which it would observe toxicity) is broader for peptide-based therapeutics than for antibody-based therapeutics. The results also demonstrate that therapeutic agents based on PD-L1 binding peptide oligonucleotide complexes show less off-target binding and lower immune response than therapeutic agents based on PD-L1 binding antibodies due to the smaller protein length (about 50 amino acids) providing fewer epitopes and less surface area for adaptive immune response.

Example 37

PD-L1 binding peptide oligonucleotide complexes for treating type 2 diabetes using siRNA

This example describes the treatment of type 2 diabetes using the PD-L1 binding peptide nucleotide complexes described herein. The transcription factor FoxO1 is necessary for beta cell identity and optimal insulin production. It is inhibited (by acetylation) by transcription cofactor p300 and/or cyclic AMP response element binding (CREB) protein (CBP) resulting in reduced β -cell dedifferentiation and insulin production capacity. While systemic destruction of p300 or CBP is detrimental because they are not specific for pancreatic β cells, targeting p300 or CBP antisense constructs to pancreatic β cells by conjugation to PD-L1 binding peptides can ameliorate symptoms or progression of type 2 diabetes. This example demonstrates this approach to targeting CBP, but equivalent strategies to target p300 are also possible. The nucleic acid portion of the peptide oligonucleotide complex comprises an siRNA targeting CBP transcripts. Short sequences in CBP mRNA (e.g., 21nt sequences in CBP mRNA) were identified, starting with AA and ending with TT (or UU in RNA), G/C content was between 30-60%, and the sequences were complementary to CBP mRNA used in the complex. For example, any 21 mer that is complementary across CBP mRNA may be used that has incomplete complementarity (e.g., no more than 85% complementarity, or at least 3 to 4 mismatches) or no complementarity or low complementarity (e.g., no more than 75%, 65%, 50%, or 30% complementarity) relative to other sequences in the transcriptome (to reduce off-target effects), with optimal lengths (e.g., 21 mer +/-up to 5 nt) appropriate for RISC complexes.

The siRNA may bind to a target molecule of any one of SEQ ID NOS.546-549. Duplex structures (e.g., dsRNA) for modulating CBP mRNA may include: exemplary CBP siRNA pairs are depicted as SEQ ID NOs 532-539 provided in Table 13. It will be appreciated that within each pair of complementary sequences described (e.g., SEQ ID NO:532 and SEQ ID NO:533, SEQ ID NO:534 and SEQ ID NO:535, etc.) together are part of the same complex and are partially complementary in reverse orientation to each other.

Table 13-examples of CBP siRNA

The flanking approximately 2-3 nucleotides are joined by Phosphodiester (PO) or Phosphorothioate (PS) linkages. All other backbones are PO bonds. Sugar chemistry is RNA and can be conventional (-OH) or 2' modification (e.g., 2' -O-Me, 2' -F).

The PD-L1 binding peptide of the peptide oligonucleotide complex and the oligonucleotide are each recombinantly expressed or chemically synthesized and then conjugated together with a linker. Optionally, the linker is cleavable. Optionally, the PD-L1 binding peptide has reduced affinity for PD-L1 at a pH below 7.4. The nucleic acid portion of the peptide oligonucleotide complex targets any portion of CBP mRNA (e.g., NCBI Refseq ID NM _001079846.1CREBBP [ organism = homo sapiens ] [ GeneID = 1387], SEQ ID NO: 546), or a functional fragment thereof. Similarly, siRNA may bind to the target molecule of SEQ ID NO. 548 or a functional fragment thereof. The PD-L1 binding peptide oligonucleotide complex is administered to a subject in need thereof. The PD-L1 binding peptide oligonucleotide complex is administered intravenously, subcutaneously, intramuscularly, by suppository or orally. The subject is a human or animal. Following administration, the PD-L1 binding peptide oligonucleotide complex accumulates in pancreatic β cells and CBP mRNA is degraded. The PD-L1 binding peptide nucleotide complex ameliorates type 2 diabetes and exhibits a reduction in symptoms of type 2 diabetes. In patients, these symptoms may include increased thirst, increased frequency of urination, increased hunger, unexpected weight loss, fatigue, blurred vision, slow healing ulcers, frequent infections, numbness or tingling of the hands and feet, diabetic retinopathy, kidney disease (nephropathy), diabetic neuropathy, and macrovascular problems.

This data demonstrates that the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are effective in treating type 2 diabetes.

Example 38

PD-L1 binding peptide oligonucleotide complexes for the treatment of type 2 diabetes using vacancy-polymers

This example describes the treatment of type 2 diabetes using the PD-L1 binding peptide nucleotide complexes described herein. The transcription factor FoxO1 is necessary for beta cell identity and optimal insulin production. Inhibition (by acetylation) of it by the transcription cofactor p300 and/or CBP leads to a decrease in the dedifferentiation and insulin production capacity of the beta cells. While systemic destruction of p300 or CBP is detrimental because they are not specific for pancreatic β cells, targeting p300 or CBP antisense constructs to pancreatic β cells by conjugation to PD-L1 binding peptides can ameliorate symptoms or progression of type 2 diabetes. This example will demonstrate this approach to targeting p300, but equivalent strategies to target CBP may also be used. The nucleic acid portion of the peptide oligonucleotide complex comprises a vacancy mer targeted to the p300 gene. Short sequences in p300mRNA (e.g., 20nt sequences in p300 mRNA) were identified with G/C content exceeding 40% and the sequences complementary to the p300mRNA used in the complex. For example, any 20 mer complementary to p300mRNA may be used that has incomplete complementarity (e.g., no more than 85% complementarity, or at least 3 to 4 mismatches) or no complementarity or low complementarity (e.g., no more than 75%, 65%, 50% or 30% complementarity) relative to other sequences in the transcriptome (e.g., a 20 mer found in the p300 gene alone) to reduce off-target effects.

The single stranded structure (e.g., ssRNA or ssDNA) used to modulate p300 mRNA can include any of SEQ ID NOS: 512-531 provided in Table 5.

Any of SEQ ID NOS.512-531 may be synthesized as the corresponding RNA sequence, wherein U replaces T. The empty multimer may bind to a target molecule (NCBI Refseq ID NG _ 009817.1) derived from any of its p300 transcribed sequences of the open reading frame, which may include sequences found in its mature transcripts, including NCBI Refseq ID NM _001429.4 homo sapiens E1A binding protein p300 (EP 300), transcript variant 1, mRNA or NM_001362843.2 homo sapiens E1A binding protein p300 (EP 300), transcript variant 2, mRNA, SEQ ID NO:548 or SEQ ID NO:549.

For this example, ASOs with complete backbone PS linkages can be constructed, where all C bases are 5-methyl-C. For this example, the middle 10nt is DNA sugar and the two sides 5nt is 2' O-MOE RNA sugar.

The peptides and oligonucleotides of the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are each recombinantly expressed or chemically synthesized and then conjugated together with a linker. Optionally, the linker is cleavable. Optionally, the PD-L1 binding peptide has reduced affinity for PD-L1 at a pH below 7.4. The nucleic acid portion of the peptide oligonucleotide complex targets any portion of the p300 pre-mRNA derived from its open reading frame (NCBI Refseq ng_ 009817.1), or a functional fragment thereof, including its mature transcript, e.g. NCBI Refseq ID NM _001429.4 homo sapiens E1A binding protein p300 (EP 300), transcript variant 1, mRNA or nm_001362843.2 homo sapiens E1A binding protein p300 (EP 300), transcript variant 2, mRNA. The PD-L1 binding peptide oligonucleotide complex is administered to a subject in need thereof. The PD-L1 binding peptide oligonucleotide complex is administered directly intracranially, intravenously, subcutaneously, intramuscularly, orally or intrathecally. The subject is a human or animal. Following administration, the PD-L1 binding peptide oligonucleotide complex accumulates in pancreatic β cells and CBP mRNA is degraded. PD-L1 binding peptide nucleotide complex ameliorates type 2 diabetes: exhibiting a symptomatic relief of type 2 diabetes. In patients, these symptoms may include increased thirst, increased frequency of urination, increased hunger, unexpected weight loss, fatigue, blurred vision, slow healing ulcers, frequent infections, numbness or tingling of the hands and feet, diabetic retinopathy, kidney disease (nephropathy), diabetic neuropathy, and macrovascular problems.

Example 39

PD-L1 binding peptide oligonucleotide complexes for treating solid tumors using anti-miR

This example describes the use of the PD-L1 binding peptide nucleotide complexes described herein to treat cancer (e.g., glioblastoma multiforme (GBM), pancreatic cancer, breast cancer, colon cancer, lung cancer, head and neck cancer). Healthy tissue can express tumor suppressor genes, such as PDCD4 and PTEN, which control cell growth and apoptosis. miRNA miR-21 is a repressor of several such tumor suppressor genes, including PDCD4 and PTEN. Thus, by restoring proper expression of a tumor suppressor gene and allowing the tumor suppression system to function, the reduction of miR-21 can play a role in cancer (e.g., GBM, pancreatic cancer, breast cancer, colon cancer, lung cancer, or head and neck cancer). The nucleic acid portion of the peptide nucleotide complex comprises an anti-miR that targets miR-21 (i.e., anti-miR-21).

The mature miRNA guide strand of miR-21 is as follows: 5'-UAGCUUAUCAGACUGAUGUUGA-3' (SEQ ID NO: 397). The anti-miR nucleotide can bind to the target molecule of SEQ ID NO. 397. Base pairing with an anti-miR sequence is as follows to generate a complementary anti-miR-21 nucleic acid:

Table 15-examples of MIR-21 miRNAs and anti-miR base pairing

The optimal anti-miRNA must be matched in the seed region, typically positions 2-7 at the 5' end of the miRNA. Thus, the truncations to be tested (to minimize length while maintaining efficacy) will truncate from the 5 'end of the anti-miR to keep the 3' end matched to the miRNA seed sequence:

TABLE 16 examples of anti-miR truncations

SEQ ID NO:	Sequence(s)	Length of
			SEQ ID NO:374	UCAACAUCAGUCUGAUAAGCUA	22 nucleotides
SEQ ID NO:375	CAACAUCAGUCUGAUAAGCUA	21 nucleotides
			SEQ ID NO:376	AACAUCAGUCUGAUAAGCUA	20 nucleotides
SEQ ID NO:377	ACAUCAGUCUGAUAAGCUA	19 nucleotides
			SEQ ID NO:378	CAUCAGUCUGAUAAGCUA	18 nucleotides
SEQ ID NO:379	AUCAGUCUGAUAAGCUA	17 nucleotides
			SEQ ID NO:380	UCAGUCUGAUAAGCUA	16 nucleotides
SEQ ID NO:381	CAGUCUGAUAAGCUA	15 nucleotides

For this exemplary anti-miR strategy, either PO or PS backbone linkages are used; optionally, 1-3 terminal bonds are PS. The sugar may be a mixture of DNA, 2'-O-Me, 2' -F and/or LNA. The C base may be 5-methyl-C.

The peptides and oligonucleotides of the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are recombinantly expressed or chemically synthesized and conjugated together with a linker. Optionally, the linker is cleavable. Optionally, the peptide has reduced affinity for PD-L1 at a pH below 7.4. The nucleic acid portion of the peptide oligonucleotide complex targets any portion of miR-21 guide strand RNA (SEQ ID NO: 397) or a functional fragment thereof. The PD-L1 binding peptide oligonucleotide complex is administered to a subject in need thereof. The PD-L1 binding peptide oligonucleotide complex is administered intravenously, subcutaneously, intramuscularly, orally, intrathecally, intravitreally, or intratumorally. The subject is a human or animal. The mouse model may include any of a variety of xenografts of human tumor lines or primary tumor cells or other related cancer models. After administration, the PD-L1 binding peptide nucleotide complex accumulates in the diseased tissue and miR-21mRNA is degraded. The PD-L1 binding peptide nucleotide complex causes a tumor or cancer cell (e.g., GBM, pancreatic, breast, colon, lung, or head and neck cancer) to grow slower, stop growing, or die. May result in a reduction of symptoms of cancer (e.g., GBM, pancreatic, breast, colon, lung, head and neck). In patients: the symptoms of cancer are reduced, the tumor mass is reduced and regrowth is prevented (disease control).

This data demonstrates that the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are effective in treating cancer (e.g., GBM, pancreatic cancer, breast cancer, colon cancer, lung cancer, or head and neck cancer).

Example 40

PD-L1 binding peptide oligonucleotide complexes for treating SARS-CoV-2 using aptamers

This example describes the use of the PD-L1 binding peptide oligonucleotide complexes described herein to treat SARS-CoV-2.SARS-CoV-2 uses ACE2 as the primary receptor for infection. Based on basal PD-L1 expression in the lung, the accumulation of PD-L1 binding agents in lung tissue may lead to the reduction of SARS-CoV-2 proliferation by the introduction of ACE2 inhibiting aptamers into the tissue and thus may be useful in the prevention and treatment of covd-19. The nucleic acid portion of the peptide oligonucleotide complex comprises an aptamer targeting ACE2 protein, optionally determined using SELEX-based screening strategies. ACE2 is entrapped in the membrane, so soluble ACE2 may be a difficult reagent to screen. However, ACE 2-overexpressing cells or membrane vesicles from ACE 2-overexpressing cells may be exposed to a library of 20-40 mer sequences flanked by primer binding sites of random nature. The cells or vesicles will be thoroughly washed and then lysed to release the bound nucleic acids, which will be amplified by PCR. Negative selection was performed in ACE2 negative material to remove sequences that are not specific for ACE 2. After several rounds of positive and negative selection and amplification, a single sequence will be synthesized and tested for its ability to bind only to ACE2 expressing cells.

For such ACE2 targeting aptamers, the backbone bond may be PO or PS; one clinical example of an aptamer, pipadatinib, uses all PO bonds. The sugar may be a mixture of DNA, RNA, 2' -O-Me, 2' -O-MOE, 2' -F, LNA, or the like. Optionally, the bases are chemically modified to promote tighter binding or even covalent binding.

The peptides and oligonucleotides of the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are recombinantly expressed or chemically synthesized and then conjugated together with a linker. The nucleic acid portion of the peptide oligonucleotide complex targets any portion of the ACE2 protein or a functional fragment thereof. The PD-L1 binding peptide oligonucleotide complex is administered to a subject in need thereof. The PD-L1 binding peptide oligonucleotide complex is administered intravenously, subcutaneously, intramuscularly, orally, intrathecally, intravitreally, or intratumorally. The subject is a human or animal. In a mouse model, mice may be humanized for ACE2 expression. After administration, the PD-L1 binding peptide oligonucleotide complex reduces the ability of SARS-CoV-2 or SARS-CoV-2 spike protein pseudotyped virus to infect or re-infect immune cells. The PD-L1 binding peptide oligonucleotide complex protects against infection and/or reduces productive infection upon exposure to SARS-CoV-2 or SARS-CoV-2-spike protein pseudotype viral particles, preventing productive infection and eventual production of symptoms of COVID-19.

This data demonstrates that the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are effective in inhibiting SARS-CoV-2 infection and the development of COVID 19.

Also, severe Acute Respiratory Syndrome (SARS) is caused by SARS-associated coronavirus (SARS-CoV-1), which also uses angiotensin converting enzyme 2 (ACE 2) as its receptor on human cells. The PD-L1 binding peptide oligonucleotide complex of the invention effectively inhibits SARS-CoV-1 or SARS-CoV-1 spike protein pseudotyped virus, thereby inhibiting SARS-CoV-1 infection and SARS development. Whereas MERS-COV uses the cell receptor dipeptidyl peptidase 4 (DPP 4), the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are effective in inhibiting MERS-COV or MERS-COV spike protein pseudotyped viruses, e.g., using an aptamer targeting DPP4, thereby inhibiting SARS-COV-1 infection and MERS development.

Example 41

PD-L1 binding peptide oligonucleotide complexes for treatment of skin cancer using U1 adaptors

This example describes the treatment of skin cancer (e.g., melanoma) using the PD-L1 binding peptide oligonucleotide complexes described herein. BCL2 is an anti-apoptotic protein associated with many solid tumors. In particular melanoma, which expresses high levels of BCL2, renders it resistant to many chemotherapeutic agents known to induce apoptosis. BCL2 gene expresses BCL2 protein, and reduction of BCL2 can be used for skin cancer (e.g., melanoma). The nucleic acid portion of the peptide oligonucleotide complex targeting the BCL2 gene comprises nucleotides complementary to BCL2 pre-mRNA attached to the U1 adapter. The 3' end of the BCL2 pre-mRNA transcript maps to chromosome 18 and polyadenylation localization software PolyASite identifies the region near base 63,126,800 (assembled on hg38 gene) as a possible polyadenylation site. Short sequences in the BCL2 gene or pre-mRNA (e.g., 20nt sequences overlapping in the BCL2 pre-mRNA within 5000 bases of the polyadenylation region) were identified with G/C content of 30% -60% and sequence complementary to BCL2 pre-mRNA and placed 5' or 3' of the U1 recognition domain used in the complex (this example demonstrates 5' placement). For example, any 20 mer complementary to a BCL2 pre-mRNA region can be tested that has incomplete complementarity (e.g., no more than 85% complementarity, or at least 3 to 4 mismatches) or no complementarity or low complementarity (e.g., no more than 75%, 65%, 50%, or 30% complementarity) relative to other sequences in the transcriptome (to reduce off-target effects).

Exemplary nucleic acid sequences contain U1 adaptors for modulating BCL2mRNA that are highly active for BCL2, may include: 5'GCCGUACAGUUCCACAAAGGGCCAGGUAAGUAU-3' (SEQ ID NO: 382), wherein the underlined part (GCCGUACAGUUCCACAAAGG (SEQ ID NO: 552)) corresponds to the BCL2 recognition sequence and the italic part (GCCAGGUAAGUAU (SEQ ID NO: 370)) corresponds to the U1 recognition sequence. The U1 adapter can bind to a target pre-mRNA molecule derived from the open reading frame of BCL2 (NCBI Refseq ID: NG_ 009361.1). Any U1 adapter in table 11 can also be ligated to BCL2 recognition sequences. Sugar modifications may include 2' -O-Me, LNA, or standard RNA or DNA, among others. The backbone linkages may include PO or PS linkages.

The peptides and oligonucleotides of the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are recombinantly expressed or chemically synthesized and then conjugated together with a linker. Optionally, the linker is cleavable. Optionally, the peptide has reduced affinity for PD-L1 at a pH below 7.4. The nucleic acid portion of the peptide oligonucleotide complex targets BCL2 pre-mRNA (NCBI Refseq ID: ng_ 009361.1) derived from BCL2 open reading frame or functional fragment thereof, including mRNA NCBI Refseq IDs NM _000633.3BCL2[ organism = homo sapiens ] [ GeneID = 596] [ transcript = α ] or nm_000657.3BCL2[ organism = homo sapiens ] [ GeneID = 596] [ transcript = β ], SEQ ID No. 411 or SEQ ID No. 412. The PD-L1 binding peptide oligonucleotide complex is administered to a subject in need thereof. The PD-L1 binding peptide oligonucleotide complex is administered intravenously, subcutaneously, intramuscularly, orally, intrathecally, intravitreally, or intratumorally. The subject is a human or animal. In the mouse model, the test will be performed in a mouse xenograft model or other related model with human melanoma cell flank tumors. Following administration, the PD-L1 binding peptide nucleotide complex accumulates in diseased tissue and reduces BCL2mRNA transcription, mRNA degradation, induces apoptosis markers and reduces tumor growth, thereby treating the animal. The PD-L1 binding peptide oligonucleotide complex can improve skin cancer (e.g., melanoma). Showing a reduction in the symptoms of skin cancer (e.g. melanoma).

This data demonstrates that the PD-L1 binding peptide oligonucleotide complexes of the present disclosure are effective in treating skin cancer (e.g., melanoma).

Example 42

Oligonucleotide sequence design of peptide oligonucleotide complexes

This example describes oligonucleotide sequence design for a target binding agent capable of binding a target molecule in a peptide oligonucleotide complex. The peptide oligonucleotide complexes of the present disclosure are optionally modulated by targeting genes: single strand (ssDNA, ssRNA) or double strand (dsDNA, dsRNA) or a combination of single and double strands (e.g., having a mismatched sequence, hairpin or other structure), antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotides (ASO), micrornas (miRNA), oligonucleotides complementary to a Natural Antisense Transcript (NAT) sequence, siRNA, snRNA, a vacancy mer, anti-miR, splice blocking ASO, or U1 adaptors. The gene can be targeted for down-regulation to ameliorate a disease condition. Short overlapping sequences complementary to the gene (e.g., 12nt, 15nt, 20nt, 21nt, 25nt, or 30nt in length) are generated and tested up to the entire length of the gene to determine which provides the most effective modulation. The selectable sequence contains 3 or more mismatches with other sequences in the transcriptome. The sequences may be selected to avoid any sequence having 14 or more matches to non-target or unwanted complementary sequences. The sequences can be selected to avoid the most common 2-8nt seed region at the 5' end of the siRNA. Chemical modifications to the oligonucleotides (either simultaneously or after sequence testing) were also tested. Chemical modifications may include modifications to the ends of the oligonucleotides to reduce exonuclease cleavage, for example by placing 1-3 phosphorothioate linkages at all ends. Chemical modifications can include 2'f bases, such as 2' f pyrimidine bases, to increase stability and binding. Chemical modifications can also include 2' -OMe or 2' -O methoxyethyl bases to reduce immune activation, including counteracting immune activation that may be increased by including a 2' f base. Chemical modifications may also include the use of BNA or LNA or any other modification of the present disclosure. Optionally, the oligonucleotides are tested in a pool, e.g., 5-10 sequences at a time, to reduce to optimal sequences. Optionally, the sequences are also tested for immune activation, for example using IFIT (interferon inducible protein with a thirty-four peptide repeat sequence) or a T cell activation assay or an innate immune activation assay, such as qRT-PCR, immune cell activation or proliferation or cytokine secretion, and sequences with lower immune activation are preferred. Optionally, a non-target AA/TT sequence is added to the end of the siRNA. Optionally, sequence overhangs are added to the ends of the siRNA. The oligonucleotide sequences may be selected to be homologous to humans and other species (e.g., mice, rats, and non-human primates). Alternatively, oligonucleotide sequences directed against the same target that are different from the oligonucleotide sequences complementary to the human target for clinical development and treatment of human patients may be used in other species (e.g., mice or rats) for preclinical development. Optionally, the siRNA sequence is designed using the following method: fakhr et al Precise and efficient siRNA design: a key point in competent gene silencing Cancer Gene treatment.2016; 23,73-82.

The ability of the test oligonucleotide or peptide oligonucleotide complex to reduce the level of intact functional RNA or the level of protein encoded by the targeted RNA. The ability of an oligonucleotide or peptide oligonucleotide complex to produce a desired phenotypic response in a cell, tissue or animal, such as a decrease in tumor growth rate, a decrease in cognitive decline or a decrease in inflammation, is tested. The oligonucleotide or peptide oligonucleotide complex was also tested for safety or adverse side effects. The test is performed in vitro, in vivo or in humans. The oligonucleotide or peptide oligonucleotide complex having the most desirable properties is selected.

Example 43

Oligonucleotide sequence design of peptide oligonucleotide complexes

Selecting a target gene for enabling the target binding agent to bind to the target molecule based on the association between the expression and the disease; this may be direct (e.g., the transcript itself or a protein encoded by the transcript is associated with or results in a disease phenotype) or indirect (e.g., the transcript itself or a different gene or transcript or protein whose protein modification activity is associated with or results in a disease phenotype). The target sequence is derived from the open reading frame of the gene. The target sequence may be present in the coding or non-coding region, or may be present in the mature mRNA (spliced, polyadenylation, capping and export to the cytosol for translation) or in the immature pre-mRNA. The target binding agent will be the complement of such an open reading frame. If the target sequence is found in the mature mRNA (e.g., when siRNA is intended to be used), searching for the appropriate sequence will begin with the identification of the appropriate transcription subtype, taking into account variables such as alternative splicing or alternative transcription initiation sites. If a target sequence is found in the immature pre-mRNA (e.g., when it is planned to use a vacancy mer, splice-blocking oligonucleotide or U1 adapter), searching for the appropriate sequence will begin with identifying the complete open reading frame of the gene in question, while taking into account variables such as alternative transcription initiation sites, but less into account alternative splice subtypes. If the target is an antisense sequence (e.g., a miRNA that is to be targeted against a miR), then the sequence will be based on the mature guide-strand sequence. These reference sequences can be found in public genome databases including, but not limited to, national Center for Biotechnology Information (NCBI) or the university of california holtz division (UCSC) genome browser. The pre-mRNA sequence is identical to the genomic sequence. Optionally, the reference sequences are as given in table 17.

TABLE 17 examples of open reading frame reference sequences

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A PD-L1 binding peptide, the PD-L1 binding peptide comprising a first PD-L1 binding motif, the first PD-L1 binding motif comprising the sequence:

(a)X ¹ X ² X ³ X ⁴ X ⁵ X ⁶ CX ⁷ X ⁸ X ⁹ c (SEQ ID NO: 361), wherein X ¹ D, E, H, K, N, Q, S, T, L, V, F, Y or P; x is X ² G, E, Q or F; x is X ³ Is D or K; x is X ⁴ G, V or P; x is X ⁵ G, H, R, V, F, W or P; x is X ⁶ A, D or K; x is X ⁷ E, H, Q, L or F; x is X ⁸ D, E, R, S, T, M, L or F; and X is ⁹ G, A, D, E, H, K, R, M, L or P; or (b)

(b)X ¹ FX ² VFX ² CLX ³ X ³ C (SEQ ID NO: 363) wherein X ¹ Is K or P; x is X ² Independently D or K; and X is ³ Independently any non-cysteine amino acid.

2. The PD-L1 binding peptide of claim 1, which comprises at least six cysteine residues.

3. The PD-L1 binding peptide of claim 2, wherein the at least six cysteine residues are located at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, wherein n corresponds to the position of the first cysteine residue of the at least six cysteine residues.

4. The PD-L1 binding peptide of claim 2 or claim 3, wherein amino acid position n corresponds to amino acid position 4, such that the at least six cysteine amino acid residues are at amino acid positions 4, 8, 18, 32, 42, and 46.

5. The PD-L1 binding peptide of any one of claims 2-4, further comprising at least three disulfide bonds that connect the at least six cysteine residues.

6. The PD-L1 binding peptide of claim 5, wherein the at least three disulfide bonds will: the first cysteine residue of the at least six cysteine residues is linked to the sixth cysteine residue of the at least six cysteine residues, the second cysteine residue of the at least six cysteine residues is linked to the fifth cysteine residue of the at least six cysteine residues, and the third cysteine residue of the at least six cysteine residues is linked to the fourth cysteine residue of the at least six cysteine residues.

7. The PD-L1 binding peptide of claim 6, wherein the first cysteine residue is at amino acid position n, the second cysteine residue is at amino acid position n+4, the third cysteine residue is at amino acid position n+14, the fourth cysteine residue is at amino acid position n+28, the fifth cysteine residue is at amino acid position n+38, and the sixth cysteine residue is at amino acid position n+42.

8. The PD-L1 binding peptide of claim 6 or claim 7, wherein the first cysteine residue is at amino acid position 4, the second cysteine residue is at amino acid position 8, the third cysteine residue is at amino acid position 18, the fourth cysteine residue is at amino acid position 32, the fifth cysteine residue is at amino acid position 42, and the sixth cysteine residue is at amino acid position 46.

9. The PD-L1 binding peptide of any one of claims 1-8, further comprising a first alpha helix comprising residues n to n+20, wherein n corresponds to the amino acid position of the first cysteine residue.

10. The PD-L1 binding peptide of any one of claims 1-9, further comprising a second alpha helix comprising residues n+34 to n+44, wherein n corresponds to the amino acid position of the first cysteine residue.

11. The PD-L1 binding peptide of claim 10, wherein the second alpha helix comprises residues n+29 to n+44.

12. The PD-L1 binding peptide of any one of claims 1-11, wherein the N-terminal amino acid residue of the first PD-L1 binding motif is located at amino acid residue position n+32, wherein N corresponds to the amino acid position of the first cysteine residue.

13. The PD-L1 binding peptide of any one of claims 1-12, wherein the C-terminal amino acid residue of the first PD-L1 binding motif is located at amino acid position n+42, wherein n corresponds to the amino acid position of the first cysteine residue.

14. The PD-L1 binding peptide of any one of claims 1-13, wherein the first PD-L1 binding motif comprises the sequence of KFDVFKCLDHC (SEQ ID NO: 365).

15. The PD-L1 binding peptide of any one of claims 1-14, further comprising a second PD-L1 binding motif comprising the sequence:

(a)CX ¹ X ² X ³ CX ⁴ X ⁵ X ⁶ X ⁷ X ⁸ X ⁹ X ¹⁰ X ¹¹ X ¹² C (SEQ ID NO: 360), wherein X ¹ K, R or V; x is X ² E, Q, S, M, L or V; x is X ³ D, E, H, K, R, N, Q, S or Y; x is X ⁴ D, M or V; x is X ⁵ A, K, R, Q, S or T; x is X ⁶ A, D, E, H, Q, S, T, M, I, L, V or W; x is X ⁷ A, E, R, Q, S, T, W or P; x is X ⁸ A, E, K, R, N, Q, T, M, I, L, V or W; x is X ⁹ G, A, E, K, N, T or Y; x is X ¹⁰ G, A, D, E, H, K, R, N, Q, S, T, M, I, L, V, W, Y or P; x is X ¹¹ D, K, R, N, L or V; and X is ¹² G, A, D, T, L, W or P; or (b)

(b)CKVX ¹ CVX ¹ X ¹ X ¹ X ¹ X ² X ³ KX ¹ C (SEQ ID NO: 362), wherein X ¹ Independently any non-cysteine amino acid; x is X ² M, I, L or V; and X is ³ Y, A, H, K, R, N, Q, S or T.

16. The PD-L1 binding peptide of claim 15, wherein the N-terminal amino acid residue of the second PD-L1 binding motif is located at amino acid residue position N, wherein N corresponds to the amino acid position of the first cysteine residue.

17. The PD-L1 binding peptide of claim 15 or claim 16, wherein the C-terminal amino acid residue of the second PD-L1 binding motif is located at amino acid position n+14, wherein n corresponds to the amino acid position of the first cysteine residue.

18. The PD-L1 binding peptide of any one of claims 15-17, wherein the second PD-L1 binding motif comprises the sequence of CKVHCVKEWMAGKAC (SEQ ID NO: 364).

19. The PD-L1 binding peptide of any one of claims 1-18, wherein the PD-L1 binding peptide comprises the amino acid sequence of SEQ ID NO:358 or SEQ ID NO:359, sequence of seq id no.

20. The PD-L1 binding peptide of any one of claims 1-19, wherein the PD-L1 binding peptide comprises an amino acid sequence that hybridizes to SEQ ID NO:1, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity.

21. The PD-L1 binding peptide of any one of claims 1-19, wherein the PD-L1 binding peptide comprises an amino acid sequence that hybridizes to SEQ ID NO:2, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity.

22. The PD-L1 binding peptide of any one of claims 1-19, wherein the PD-L1 binding peptide comprises an amino acid sequence that hybridizes to SEQ ID NO:3, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity.

23. The PD-L1 binding peptide of any one of claims 1-19, wherein the PD-L1 binding peptide comprises an amino acid sequence that hybridizes to SEQ ID NO:4, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity.

24. A PD-L1 binding peptide, which comprises at least six cysteine residues and which hybridizes to SEQ ID NO:57 or SEQ ID NO:59 having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% sequence identity, said at least six cysteine residues being located at amino acid positions n, n+4, n+14, n+28, n+38 and n+42, wherein n corresponds to the position of the first cysteine residue of said at least six cysteine residues.

25. A PD-L1 binding peptide, which comprises at least eight cysteine residues and which hybridizes to SEQ ID NO:58, said at least eight cysteine residues being located at amino acid positions n, n+11, n+17, n+21, n+31, n+38, n+40 or n+44, wherein n corresponds to the position of the first cysteine residue of said at least six cysteine residues.

26. A PD-L1 binding peptide, said PD-L1 binding peptide comprising an amino acid sequence that hybridizes to SEQ ID NO:1-SEQ ID NO: 118. SEQ ID NO: 435. SEQ ID NO: 436. SEQ ID NO:437 or SEQ ID NO:554-SEQ ID NO:567 has a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity.

27. The PD-L1 binding peptide of claim 26, wherein the PD-L1 binding peptide comprises the amino acid sequence of SEQ ID NO:1-SEQ ID NO: 118. SEQ ID NO: 435. SEQ ID NO: 436. SEQ ID NO:437 or SEQ ID NO:554-SEQ ID NO:567, and a sequence of any of seq id no.

28. The PD-L1 binding peptide of any one of claims 1-27, wherein the PD-L1 binding peptide is capable of having an equilibrium dissociation constant (K) of no greater than 100nM, no greater than 50nM, no greater than 1nM, no greater than 500pM, no greater than 300pM, no greater than 250pM, or no greater than 200pM _D ) Binds to PD-L1.

29. The PD-L1 binding peptide of claim 28, wherein the PD-L1 binding peptide is capable of binding to a target antigen with an equilibrium dissociation constant (K _D ) Binds to PD-L1.

30. The PD-L1 binding peptide of any one of claims 1-29, wherein the PD-L1 binding peptide is capable of undergoing a change in equilibrium dissociation constant (K) that differs by no more than 1.5-fold, no more than 2-fold, no more than 5-fold, or no more than 10-fold _D ) Binding to human PD-L1 and cynomolgus PD-L1.

31. The PD-L1 binding peptide of any one of claims 1-30, wherein the PD-L1 binding peptide comprises at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, or at least 49 amino acid residues.

32. The PD-L1 binding peptide of any one of claims 1-31, wherein the PD-L1 binding peptide comprises 43 to 51 amino acid residues.

33. The PD-L1 binding peptide of any one of claims 1-32, wherein the PD-L1 binding peptide comprises no more than 50 amino acid residues.

34. The PD-L1 binding peptide of any one of claims 1-33, wherein the PD-L1 binding peptide comprises 43 to 49 amino acid residues.

35. The PD-L1 binding peptide of any one of claims 1-34, further comprising a half-life modulator.

36. The PD-L1 binding peptide of claim 35, wherein the half-life modulator is selected from the group consisting of: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly (amino acids), water-soluble polymers of proline, alanine and serine, water-soluble polymers containing glycine, glutamic acid and serine, fc regions, fatty acids, palmitic acid, albumin and molecules bound to albumin.

37. The PD-L1 binding peptide of claim 36, wherein the half-life modulator is an albumin binding peptide.

38. The PD-L1 binding peptide of claim 36, wherein the half-life modulator is an Fc domain.

39. The PD-L1 binding peptide of claim 36, wherein the half-life modulator is polyethylene glycol.

40. The PD-L1 binding peptide of claim 36, wherein the half-life modulator is a fatty acid.

41. A peptide complex comprising a PD-L1 binding peptide complexed with an active agent, wherein the PD-L1 binding peptide comprises:

at least six cysteine residues at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, wherein n corresponds to the amino acid position of the first cysteine residue of the at least six cysteine residues;

at least three disulfide bonds connecting the first cysteine residue to a sixth cysteine residue of the at least six cysteine residues, a second cysteine residue of the at least six cysteine residues to a fifth cysteine residue of the at least six cysteine residues, a third cysteine residue of the at least six cysteine residues to a fourth cysteine residue of the at least six cysteine residues.

42. The peptide complex of claim 41, wherein amino acid position n corresponds to amino acid position 4, such that the at least six cysteine amino acid residues are at amino acid positions 4, 8, 18, 32, 42, and 46.

43. The peptide complex of claim 41 or claim 42, wherein the PD-L1 binding peptide further comprises a first alpha helix comprising residues n to n+20, wherein n corresponds to the amino acid position of the first cysteine residue.

44. The peptide complex of any one of claims 41-43, wherein the PD-L1 binding peptide further comprises a second alpha helix comprising residues n+34 to n+44, wherein n corresponds to the amino acid position of the first cysteine residue.

45. The PD-L1 binding peptide of claim 44, wherein the second alpha helix comprises residues n+29 to n+44.

46. The peptide complex of any one of claims 41-45, wherein the PD-L1 binding peptide comprises a first PD-L1 binding motif comprising the sequence:

47. The peptide complex of claim 46, wherein the N-terminal amino acid residue of the first PD-L1 binding motif is located at amino acid residue position n+32.

48. The peptide complex of claim 46 or claim 47, wherein the C-terminal amino acid residue of the first PD-L1 binding motif is at amino acid position n+42.

49. The peptide complex of any one of claims 46-48, wherein the first PD-L1 binding motif comprises the sequence of KFDVFKCLDHC (SEQ ID NO: 365).

50. The peptide complex of any one of claims 41-49, wherein the PD-L1 binding peptide further comprises a second PD-L1 binding motif, the second PD-L1 binding motif comprising the sequence:

51. The peptide complex of claim 50, wherein the N-terminal amino acid residue of the second PD-L1 binding motif is located at amino acid residue position N.

52. The peptide complex of claim 50 or claim 51, wherein the C-terminal amino acid residue of the first PD-L1 binding motif is at amino acid position n+14.

53. The peptide complex of any one of claims 50-52, wherein the second PD-L1 binding motif comprises the sequence of CKVHCVKEWMAGKAC (SEQ ID NO: 364).

54. The peptide complex of any one of claims 41-53, wherein amino acid position n corresponds to amino acid position 4 of the PD-L1 binding peptide, such that the at least six cysteine amino acid residues are located at amino acid positions 4, 8, 18, 32, 42, and 46 of the PD-L1 binding peptide.

55. The peptide complex of any one of claims 41-54, wherein the PD-L1 binding peptide is capable of being cleaved at an equilibrium dissociation constant (K) of no greater than 100nM, no greater than 50nM, no greater than 30nM, no greater than 20nM, no greater than 1nM, no greater than 500pM, no greater than 300pM, no greater than 250pM, or no greater than 200pM _D ) Binds to PD-L1.

56. The peptide complex of any one of claims 41-55, wherein the PD-L1 binding peptide comprises at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, or at least 49 amino acid residues.

57. The peptide complex of any one of claims 41-56, wherein the PD-L1 binding peptide comprises 43 to 51 amino acid residues.

58. The peptide complex of any one of claims 41-57, wherein the PD-L1 binding peptide comprises 43 to 49 amino acid residues.

59. A peptide complex comprising the PD-L1 binding peptide of any one of claims 1-40 complexed with an active agent.

60. The peptide complex of any one of claims 41-59, wherein the active agent comprises an immune cell targeting agent.

61. The peptide complex of claim 60, wherein the immune cell targeting agent is an immune cell targeting peptide.

62. The peptide complex of claim 60 or claim 61, wherein the immune cell targeting agent comprises a single chain variable fragment (scFv), a cysteine dense peptide, a high affinity multimer, a Kong Nici domain, an affibody, an adestine protein, a nano-phenanthrene Ding Danbai, a fenobody, a β -hairpin, a staple peptide, a bicyclic peptide, an antibody fragment, a protein, a peptide fragment, a binding domain, a small molecule, or a nanobody capable of binding to an immune cell.

63. The peptide complex of any one of claims 60-62, wherein the immune cell targeting agent is capable of binding T cells, B cells, macrophages, natural killer cells, fibroblasts, regulatory T cells, regulatory immune cells, neural stem cells, or mesenchymal stem cells.

64. The peptide complex of claim 63, wherein the immune cell targeting agent is capable of binding T cells.

65. The peptide complex of claim 63, wherein the immune cell targeting agent is capable of binding regulatory T cells.

66. The peptide complex of any one of claims 60-65, wherein the immune cell targeting agent is capable of binding CD3, 4-1BB, CD28, CD137, CD89, CD16, CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX40, PD-1, CTLA-4, or STRO-1.

67. The peptide complex of claim 66, wherein the immune cell targeting agent is capable of binding CD3.

68. The peptide complex of claim 66, wherein the immune cell targeting agent is capable of binding CD25.

69. The peptide complex of claim 66, wherein the immune cell targeting agent is capable of binding 4-1BB.

70. The peptide complex of claim 66, wherein the immune cell targeting agent is capable of binding CD28.

71. The peptide complex of any one of claims 60-70, wherein the immune cell targeting agent comprises an amino acid sequence that hybridizes to SEQ ID NO:122 or SEQ ID NO:442-SEQ ID NO:491, wherein any of the sequences has at least 90% sequence identity.

72. The peptide complex of any one of claims 60-71, wherein the immune cell targeting agent is fused to a first heterodimerization domain and the PD-L1 binding peptide is fused to a second heterodimerization domain.

73. The peptide complex of any one of claims 60-71, wherein the immune cell targeting agent is fused to a second heterodimerization domain and the PD-L1 binding peptide is fused to a first heterodimerization domain.

74. The peptide complex of claim 72 or claim 73, wherein the first heterodimerization domain complexes with the second heterodimerization domain to form a heterodimer.

75. The peptide complex of any one of claims 72-74, wherein the first heterodimerization domain, the second dimerization domain, or both comprise an Fc domain.

76. The peptide complex of any one of claims 72-74, wherein the first heterodimerization domain, the second dimerization domain, or both comprise the amino acid sequence of SEQ ID NO:124-SEQ ID NO:153, or a sequence of any one of seq id no.

77. The peptide complex of any one of claims 72-76, wherein the first heterodimerization domain comprises the amino acid sequence of SEQ ID NO: 124. SEQ ID NO: 126. SEQ ID NO: 128. SEQ ID NO: 130. SEQ ID NO: 132. SEQ ID NO: 134. SEQ ID NO: 136. SEQ ID NO: 138. SEQ ID NO: 140. SEQ ID NO: 142. SEQ ID NO: 144. SEQ ID NO: 146. SEQ ID NO: 148. SEQ ID NO:150 or SEQ ID NO:152 and the second heterodimerization domain comprises the sequence of any one of SEQ ID NOs: 125. SEQ ID NO: 127. SEQ ID NO: 129. SEQ ID NO: 131. SEQ ID NO: 133. SEQ ID NO: 135. SEQ ID NO: 137. SEQ ID NO: 139. SEQ ID NO: 141. SEQ ID NO: 143. SEQ ID NO: 145. SEQ ID NO: 147. SEQ ID NO: 149. SEQ ID NO:151 or SEQ ID NO:153, or a sequence of any one of seq id no.

78. The peptide complex of any one of claims 72-76, wherein the first heterodimerization domain comprises chain 1 of the heterodimerization pair provided in table 3.

79. The peptide complex of any one of claims 72-76, wherein the second heterodimerization domain comprises chain 2 of the heterodimerization pair provided in table 3.

80. The peptide complex of any one of claims 72-76, wherein the second heterodimerization domain comprises the amino acid sequence of SEQ ID NO: 124. SEQ ID NO: 126. SEQ ID NO: 128. SEQ ID NO: 130. SEQ ID NO: 132. SEQ ID NO: 134. SEQ ID NO: 136. SEQ ID NO: 138. SEQ ID NO: 140. SEQ ID NO: 142. SEQ ID NO: 144. SEQ ID NO: 146. SEQ ID NO: 148. SEQ ID NO:150 or SEQ ID NO:152 and the first heterodimerization domain comprises the sequence of any one of SEQ ID NOs: 125. SEQ ID NO: 127. SEQ ID NO: 129. SEQ ID NO: 131. SEQ ID NO: 133. SEQ ID NO: 135. SEQ ID NO: 137. SEQ ID NO: 139. SEQ ID NO: 141. SEQ ID NO: 143. SEQ ID NO: 145. SEQ ID NO: 147. SEQ ID NO: 149. SEQ ID NO:151 or SEQ ID NO:153, or a sequence of any one of seq id no.

81. The peptide complex of any one of claims 72-76, wherein the first heterodimerization domain comprises chain 2 of the heterodimerization pair provided in table 3.

82. The peptide complex of any one of claims 72-76, wherein the second heterodimerization domain comprises chain 1 of the heterodimerization pair provided in table 3.

83. The peptide complex of any one of claims 60-82, comprising an amino acid sequence that hybridizes to SEQ ID NO:119 or SEQ ID NO:120 has a sequence of at least 90% sequence identity.

84. The peptide complex of any one of claims 60-82, comprising an amino acid sequence that hybridizes to SEQ ID NO:123 has a sequence of at least 90% sequence identity.

85. The peptide complex of any one of claims 60-71, wherein the immune cell targeting agent and the PD-L1 binding peptide are fused to a homodimerization domain.

86. The peptide complex of any one of claims 60-85, wherein the immune cell targeting agent and the PD-L1 binding peptide form a single polypeptide chain.

87. The peptide complex of claim 86, comprising an amino acid sequence identical to SEQ ID NO:121 or SEQ ID NO:438-SEQ ID NO:441 has a sequence of at least 90% sequence identity.

88. The peptide complex of any one of claims 60-87, wherein the immune cell targeting agent is linked to the PD-L1 binding peptide via a linker.

89. The peptide complex of claim 88, wherein the linker comprises a peptide linker.

90. The peptide complex of claim 88, wherein the linker comprises a small molecule linker.

91. The peptide complex of claim 88, wherein the linker comprises an Fc domain.

92. The peptide complex of any one of claims 60-91, further comprising an albumin binding domain, polyethylene glycol, or both.

93. The peptide complex of any one of claims 41-59, wherein the active agent comprises a transmembrane domain, an cytoplasmic domain, or a combination thereof.

94. The peptide complex of claim 93, wherein the active agent comprises a chimeric antigen receptor.

95. The peptide complex of claim 93 or claim 94, further comprising a T cell.

96. The peptide complex of any one of claims 41-59, wherein the active agent comprises a therapeutic agent, a detectable agent, or a combination thereof.

97. The peptide complex of claim 96, wherein the detectable agent comprises a fluorophore, a near infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radionuclide, or a radionuclide chelator.

98. The peptide complex of claim 96 or claim 97, wherein the therapeutic agent comprises an anticancer agent, a chemotherapeutic agent, a radiotherapeutic agent, an anti-inflammatory agent, a pro-inflammatory cytokine, an oligonucleotide, an immune tumor agent, or a combination thereof.

99. The peptide complex of any one of claims 96-98, wherein the active agent comprises a radioisotope.

100The peptide complex of claim 99, wherein the radioisotope comprises an alpha emitter, a beta emitter, a positron emitter, a gamma emitter, a metal, an actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, yttrium, actinium-225, lead-212, ¹¹ C or ¹⁴ C、 ¹³ N、 ¹⁸ F、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁸⁹ Zr、 ¹⁷⁷ Lu, indium-111, technetium-99 m, yttrium-90, iodine-131, iodine-123, or astatine-211.

101. The peptide complex of claim 98, wherein the oligonucleotide comprises DNA, RNA, antisense oligonucleotide, aptamer, miRNA, siRNA, alternative splicing modulator, mRNA binding sequence, miRNA binding sequence, siRNA binding sequence, RNaseH1 binding oligonucleotide, RISC binding oligonucleotide, polyadenylation modulator, or a combination thereof.

102. The peptide complex of claim 98 or claim 101, wherein the oligonucleotide comprises the amino acid sequence of SEQ ID NO:366-SEQ ID NO: 396. SEQ ID NO:492-SEQ ID NO:545 or SEQ ID NO: 552.

103. The peptide complex of claim 98 or claim 101, wherein the oligonucleotide binds a polypeptide comprising SEQ ID NO:397-SEQ ID NO:430 or SEQ ID NO:546-SEQ ID NO: 549.

104. The peptide complex of any one of claims 101-103, wherein the peptide complex remains intact after incubation in human serum.

105. The peptide complex of any one of claims 101-104, wherein at least 5% -10%, at least 10% -20%, at least 20% -30%, at least 30% -40%, at least 40% -50%, at least 50% -60%, at least 60% -70%, at least 70% -80%, at least 80% -90% or at least 90% -100% remain intact after incubation in human serum.

106. The peptide complex of any one of claims 101-105, wherein the PD-L1 binding peptide, when complexed with the oligonucleotide, has an equilibrium dissociation constant (K) for PD-L1 _D ) No greater than 10nM, 5nM, 1nM, 800pM, 600pM, 500pM, 400pM, 300pM, 250pM or 200pM.

107. The peptide complex of any one of claims 101-106, wherein the PD-L1 binding peptide has a lower affinity for PD-L1 at pH 5.5, 6.0, or 6.5 than at pH 7.4.

108. The peptide complex of claim 98, wherein the anti-inflammatory agent comprises an anti-inflammatory cytokine, a steroid, a glucocorticoid, a corticosteroid, a cytokine inhibitor, a rory inhibitor, a JAK inhibitor, a tyrosine kinase inhibitor, or a non-steroidal anti-inflammatory drug.

109. The peptide complex of claim 98, wherein the anti-cancer agent comprises an anti-tumor agent, a cytotoxic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, a retinoid, a microtubule polymerization inhibitor, a pyridobenzodiazepine dimer, or an anti-cancer antibody.

110. The peptide complex of claim 98, wherein the proinflammatory cytokine comprises tnfα, IL-2, IL-6, IL-12, IL-15, IL-21, or ifnγ.

111. The peptide complex of any one of claims 96-110, wherein the therapeutic agent comprises an oncolytic viral vector.

112. The peptide complex of any one of claims 41-111, further comprising a half-life modulator.

113. The peptide complex of claim 112, wherein the half-life modulator is selected from the group consisting of: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly (amino acids), water-soluble polymers of proline, alanine and serine, water-soluble polymers containing glycine, glutamic acid and serine, fc regions, fatty acids, palmitic acid and molecules bound to albumin.

114. The peptide complex of claim 113, wherein the albumin-binding molecule is a serum albumin binding peptide.

115. The peptide complex of any one of claims 41-114, further comprising a cell penetrating peptide.

116. The peptide complex of claim 115, wherein the cell penetrating peptide comprises SEQ ID NO:249-SEQ ID NO:341 of any one of the sequences of seq id no.

117. A pharmaceutical composition comprising the PD-L1 binding peptide of any one of claims 1-38, or the peptide complex of any one of claims 41-116, and a pharmaceutically acceptable carrier.

118. A method of inhibiting PD-L1 in a subject, the method comprising:

administering to the subject a composition comprising a PD-L1 binding peptide, the PD-L1 binding peptide comprising at least six cysteine residues, and at least three disulfide bonds connecting the at least six cysteine residues;

binding the PD-L1 binding peptide to PD-L1 on a PD-L1 positive cell; and

inhibiting the PD-L1.

119. The method of claim 118, wherein the at least six cysteine residues are at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, wherein n corresponds to the amino acid position of the first cysteine residue of the at least six cysteine residues.

120. The method of claim 118 or claim 119, wherein amino acid position n corresponds to amino acid position 4 such that the at least six cysteine amino acid residues are at amino acid positions 4, 8, 18, 32, 42, and 46.

121. A method of inhibiting PD-L1 in a subject, the method comprising:

administering to the subject a composition comprising the PD-L1 binding peptide of any one of claims 1-40;

binding the PD-L1 binding peptide to PD-L1 on a PD-L1 positive cell; and

inhibiting the PD-L1.

122. The method of any one of claims 118-121, wherein inhibiting PD-L1 comprises inhibiting the binding of PD-1 to PD-L1.

123. The method of any one of claims 118-122, further comprising reducing immunosuppression, reducing T cell depletion, restoring immune function, or a combination thereof.

124. The method of any one of claims 118-123, further comprising treating the subject for a disorder.

125. The method of claim 124, wherein the disorder is cancer, and wherein the PD-L1 positive cells are cancer cells.

126. The method of claim 125, wherein treating the cancer comprises enhancing an immune response against the cancer cells.

127. A method of delivering an active agent to a PD-L1 positive cell of a subject, the method comprising:

administering to the subject a peptide complex comprising a PD-L1 binding peptide complexed with an active agent, the PD-L1 binding peptide comprising at least six cysteine residues, and at least three disulfide bonds linking the at least six cysteine residues;

binding the PD-L1 binding peptide to a PD-L1 positive cell; and

delivering the active agent to the PD-L1 positive cells.

128. The method of claim 127, wherein the at least six cysteine residues are at amino acid positions n, n+4, n+14, n+28, n+38, and n+42, wherein n corresponds to the amino acid position of the first cysteine residue of the at least six cysteine residues.

129. The method of claim 127 or claim 128, wherein amino acid position n corresponds to amino acid position 4, such that the at least six cysteine amino acid residues are at amino acid positions 4, 8, 18, 32, 42, and 46.

130. A method of delivering an active agent to a PD-L1 positive cell of a subject, the method comprising:

administering to the subject a peptide complex comprising the PD-L1 binding peptide of any one of claims 1-40, or the peptide complex of any one of claims 41-116, complexed with an active agent;

Binding the PD-L1 binding peptide to a PD-L1 positive cell; and

delivering the active agent to the PD-L1 positive cells.

131. The method of any one of claims 118-130, wherein the active agent comprises an anticancer agent, a chemotherapeutic agent, a radiotherapeutic agent, or a pro-inflammatory cytokine.

132. The method of any one of claims 118-131, wherein the active agent comprises an oligonucleotide.

133. The method of claim 132, wherein the peptide complex remains intact after incubation in human serum.

134. The method of claim 132 or claim 133, wherein the PD-L1 binding peptide, when complexed with the oligonucleotide, has an equilibrium dissociation constant (K) of no greater than 10nM, 5nM, 1nM, 800pM, 600pM, 500pM, 400pM, 300pM, 250pM, or 200pM _D ) Binds to PD-L1.

135. The method of any one of claims 132-134, further comprising binding the oligonucleotide to a target sequence after delivery to the PD-L1 positive cell.

136. The method of claim 135, further comprising modulating alternative splicing of the target sequence, indicating the position of a polyadenylation site of the target sequence, inhibiting translation of the target sequence, inhibiting binding of the target sequence to a secondary target sequence, recruiting RISC to the target sequence, recruiting RNaseH1 to the target sequence, inducing cleavage of the target sequence, or modulating the target sequence upon binding of the oligonucleotide to the target sequence.

137. The method of claim 136, wherein the active agent comprises an anti-inflammatory cytokine, steroid, glucocorticoid, corticosteroid, or a non-steroidal anti-inflammatory drug.

138. The method of any one of claims 118-137, wherein the active agent comprises an immune cell targeting agent.

139. The method of claim 138, further comprising binding the immune cell targeting agent to an immune cell and recruiting the immune cell to the PD-L1 positive cell.

140. The method of claim 139, wherein recruiting the immune cell to the PD-L1 positive cell comprises forming an immune synapse.

141. The method of claim 140, wherein the width of the immune synapse is 3nm to 25nm, 5nm to 20nm, or 10nm to 15nm.

142. The method of claim 140 or claim 141, wherein the width of the immune synapse is no greater than 3nm, no greater than 5nm, no greater than 8nm, no greater than 10nm, no greater than 13nm, no greater than 15nm, no greater than 18nm, no greater than 20nm, no greater than 23nm, no greater than 25nm, no greater than 30nm, no greater than 35nm, no greater than 40nm, no greater than 45nm, or no greater than 50nm.

143. The method of any one of claims 139-142, wherein the immune cell comprises a T cell, B cell, macrophage, natural killer cell, fibroblast, regulatory T cell, regulatory immune cell, neural stem cell, or mesenchymal stem cell.

144. The method of any one of claims 138-143, wherein the immune cell targeting agent binds CD3, 4-1BB, CD28, CD137, CD89, CD16, CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, CD27, GITR, TIGIT, LAG3, TCR, CD40L, OX40, PD-1, CTLA-4, or STRO-1.

145. The method of claim 144, wherein the immune cell targeting agent binds CD3.

146. The method of claim 144, wherein the immune cell targeting agent binds CD25.

147. The method of claim 144, wherein the immune cell targeting agent binds 4-1BB.

148. The method of claim 144, wherein the immune cell targeting agent binds CD28.

149. The method of any one of claims 139-148, further comprising killing the PD-L1 positive cell after the immune cell is delivered to the PD-L1 positive cell.

150. The method of any one of claims 139-149, further comprising inhibiting the PD-L1 positive cell after the immune cell is delivered to the PD-L1 positive cell.

151. The method of any one of claims 138-150, wherein the immune cell targeting agent comprises a single chain variable fragment (scFv), a cysteine dense peptide, a high affinity multimer, a Kong Nici domain, an affibody, an adestin protein, a nano-phenanthrene Ding Danbai, a fenobody, a β -hairpin, a staple peptide, a bicyclic peptide, an antibody fragment, a protein, a peptide fragment, a binding domain, a small molecule, or a nano-antibody.

152. The method of any one of claims 138-151, wherein the immune cell targeting agent is fused to a first heterodimerization domain and the PD-L1 binding peptide is fused to a second heterodimerization domain.

153. The method of claim 152, wherein the first heterodimerization domain is complexed with the second heterodimerization domain to form a heterodimer.

154. The method of claim 152 or claim 153, wherein the first heterodimerization domain, the second dimerization domain, or both comprise an Fc domain.

155. The method of any one of claims 138-154, wherein the immune cell targeting agent is linked to the PD-L1 binding peptide via a linker.

156. The method of any one of claims 138-155, wherein the immune cell targeting agent is linked to the PD-L1 binding peptide via an Fc domain.

157. The method of any one of claims 138-155, wherein the immune cell targeting agent and the PD-L1 binding peptide form a single polypeptide chain.

158. The method of any one of claims 118-157, wherein the peptide complex comprises a chimeric antigen receptor.

159. The method of claim 158, wherein the active agent comprises a transmembrane domain, cytoplasmic domain, or a combination thereof.

160. The method of claim 158 or claim 159, wherein the peptide complex further comprises a T cell.

161. The method of claim 160, further comprising delivering the T cell to the PD-L1 positive cell.

162. The method of claim 161, further comprising killing the PD-L1 positive cells.

163. The method of any one of claims 118-162, further comprising treating the subject for a disorder.

164. The method of claim 163, wherein the disorder is cancer.

165. The method of claim 164, wherein the PD-L1 positive cell is a cancer cell.

166. The method of claim 164 or claim 165, wherein the cancer comprises melanoma, skin cancer, non-small cell lung cancer, kidney cancer, esophageal cancer, oral cancer, hepatocellular cancer, ovarian cancer, cervical cancer, colorectal cancer, colon cancer, rectal cancer, head and neck cancer, lymphoma, bladder cancer, liver cancer, gastric cancer, stomach cancer, breast cancer, triple negative breast cancer, pancreatic cancer, prostate cancer, mercker cell cancer, mesothelioma, brain cancer, or PD-L1 expressing cancer.

167. The method of claim 166, wherein the brain cancer comprises glioblastoma, astrocytoma, meningioma, primary brain cancer, metastatic brain cancer, PDL1 expressing cancer, or metastatic brain cancer.

168. The method of claim 163, wherein the disorder is hyperglycemia, type 1 diabetes, or type 2 diabetes.

169. The method of claim 168, wherein the PD-L1 positive cells comprise pancreatic beta cells.

170. The method of claim 168 or claim 169, wherein the immune cells are regulatory T cells, and wherein recruitment of the regulatory T cells to the pancreatic β cells protects the pancreatic β cells and prevents, reduces the effects of, reduces the symptoms of, slows the onset of, and thereby treats the hyperglycemia, the type 1 diabetes, or the type 2 diabetes in the subject.

171. The method of claim 163, wherein the disorder is an autoimmune or inflammatory disorder.

172. The method of claim 171, wherein the PD-L1 positive cells comprise pancreatic beta cells.

173. The method of claim 171 or claim 172, wherein the immune cells comprise regulatory T cells or mesenchymal stem cells.

174. The method of any one of claims 171-173, wherein the immune cell targeting agent binds CD25, CD13, CD29, CD44, CD71, CD73, CD90, CD105, CD166, or STRO-1.

175. The method of any one of claims 171-174, wherein upon recruitment to a PD-L1 positive cell, the immune cell inhibits an autoimmune or inflammatory response, thereby treating the autoimmune or inflammatory disorder.

176. The method of any of claims 171-175, wherein the autoimmune or inflammatory disorder comprises rheumatoid arthritis, atherosclerosis, ischemia reperfusion injury, colitis, psoriasis, lupus, inflammatory bowel disease, crohn's disease, ulcerative colitis, multiple sclerosis, type 1 diabetes, type 2 diabetes, or neuroinflammation.

177. The method of any one of claims 118-176, wherein the PD-L1 binding peptide has an equilibrium dissociation constant (K) of no greater than 100nM, no greater than 50nM, no greater than 1nM, no greater than 500pM, no greater than 300pM, no greater than 250pM, or no greater than 200pM _D ) Binds to PD-L1.